Down-regulation of voltage-dependent sodium channels initiated by sodium influx in developing neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dargent, B.; Couraud, F.

1990-08-01

To address the issue of whether regulatory feedback exists between the electrical activity of a neuron and ion-channel density, the authors investigated the effect of Na{sup +}-channel activators (scorpion {alpha} toxin, batrachotoxin, and veratridine) on the density of Na{sup +} channels in fetal rat brain neurons in vitro. A partial but rapid (t{sub 1/2}, 15 min) disappearance of surface Na{sup +} channels was observed as measured by a decrease in the specific binding of ({sup 3}H)saxitoxin and {sup 125}I-labeled scorpion {beta} toxin and a decrease in specific {sup 22}Na{sup +} uptake. Moreover, the increase in the number of Na{sup +}more » channels that normally occurs during neuronal maturation in vitro was inhibited by chronic channel activator treatment. The induced disappearance of Na{sup +} channels was abolished by tetrodotoxin, was found to be dependent on the external Na{sup +} concentration, and was prevented when either choline (a nonpermeant ion) or Li{sup +} (a permeant ion) was substituted for Na{sup +}. Amphotericin B, a Na{sup +} ionophore, and monensin were able to mimick the effect of Na{sup +}-channel activators, while a KCl depolarization failed to do this. This feedback regulation seems to be a neuronal property since Na{sup +}-channel density in cultured astrocytes was not affected by channel activator treatment or by amphotericin B. The present evidence suggests that an increase in intracellular Na{sup +} concentration, whether elicited by Na{sup +}-channel activators or mediated by a Na{sup +} ionophore, can induce a decrease in surface Na{sup +} channels and therefore is involved in down-regulation of Na{sup +}-channel density in fetal rat brain neurons in vitro.« less

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase

NASA Astrophysics Data System (ADS)

Wagner, John A.; Cozens, Alison L.; Schulman, Howard; Gruenert, Dieter C.; Stryer, Lubert; Gardner, Phyllis

1991-02-01

CYSTIC fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase1,2 and protein kinase C3,4. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels1-4. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2+-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2+-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.

Vascular ATP-sensitive potassium channels are over-expressed and partially regulated by nitric oxide in experimental septic shock.

PubMed

Collin, Solène; Sennoun, Nacira; Dron, Anne-Gaëlle; de la Bourdonnaye, Mathilde; Montemont, Chantal; Asfar, Pierre; Lacolley, Patrick; Meziani, Ferhat; Levy, Bruno

2011-05-01

To study the activation and expression of vascular (aorta and small mesenteric arteries) potassium channels during septic shock with or without modulation of the NO pathway. Septic shock was induced in rats by peritonitis. Selective inhibitors of vascular K(ATP) (PNU-37883A) or BK(Ca) [iberiotoxin (IbTX)] channels were used to demonstrate their involvement in vascular hyporeactivity. Vascular response to phenylephrine was measured on aorta and small mesenteric arteries mounted on a wire myograph. Vascular expression of potassium channels was studied by PCR and Western blot, in the presence or absence of 1400W, an inducible NO synthase (iNOS) inhibitor. Aortic activation of the transcriptional factor nuclear factor-kappaB (NF-κB) was assessed by electrophoretic mobility shift assay. Arterial pressure as well as in vivo and ex vivo vascular reactivity were reduced by sepsis and improved by PNU-37883A but not by IbTX. Sepsis was associated with an up-regulation of mRNA and protein expression of vascular K(ATP) channels, while expression of vascular BK(Ca) channels remained unchanged. Selective iNOS inhibition blunted the sepsis-induced increase in aortic NO, decreased NF-κB activation, and down-regulated vascular K(ATP) channel expression. Vascular K(ATP) but not BK(Ca) channels are activated, over-expressed, and partially regulated by NO via NF-κB activation during septic shock. Their selective inhibition restores arterial pressure and vascular reactivity and decreases lactate concentration. The present data suggest that selective vascular K(ATP) channel inhibitors offer potential therapeutic perspectives for septic shock.

The human red cell voltage-regulated cation channel. The interplay with the chloride conductance, the Ca(2+)-activated K(+) channel and the Ca(2+) pump.

PubMed

Bennekou, P; Kristensen, B I; Christophersen, P

2003-09-01

The activation/deactivation kinetics of the human erythrocyte voltage-dependent cation channel was characterized at the single-channel level using inside-out patches. It was found that the time dependence for voltage activation after steps to positive membrane potentials was slow ( t(1/2) about 30 s), whereas the deactivation was fast ( t(1/2) about 15 ms). Both activation and deactivation of this channel were also demonstrated in intact red cells in suspension. At very positive membrane potentials generated by suspension in extracellular low Cl(-) concentrations, the cation conductance switched on with a time constant of about 2 min. Deactivation of the cation channel was clearly demonstrated during transient activation of the Gárdos channel elicited by Ca(2+) influx via the cation channel and ensuing efflux via the Ca(2+) pump. Thus, the voltage-dependent cation channel, the Gárdos channel and the Ca(2+) pump constitute a coupled feedback-regulated system that may become operative under physiological conditions.

Regulation of Transient Receptor Potential channels by the phospholipase C pathway

PubMed Central

Rohacs, Tibor

2013-01-01

Transient Receptor Potential (TRP) channels were discovered while analyzing visual mutants in drosophila. The protein encoded by the transient receptor potential (trp) gene is a Ca2+ permeable cation channel activated downstream of the phospholipase C (PLC) pathway. While searching for homologues in other organisms, a surprisingly large number of mammalian TRP channels were cloned. The regulation of TRP channels is quite diverse, but many of them are either activated downstream of the PLC pathway, or modulated by it. This review will summarize the current knowledge on regulation of TRP channels by the PLC pathway, with special focus on TRPC-s, which can be considered as effectors of the PLC pathway, and the heat and capsaicin sensitive TRPV1, which is modulated by the PLC pathway in a complex manner. PMID:23916247

A unique alkaline pH-regulated and fatty acid-activated tandem pore domain potassium channel (K2P) from a marine sponge

PubMed Central

Wells, Gregory D.; Tang, Qiong-Yao; Heler, Robert; Tompkins-MacDonald, Gabrielle J.; Pritchard, Erica N.; Leys, Sally P.; Logothetis, Diomedes E.; Boland, Linda M.

2012-01-01

SUMMARY A cDNA encoding a potassium channel of the two-pore domain family (K2P, KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK2P cannot be placed into any of the established functional groups of mammalian K2P channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK2P. In whole cells, non-inactivating, voltage-independent, outwardly rectifying K+ currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC50 ∼30 μmol l–1), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK2P but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K2P channels, the sponge K2P channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pKa 8.18) activated the AquK2P channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K2P channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K2P channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA. PMID:22723483

A unique alkaline pH-regulated and fatty acid-activated tandem pore domain potassium channel (K₂P) from a marine sponge.

PubMed

Wells, Gregory D; Tang, Qiong-Yao; Heler, Robert; Tompkins-MacDonald, Gabrielle J; Pritchard, Erica N; Leys, Sally P; Logothetis, Diomedes E; Boland, Linda M

2012-07-15

A cDNA encoding a potassium channel of the two-pore domain family (K(2P), KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK(2P) cannot be placed into any of the established functional groups of mammalian K(2P) channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK(2P). In whole cells, non-inactivating, voltage-independent, outwardly rectifying K(+) currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC(50) ∼30 μmol l(-1)), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK(2P) but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K(2P) channels, the sponge K(2P) channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pK(a) 8.18) activated the AquK(2P) channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K(2P) channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K(2P) channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA.

Lysophospholipids stimulate prostate cancer cell migration via TRPV2 channel activation.

PubMed

Monet, Michaël; Gkika, Dimitra; Lehen'kyi, V'yacheslav; Pourtier, Albin; Vanden Abeele, Fabien; Bidaux, Gabriel; Juvin, Véronique; Rassendren, François; Humez, Sandrine; Prevarsakaya, Natalia

2009-03-01

The physiological role, the mechanisms of activation, as well as the endogenous regulators for the non-selective cationic channel TRPV2 are not known so far. In the present work we report that endogenous lysophospholipids such as lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) induce a calcium influx via TRPV2 channel. This activation is dependent on the length of the side-chain and the nature of the lysophospholipid head-group. TRPV2-mediated calcium uptake stimulated by LPC and LPI occurred via Gq/Go-protein and phosphatidylinositol-3,4 kinase (PI3,4K) signalling. We have shown that the mechanism of TRPV2 activation induced by LPC and LPI is due to the TRPV2 channel translocation to the plasma membrane. The activation of TRPV2 channel by LPC and LPI leads to an increase in the cell migration of the prostate cancer cell line PC3. We have demonstrated that TRPV2 is directly involved in both steady-state and lysophospholipid-stimulated cancer cell migration. Thus, for the first time, we have identified one of the natural regulators of TRPV2 channel, one of the mechanisms of TRPV2 activation and regulation, as well as its pathophysiological role in cancer.

Insulin receptor regulates photoreceptor CNG channel activity

PubMed Central

Gupta, Vivek K.; Rajala, Ammaji

2012-01-01

Photoreceptor cyclic nucleotide gated (CNG) channels are critical elements in phototransduction and light adaptation. Here we report that insulin receptor (IR), an integral membrane protein, directly phosphorylates the CNGA1 subunit of CNG channels that in turn affects the function of these channels negatively. The IR phosphorylates Tyr498 and Tyr503 residues on CNGA1 that are situated at the membrane-cytoplasmic interface. The IR tyrosine kinase activity is essential for the inhibition of CNG channel. To maintain the channels in an off state, it is necessary not only to have a precise balance of the cGMP levels but also to have a control on the cGMP sensitivity of the CNG channels itself. In this study, we observed that the channel opens at a lower concentration of cGMP in IR−/− mice. These studies suggest that IR regulates the modulation of CNG channel activity in vivo. PMID:23032687

Insulin receptor regulates photoreceptor CNG channel activity.

PubMed

Gupta, Vivek K; Rajala, Ammaji; Rajala, Raju V S

2012-12-01

Photoreceptor cyclic nucleotide gated (CNG) channels are critical elements in phototransduction and light adaptation. Here we report that insulin receptor (IR), an integral membrane protein, directly phosphorylates the CNGA1 subunit of CNG channels that in turn affects the function of these channels negatively. The IR phosphorylates Tyr(498) and Tyr(503) residues on CNGA1 that are situated at the membrane-cytoplasmic interface. The IR tyrosine kinase activity is essential for the inhibition of CNG channel. To maintain the channels in an off state, it is necessary not only to have a precise balance of the cGMP levels but also to have a control on the cGMP sensitivity of the CNG channels itself. In this study, we observed that the channel opens at a lower concentration of cGMP in IR(-/-) mice. These studies suggest that IR regulates the modulation of CNG channel activity in vivo.

pH and external Ca(2+) regulation of a small conductance Cl(-) channel in kidney distal tubule.

PubMed

Sauvé, R; Cai, S; Garneau, L; Klein, H; Parent, L

2000-12-20

A single channel characterization of the Cl(-) channels in distal nephron was undertaken using vesicles prepared from plasma membranes of isolated rabbit distal tubules. The presence in this vesicle preparation of ClC-K type Cl(-) channels was first established by immunodetection using an antibody raised against ClC-K isoforms. A ClC-K1 based functional characterization was next performed by investigating the pH and external Ca(2+) regulation of a small conductance Cl(-) channel which we identified previously by channel incorporation experiments. Acidification of the cis (external) solution from pH 7.4 to 6.5 led to a dose-dependent inhibition of the channel open probability P(O). Similarly, changing the trans pH from 7.4 to 6.8 resulted in a 4-fold decrease of the channel P(O) with no effect on the channel conductance. Channel activity also appeared to be regulated by cis (external) Ca(2+) concentration, with a dose-dependent increase in channel activity as a function of the cis Ca(2+) concentration. It is concluded on the basis of these results that the small conductance Cl(-) channel present in rabbit distal tubules is functionally equivalent to the ClC-K1 channel in the rat. In addition, the present work constitutes the first single channel evidence for a chloride channel regulated by external Ca(2+).

The lysosomal Ca2+ release channel TRPML1 regulates lysosome size by activating calmodulin

PubMed Central

Cao, Qi; Yang, Yiming; Zhong, Xi Zoë; Dong, Xian-Ping

2017-01-01

Intracellular lysosomal membrane trafficking, including fusion and fission, is crucial for cellular homeostasis and normal cell function. Both fusion and fission of lysosomal membrane are accompanied by lysosomal Ca2+ release. We recently have demonstrated that the lysosomal Ca2+ release channel P2X4 regulates lysosome fusion through a calmodulin (CaM)-dependent mechanism. However, the molecular mechanism underlying lysosome fission remains uncertain. In this study, we report that enlarged lysosomes/vacuoles induced by either vacuolin-1 or P2X4 activation are suppressed by up-regulating the lysosomal Ca2+ release channel transient receptor potential mucolipin 1 (TRPML1) but not the lysosomal Na+ release channel two-pore channel 2 (TPC2). Activation of TRPML1 facilitated the recovery of enlarged lysosomes/vacuoles. Moreover, the effects of TRPML1 on lysosome/vacuole size regulation were eliminated by Ca2+ chelation, suggesting a requirement for TRPML1-mediated Ca2+ release. We further demonstrate that the prototypical Ca2+ sensor CaM is required for the regulation of lysosome/vacuole size by TRPML1, suggesting that TRPML1 may promote lysosome fission by activating CaM. Given that lysosome fission is implicated in both lysosome biogenesis and reformation, our findings suggest that TRPML1 may function as a key lysosomal Ca2+ channel controlling both lysosome biogenesis and reformation. PMID:28360104

Methamphetamine Regulation of Firing Activity of Dopamine Neurons

PubMed Central

Lin, Min; Sambo, Danielle

2016-01-01

Methamphetamine (METH) is a substrate for the dopamine transporter that increases extracellular dopamine levels by competing with dopamine uptake and increasing reverse transport of dopamine via the transporter. METH has also been shown to alter the excitability of dopamine neurons. The mechanism of METH regulation of the intrinsic firing behaviors of dopamine neurons is less understood. Here we identified an unexpected and unique property of METH on the regulation of firing activity of mouse dopamine neurons. METH produced a transient augmentation of spontaneous spike activity of midbrain dopamine neurons that was followed by a progressive reduction of spontaneous spike activity. Inspection of action potential morphology revealed that METH increased the half-width and produced larger coefficients of variation of the interspike interval, suggesting that METH exposure affected the activity of voltage-dependent potassium channels in these neurons. Since METH has been shown to affect Ca2+ homeostasis, the unexpected findings that METH broadened the action potential and decreased the amplitude of afterhyperpolarization led us to ask whether METH alters the activity of Ca2+-activated potassium (BK) channels. First, we identified BK channels in dopamine neurons by their voltage dependence and their response to a BK channel blocker or opener. While METH suppressed the amplitude of BK channel-mediated unitary currents, the BK channel opener NS1619 attenuated the effects of METH on action potential broadening, afterhyperpolarization repression, and spontaneous spike activity reduction. Live-cell total internal reflection fluorescence microscopy, electrophysiology, and biochemical analysis suggest METH exposure decreased the activity of BK channels by decreasing BK-α subunit levels at the plasma membrane. SIGNIFICANCE STATEMENT Methamphetamine (METH) competes with dopamine uptake, increases dopamine efflux via the dopamine transporter, and affects the excitability of dopamine neurons. Here, we identified an unexpected property of METH on dopamine neuron firing activity. METH transiently increased the spontaneous spike activity of dopamine neurons followed by a progressive reduction of the spontaneous spike activity. METH broadened the action potentials, increased coefficients of variation of the interspike interval, and decreased the amplitude of afterhyperpolarization, which are consistent with changes in the activity of Ca2+-activated potassium (BK) channels. We found that METH decreased the activity of BK channels by stimulating BK-α subunit trafficking. Thus, METH modulation of dopamine neurotransmission and resulting behavioral responses is, in part, due to METH regulation of BK channel activity. PMID:27707972

Sub-cellular distribution and translocation of TRP channels.

PubMed

Toro, Carlos A; Arias, Luis A; Brauchi, Sebastian

2011-01-01

Cellular electrical activity is the result of a highly complex processes that involve the activation of ion channel proteins. Ion channels make pores on cell membranes that rapidly transit between conductive and non-conductive states, allowing different ions to flow down their electrochemical gradients across cell membranes. In the case of neuronal cells, ion channel activity orchestrates action potentials traveling through axons, enabling electrical communication between cells in distant parts of the body. Somatic sensation -our ability to feel touch, temperature and noxious stimuli- require ion channels able to sense and respond to our peripheral environment. Sensory integration involves the summing of various environmental cues and their conversion into electrical signals. Members of the Transient Receptor Potential (TRP) family of ion channels have emerged as important mediators of both cellular sensing and sensory integration. The regulation of the spatial and temporal distribution of membrane receptors is recognized as an important mechanism for controlling the magnitude of the cellular response and the time scale on which cellular signaling occurs. Several studies have shown that this mechanism is also used by TRP channels to modulate cellular response and ultimately fulfill their physiological function as sensors. However, the inner-working of this mode of control for TRP channels remains poorly understood. The question of whether TRPs intrinsically regulate their own vesicular trafficking or weather the dynamic regulation of TRP channel residence on the cell surface is caused by extrinsic changes in the rates of vesicle insertion or retrieval remain open. This review will examine the evidence that sub-cellular redistribution of TRP channels plays an important role in regulating their activity and explore the mechanisms that control the trafficking of vesicles containing TRP channels.

Effects of nitric oxide on expressions of nitrosocysteine and calcium-activated potassium channels in the supraoptic nuclei and neural lobe of dehydrated rats

PubMed Central

Kadekaro, Massako; Su, Guangxiao; Chu, Rong; Lei, Yongzhong; Li, Junfa; Fang, Li

2007-01-01

Nitric oxide (NO) is an important gas mediator in the signal transduction cascade regulating osmotic function in the hypothalamo-neurohypophysial system. We previously found that increased nitric oxide synthase (NOS) activity in the supraoptic nuclei (SON) and neural lobe following osmotic stimulation and NO could regulate the expression of Ca2+-activated K+ channel (BK channels) protein in the magnocellular system during dehydration. The aim of the current study is to examine the role of NO in the regulation of nitrosocysteine and BK channel protein in the magnocellular system in dehydrated animals. Using Western blot analysis and quantitative immunofluorescent staining study, we found that water deprivation in rats significantly enhanced the expression of nitrosocysteine protein in SON and neural lobes. Immunohistochemistry study indicated that dehydration significantly increased the profiles of SON neurons co-expressing nitrosocysteine with BK-channel protein. Intracerebroventricular administration of L-NAME (an inhibitor of NO synthase) significantly reduced the neuronal profiles of nitrosocysteine, as well as their co-expression with BK-channel in SON of dehydrated rats. However, treatment of sodium nitroprusside (a donor of NO) increased this co-expression. Our results indicate that NO signaling cascade may control the expression of BK channels through the regulation of nitrosocysteine in SON and neural lobe of rats during osmotic regulation. PMID:17098363

Regulation of murine cystic fibrosis transmembrane conductance regulator Cl− channels expressed in Chinese hamster ovary cells

PubMed Central

Lansdell, K A; Kidd, J F; Delaney, S J; Wainwright, B J; Sheppard, D N

1998-01-01

We investigated the effect of protein kinases and phosphatases on murine cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels, expressed in Chinese hamster ovary (CHO) cells, using iodide efflux and the excised inside-out configuration of the patch-clamp technique.The protein kinase C (PKC) activator, phorbol dibutyrate, enhanced cAMP-stimulated iodide efflux. However, PKC did not augment the single-channel activity of either human or murine CFTR Cl− channels that had previously been activated by protein kinase A.Fluoride, a non-specific inhibitor of protein phosphatases, stimulated both human and murine CFTR Cl− channels. However, calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, did not enhance cAMP-stimulated iodide efflux.The alkaline phosphatase inhibitor, (−)-bromotetramisole augmented cAMP-stimulated iodide efflux and, by itself, stimulated a larger efflux than that evoked by cAMP agonists. However, (+)-bromotetramisole, the inactive enantiomer, had the same effect. For murine CFTR, neither enantiomer enhanced single-channel activity. In contrast, both enantiomers increased the open probability (Po) of human CFTR, suggesting that bromotetramisole may promote the opening of human CFTR.As murine CFTR had a low Po and was refractory to stimulation by activators of human CFTR, we investigated whether murine CFTR may open to a subconductance state. When single-channel records were filtered at 50 Hz, a very small subconductance state of murine CFTR was observed that had a Po greater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR. PMID:9769419

Differential regulation of a CLC anion channel by SPAK kinase ortholog-mediated multisite phosphorylation

PubMed Central

Miyazaki, Hiroaki

2012-01-01

Shrinkage-induced inhibition of the Caenorhabditis elegans cell volume and cell cycle-dependent CLC anion channel CLH-3b occurs by concomitant phosphorylation of S742 and S747, which are located on a 175 amino acid linker domain between cystathionine-β-synthase 1 (CBS1) and CBS2. Phosphorylation is mediated by the SPAK kinase homolog GCK-3 and is mimicked by substituting serine residues with glutamate. Type 1 serine/threonine protein phosphatases mediate swelling-induced channel dephosphorylation. S742E/S747E double mutant channels are constitutively inactive and cannot be activated by cell swelling. S742E and S747E mutant channels were fully active in the absence of GCK-3 and were inactive when coexpressed with the kinase. Both channels responded to cell volume changes. However, the S747E mutant channel activated and inactivated in response to cell swelling and shrinkage, respectively, much more slowly than either wild-type or S742E mutant channels. Slower activation and inactivation of S747E was not due to altered rates of dephosphorylation or dephosphorylation-dependent conformational changes. GCK-3 binds to the 175 amino acid inter-CBS linker domain. Coexpression of wild-type CLH-3b and GCK-3 with either wild-type or S742E linkers gave rise to similar channel activity and regulation. In contrast, coexpression with the S747E linker greatly enhanced basal channel activity and increased the rate of shrinkage-induced channel inactivation. Our findings suggest the intriguing possibility that the phosphorylation state of S742 in S747E mutant channels modulates GCK-3/channel interaction and hence channel phosphorylation. These results provide a foundation for further detailed studies of the role of multisite phosphorylation in regulating CLH-3b and GCK-3 activity. PMID:22357738

Plasma Membrane Ca2+-Permeable Channels are Differentially Regulated by Ethylene and Hydrogen Peroxide to Generate Persistent Plumes of Elevated Cytosolic Ca2+ During Transfer Cell Trans-Differentiation.

PubMed

Zhang, Hui-ming; van Helden, Dirk F; McCurdy, David W; Offler, Christina E; Patrick, John W

2015-09-01

The enhanced transport capability of transfer cells (TCs) arises from their ingrowth wall architecture comprised of a uniform wall on which wall ingrowths are deposited. The wall ingrowth papillae provide scaffolds to amplify plasma membranes that are enriched in nutrient transporters. Using Vicia faba cotyledons, whose adaxial epidermal cells spontaneously and rapidly (hours) undergo a synchronous TC trans-differentiation upon transfer to culture, has led to the discovery of a cascade of inductive signals orchestrating deposition of ingrowth wall papillae. Auxin-induced ethylene biosynthesis initiates the cascade. This in turn drives a burst in extracellular H2O2 production that triggers uniform wall deposition. Thereafter, a persistent and elevated cytosolic Ca(2+) concentration, resulting from Ca(2+) influx through plasma membrane Ca(2+)-permeable channels, generates a Ca(2+) signal that directs formation of wall ingrowth papillae to specific loci. We now report how these Ca(2+)-permeable channels are regulated using the proportionate responses in cytosolic Ca(2+) concentration as a proxy measure of their transport activity. Culturing cotyledons on various combinations of pharmacological agents allowed the regulatory influence of each upstream signal on Ca(2+) channel activity to be evaluated. The findings demonstrated that Ca(2+)-permeable channel activity was insensitive to auxin, but up-regulated by ethylene through two independent routes. In one route ethylene acts directly on Ca(2+)-permeable channel activity at the transcriptional and post-translational levels, through an ethylene receptor-dependent pathway. The other route is mediated by an ethylene-induced production of extracellular H2O2 which then acts translationally and post-translationally to up-regulate Ca(2+)-permeable channel activity. A model describing the differential regulation of Ca(2+)-permeable channel activity is presented. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface*

PubMed Central

Chin, Stephanie; Yang, Donghe; Miles, Andrew J.; Eckford, Paul D. W.; Molinski, Steven; Wallace, B. A.; Bear, Christine E.

2017-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation. PMID:28003367

Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels.

PubMed

Gschwentner, M; Nagl, U O; Wöll, E; Schmarda, A; Ritter, M; Paulmichl, M

1995-08-01

Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

Interleukin-4 activates large-conductance, calciumactivated potassium (BKCa) channels in human airway smooth muscle cells

PubMed Central

Martin, Gilles; O’Connell, Robert J.; Pietrzykowski, Andrzej Z.; Treistman, Steven N.; Ethier, Michael F.; Madison, J. Mark

2014-01-01

Large-conductance, calcium-activated potassium (BKCa) channels are regulated by voltage and near-membrane calcium concentrations and are determinants of membrane potential and excitability in airway smooth muscle cells. Since the T helper–2 (Th2) cytokine, interleukin (IL)-4, is an important mediator of airway inflammation, we investigated whether IL-4 rapidly regulated BKCa activity in normal airway smooth muscle cells. On-cell voltage clamp recordings were made on subconfluent, cultured human bronchial smooth muscle cells (HBSMC). Interleukin-4 (50 ng ml−1), IL-13 (50 ng ml−1) or histamine (10 μm) was added to the bath during the recordings. Immunofluorescence studies with selective antibodies against the α and β1 subunits of BKCa were also performed. Both approaches demonstrated that HBSMC membranes contained large-conductance channels (>200 pS) with both calcium and voltage sensitivity, all of which is characteristic of the BKCa channel. Histamine caused a rapid increase in channel activity, as expected. A new finding was that perfusion with IL-4 stimulated rapid, large increases in BKCa channel activity (77.2 ± 63.3-fold increase, P < 0.05, n = 18). This large potentiation depended on the presence of external calcium. In contrast, IL-13 (50 ng ml−1) had little effect on BKCa channel activity, but inhibited the effect of IL-4. Thus, HBSMC contain functional BKCa channels whose activity is rapidly potentiated by the cytokine, IL-4, but not by IL-13.These findings are consistent with a model in which IL-4 rapidly increases near-membrane calcium concentrations to regulate BKCa activity. PMID:18403443

Coupling of SK channels, L-type Ca2+ channels, and ryanodine receptors in cardiomyocytes.

PubMed

Zhang, Xiao-Dong; Coulibaly, Zana A; Chen, Wei Chun; Ledford, Hannah A; Lee, Jeong Han; Sirish, Padmini; Dai, Gu; Jian, Zhong; Chuang, Frank; Brust-Mascher, Ingrid; Yamoah, Ebenezer N; Chen-Izu, Ye; Izu, Leighton T; Chiamvimonvat, Nipavan

2018-03-16

Small-conductance Ca 2+ -activated K + (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca 2+ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca 2+ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca 2+ microdomains necessary for SK channel activation. SK currents coupled with Ca 2+ influx via L-type Ca 2+ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca 2+ store, suggesting that LTCCs provide the immediate Ca 2+ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Ca v 1.2 Ca 2+ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Ca v 1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca 2+ signaling.

Chemico-Genetic Identification of Drebrin as a Regulator of Calcium Responses

PubMed Central

Mercer, Jason C.; Qi, Qian; Mottram, Laurie F.; Law, Mankit; Bruce, Danny; Iyer, Archana; Morales, J. Luis; Yamazaki, Hiroyuki; Shirao, Tomoaki; Peterson, Blake R.; August, Avery

2009-01-01

Store-operated calcium channels are plasma membrane Ca2+ channels that are activated by depletion of intracellular Ca2+ stores, resulting in an increase in intracellular Ca2+ concentration, which is maintained for prolonged periods in some cell types. Increases in intracellular Ca2+ concentration serve as signals that activate a number of cellular processes, however, little is known about the regulation of these channels. We have characterized the immuno-suppressant compound BTP, which blocks store-operated channel mediated calcium influx into cells. Using an affinity purification scheme to identify potential targets of BTP, we identified the actin reorganizing protein, drebrin, and demonstrated that loss of drebrin protein expression prevents store-operated channel mediated Ca2+ entry, similar to BTP treatment. BTP also blocks actin rearrangements induced by drebrin. While actin cytoskeletal reorganization has been implicated in store-operated calcium channel regulation, little is known about actin binding proteins that are involved in this process, or how actin regulates channel function. The identification of drebrin as a mediator of this process should provide new insight into the interaction between actin rearrangement and tore-operated channel mediated calcium influx. PMID:19948240

Complex role of STIM1 in the activation of store-independent Orai1/3 channels

PubMed Central

Zhang, Wei; González-Cobos, José C.; Jardin, Isaac; Romanin, Christoph; Matrougui, Khalid

2014-01-01

Orai proteins contribute to Ca2+ entry into cells through both store-dependent, Ca2+ release–activated Ca2+ (CRAC) channels (Orai1) and store-independent, arachidonic acid (AA)-regulated Ca2+ (ARC) and leukotriene C4 (LTC4)-regulated Ca2+ (LRC) channels (Orai1/3 heteromultimers). Although activated by fundamentally different mechanisms, CRAC channels, like ARC and LRC channels, require stromal interacting molecule 1 (STIM1). The role of endoplasmic reticulum–resident STIM1 (ER-STIM1) in CRAC channel activation is widely accepted. Although ER-STIM1 is necessary and sufficient for LRC channel activation in vascular smooth muscle cells (VSMCs), the minor pool of STIM1 located at the plasma membrane (PM-STIM1) is necessary for ARC channel activation in HEK293 cells. To determine whether ARC and LRC conductances are mediated by the same or different populations of STIM1, Orai1, and Orai3 proteins, we used whole-cell and perforated patch-clamp recording to compare AA- and LTC4-activated currents in VSMCs and HEK293 cells. We found that both cell types show indistinguishable nonadditive LTC4- and AA-activated currents that require both Orai1 and Orai3, suggesting that both conductances are mediated by the same channel. Experiments using a nonmetabolizable form of AA or an inhibitor of 5-lipooxygenase suggested that ARC and LRC currents in both cell types could be activated by either LTC4 or AA, with LTC4 being more potent. Although PM-STIM1 was required for current activation by LTC4 and AA under whole-cell patch-clamp recordings in both cell types, ER-STIM1 was sufficient with perforated patch recordings. These results demonstrate that ARC and LRC currents are mediated by the same cellular populations of STIM1, Orai1, and Orai3, and suggest a complex role for both ER-STIM1 and PM-STIM1 in regulating these store-independent Orai1/3 channels. PMID:24567509

A Sodium Leak Current Regulates Pacemaker Activity of Adult Central Pattern Generator Neurons in Lymnaea Stagnalis

PubMed Central

Lu, Tom Z.; Feng, Zhong-Ping

2011-01-01

The resting membrane potential of the pacemaker neurons is one of the essential mechanisms underlying rhythm generation. In this study, we described the biophysical properties of an uncharacterized channel (U-type channel) and investigated the role of the channel in the rhythmic activity of a respiratory pacemaker neuron and the respiratory behaviour in adult freshwater snail Lymnaea stagnalis. Our results show that the channel conducts an inward leak current carried by Na+ (ILeak-Na). The ILeak-Na contributed to the resting membrane potential and was required for maintaining rhythmic action potential bursting activity of the identified pacemaker RPeD1 neurons. Partial knockdown of the U-type channel suppressed the aerial respiratory behaviour of the adult snail in vivo. These findings identified the Na+ leak conductance via the U-type channel, likely a NALCN-like channel, as one of the fundamental mechanisms regulating rhythm activity of pacemaker neurons and respiratory behaviour in adult animals. PMID:21526173

Dual regulation of the native ClC-K2 chloride channel in the distal nephron by voltage and pH

PubMed Central

Pinelli, Laurent; Nissant, Antoine; Edwards, Aurélie; Paulais, Marc

2016-01-01

ClC-K2, a member of the ClC family of Cl− channels and transporters, forms the major basolateral Cl− conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl− absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl−, and Ca2+ on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca2+ strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl− has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl−/HCO3− exchange in type B intercalated cells. PMID:27574292

Dual regulation of the native ClC-K2 chloride channel in the distal nephron by voltage and pH.

PubMed

Pinelli, Laurent; Nissant, Antoine; Edwards, Aurélie; Lourdel, Stéphane; Teulon, Jacques; Paulais, Marc

2016-09-01

ClC-K2, a member of the ClC family of Cl(-) channels and transporters, forms the major basolateral Cl(-) conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl(-) absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl(-), and Ca(2+) on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca(2+) strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl(-) has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl(-)/HCO3 (-) exchange in type B intercalated cells. © 2016 Pinelli et al.

Altered Regulation of Airway Epithelial Cell Chloride Channels in Cystic Fibrosis

NASA Astrophysics Data System (ADS)

Frizzell, Raymond A.; Rechkemmer, Gerhard; Shoemaker, Richard L.

1986-08-01

In many epithelial cells the chloride conductance of the apical membrane increases during the stimulation of electrolyte secretion. Single-channel recordings from human airway epithelial cells showed that β -adrenergic stimulation evoked apical membrane chloride channel activity, but this response was absent in cells from patients with cystic fibrosis (CF). However, when membrane patches were excised from CF cells into media containing sufficient free calcium (approximately 180 nanomolar), chloride channels were activated. The chloride channels of CF cells were similar to those of normal cells as judged by their current-voltage relations, ion selectivity, and kinetic behavior. These findings demonstrate the presence of chloride channels in the apical membranes of CF airway cells. Their regulation by calcium appears to be intact, but cyclic adenosine monophosphate (cAMP)-dependent control of their activity is defective.

The temperature dependence of the BK channel activity - kinetics, thermodynamics, and long-range correlations.

PubMed

Wawrzkiewicz-Jałowiecka, Agata; Dworakowska, Beata; Grzywna, Zbigniew J

2017-10-01

Large-conductance, voltage dependent, Ca 2+ -activated potassium channels (BK) are transmembrane proteins that regulate many biological processes by controlling potassium flow across cell membranes. Here, we investigate to what extent temperature (in the range of 17-37°C with ΔT=5°C step) is a regulating parameter of kinetic properties of the channel gating and memory effect in the series of dwell-time series of subsequent channel's states, at membrane depolarization and hyperpolarization. The obtained results indicate that temperature affects strongly the BK channels' gating, but, counterintuitively, it exerts no effect on the long-range correlations, as measured by the Hurst coefficient. Quantitative differences between dependencies of appropriate channel's characteristics on temperature are evident for different regimes of voltage. Examining the characteristics of BK channel activity as a function of temperature allows to estimate the net activation energy (E act ) and changes of thermodynamic parameters (ΔH, ΔS, ΔG) by channel opening. Larger E act corresponds to the channel activity at membrane hyperpolarization. The analysis of entropy and enthalpy changes of closed to open channel's transition suggest the entropy-driven nature of the increase of open state probability during voltage activation and supports the hypothesis about the voltage-dependent geometry of the channel vestibule. Copyright © 2017 Elsevier B.V. All rights reserved.

Redox regulation of neuronal voltage-gated calcium channels.

PubMed

Todorovic, Slobodan M; Jevtovic-Todorovic, Vesna

2014-08-20

Voltage-gated calcium channels are ubiquitously expressed in neurons and are key regulators of cellular excitability and synaptic transmitter release. There is accumulating evidence that multiple subtypes of voltage-gated calcium channels may be regulated by oxidation and reduction. However, the redox mechanisms involved in the regulation of channel function are not well understood. Several studies have established that both T-type and high-voltage-activated subtypes of voltage-gated calcium channel can be redox-regulated. This article reviews different mechanisms that can be involved in redox regulation of calcium channel function and their implication in neuronal function, particularly in pain pathways and thalamic oscillation. A current critical issue in the field is to decipher precise mechanisms of calcium channel modulation via redox reactions. In this review we discuss covalent post-translational modification via oxidation of cysteine molecules and chelation of trace metals, and reactions involving nitric oxide-related molecules and free radicals. Improved understanding of the roles of redox-based reactions in regulation of voltage-gated calcium channels may lead to improved understanding of novel redox mechanisms in physiological and pathological processes. Identification of redox mechanisms and sites on voltage-gated calcium channel may allow development of novel and specific ion channel therapies for unmet medical needs. Thus, it may be possible to regulate the redox state of these channels in treatment of pathological process such as epilepsy and neuropathic pain.

Dopamine negatively modulates the NCA ion channels in C. elegans

PubMed Central

Topalidou, Irini; Pereira, Laura

2017-01-01

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels. PMID:28968387

Dopamine negatively modulates the NCA ion channels in C. elegans.

PubMed

Topalidou, Irini; Cooper, Kirsten; Pereira, Laura; Ailion, Michael

2017-10-01

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels.

The Control of Male Fertility by Spermatozoan Ion Channels

PubMed Central

Lishko, Polina V.; Kirichok, Yuriy; Ren, Dejian; Navarro, Betsy; Chung, Jean-Ju

2014-01-01

Ion channels control the sperm ability to fertilize the egg by regulating sperm maturation in the female reproductive tract and by triggering key sperm physiological responses required for successful fertilization such as hyperactivated motility, chemotaxis, and the acrosome reaction. CatSper, a pH-regulated, calcium-selective ion channel, and KSper (Slo3) are core regulators of sperm tail calcium entry and sperm hyperactivated motility. Many other channels had been proposed as regulating sperm activity without direct measurements. With the development of the sperm patch-clamp technique, CatSper and KSper have been confirmed as the primary spermatozoan ion channels. In addition, the voltage-gated proton channel Hv1 has been identified in human sperm tail, and the P2X2 ion channel has been identified in the midpiece of mouse sperm. Mutations and deletions in sperm-specific ion channels affect male fertility in both mice and humans without affecting other physiological functions. The uniqueness of sperm ion channels makes them ideal pharmaceutical targets for contraception. In this review we discuss how ion channels regulate sperm physiology. PMID:22017176

TMEM150C/Tentonin3 Is a Regulator of Mechano-gated Ion Channels.

PubMed

Anderson, Evan O; Schneider, Eve R; Matson, Jon D; Gracheva, Elena O; Bagriantsev, Sviatoslav N

2018-04-17

Neuronal mechano-sensitivity relies on mechano-gated ion channels, but pathways regulating their activity remain poorly understood. TMEM150C was proposed to mediate mechano-activated current in proprioceptive neurons. Here, we studied functional interaction of TMEM150C with mechano-gated ion channels from different classes (Piezo2, Piezo1, and the potassium channel TREK-1) using two independent methods of mechanical stimulation. We found that TMEM150C significantly prolongs the duration of the mechano-current produced by all three channels, decreases apparent activation threshold in Piezo2, and induces persistent current in Piezo1. We also show that TMEM150C is co-expressed with Piezo2 in trigeminal neurons, expanding its role beyond proprioceptors. Finally, we cloned TMEM150C from the trigeminal neurons of the tactile-foraging domestic duck and showed that it functions similarly to the mouse ortholog, demonstrating evolutionary conservation among vertebrates. Our studies reveal TMEM150C as a general regulator of mechano-gated ion channels from different classes. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Big-conductance Ca2+-activated K+ channels in physiological and pathophysiological urinary bladder smooth muscle cells

PubMed Central

Parajuli, Shankar P.; Zheng, Yun-Min; Levin, Robert; Wang, Yong-Xiao

2016-01-01

ABSTRACT Contraction and relaxation of urinary bladder smooth muscle cells (UBSMCs) represent the important physiological functions of the bladder. Contractile responses in UBSMCs are regulated by a number of ion channels including big-conductance Ca2+- activated K+ (BK) channels. Great progress has been made in studies of BK channels in UBSMCs. The intent of this review is to summarize recent exciting findings with respect to the functional interactions of BK channels with muscarinic receptors, ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) as well as their functional importance under normal and pathophysiological conditions. BK channels are highly expressed in UBSMCs. Activation of muscarinic M3 receptors inhibits the BK channel activity, facilitates opening of voltage-dependent Ca2+ (CaV) channels, and thereby enhances excitability and contractility of UBSMCs. Signaling molecules and regulatory mechanisms involving RyRs and IP3Rs have a significant effect on functions of BK channels and thereby regulate cellular responses in UBSMCs under normal and pathophysiological conditions including overactive bladders. Moreover, BK channels may represent a novel target for the treatment of bladder dysfunctions. PMID:27101440

A serine residue in ClC-3 links phosphorylation-dephosphorylation to chloride channel regulation by cell volume.

PubMed

Duan, D; Cowley, S; Horowitz, B; Hume, J R

1999-01-01

In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, ICl.vol.

Calcium feedback mechanisms regulate oscillatory activity of a TRP-like Ca2+ conductance in C. elegans intestinal cells

PubMed Central

Estevez, Ana Y; Strange, Kevin

2005-01-01

Inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations in Caenorhabditis elegans intestinal epithelial cells regulate the nematode defecation cycle. The role of plasma membrane ion channels in intestinal cell oscillatory Ca2+ signalling is unknown. We have shown previously that cultured intestinal cells express a Ca2+-selective conductance, IORCa, that is biophysically similar to TRPM7 currents. IORCa activates slowly and stabilizes when cells are patch clamped with pipette solutions containing 10 mm BAPTA and free Ca2+ concentrations of ∼17 nm. However, when BAPTA concentration is lowered to 1 mm, IORCa oscillates. Oscillations in channel activity induced simultaneous oscillations in cytoplasmic Ca2+ levels. Removal of extracellular Ca2+ inhibited IORCa oscillations, whereas readdition of Ca2+ to the bath caused a rapid and transient reactivation of the current. Experimental manoeuvres that elevated intracellular Ca2+ blocked current oscillations. Elevation of intracellular Ca2+ in the presence of 10 mm BAPTA to block IORCa oscillations led to a dose-dependent increase in the rate of current activation. At intracellular Ca2+ concentrations of 250 nm, current activation was transient. Patch pipette solutions buffered with 1–4 mm of either BAPTA or EGTA gave rise to similar patterns of IORCa oscillations. We conclude that changes in Ca2+ concentration close to the intracellular opening of the channel pore regulate channel activity. Low concentrations of Ca2+ activate the channel. As Ca2+ enters and accumulates near the pore mouth, channel activity is inhibited. Oscillating plasma membrane Ca2+ entry may play a role in generating intracellular Ca2+ oscillations that regulate the C. elegans defecation rhythm. PMID:15961418

PIP₂ modulation of Slick and Slack K⁺ channels.

PubMed

de los Angeles Tejada, Maria; Jensen, Lars Jørn; Klaerke, Dan A

2012-07-27

Slick and Slack are members of the Slo family of high-conductance potassium channels. These channels are activated by Na(+) and Cl(-) and are highly expressed in the CNS, where they are believed to contribute to the resting membrane potential of neurons and the control of excitability. Herein, we provide evidence that Slick and Slack channels are regulated by the phosphoinositide PIP(2). Two stereoisomers of PIP(2) were able to exogenously activate Slick and Slack channels expressed in Xenopus oocytes, and in addition, it is shown that Slick and Slack channels are modulated by endogenous PIP(2). The activating effect of PIP(2) appears to occur by direct interaction with lysine 306 in Slick and lysine 339 in Slack, located at the proximal C-termini of both channels. Overall, our data suggest that PIP(2) is an important regulator of Slick and Slack channels, yet it is not involved in the recently described cell volume sensitivity of Slick channels, since mutated PIP(2)-insensitive Slick channels retained their sensitivity to cell volume. Copyright © 2012 Elsevier Inc. All rights reserved.

Kv7 channels critically determine coronary artery reactivity: left-right differences and down-regulation by hyperglycaemia.

PubMed

Morales-Cano, Daniel; Moreno, Laura; Barreira, Bianca; Pandolfi, Rachele; Chamorro, Virginia; Jimenez, Rosario; Villamor, Eduardo; Duarte, Juan; Perez-Vizcaino, Francisco; Cogolludo, Angel

2015-04-01

Voltage-gated potassium channels encoded by KCNQ genes (Kv7 channels) are emerging as important regulators of vascular tone. In this study, we analysed the contribution of Kv7 channels to the vasodilation induced by hypoxia and the cyclic AMP pathway in the coronary circulation. We also assessed their regional distribution and possible impairment by diabetes. We examined the effects of Kv7 channel modulators on K+ currents and vascular reactivity in rat left and right coronary arteries (LCAs and RCAs, respectively). Currents from LCA were more sensitive to Kv7 channel inhibitors (XE991, linopirdine) and activators (flupirtine, retigabine) than those from RCA. Accordingly, LCAs were more sensitive than RCAs to the relaxation induced by Kv7 channel enhancers. Likewise, relaxation induced by the adenylyl cyclase activator forskolin and hypoxia, which were mediated through Kv7 channel activation, were greater in LCA than in RCA. KCNQ1 and KCNQ5 expression was markedly higher in LCA than in RCA. After incubation with high glucose (HG, 30 mmol/L), myocytes from LCA, but not from RCA, were more depolarized and showed reduced Kv7 currents. In HG-incubated LCA, the effects of Kv7 channel modulators and forskolin were diminished, and the expression of KCNQ1 and KCNQ5 was reduced. Finally, vascular responses induced by Kv7 channel modulators were impaired in LCA, but not in RCA, from type 1 diabetic rats. Our results reveal that the high expression and function of Kv7 channels in the LCA and their down-regulation by diabetes critically determine the sensitivity to key regulators of coronary tone. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

KCNQ channels determine serotonergic modulation of ventral surface chemoreceptors and respiratory drive

PubMed Central

Hawryluk, Joanna M.; Moreira, Thiago S.; Takakura, Ana C.; Wenker, Ian C.; Tzingounis, Anastasios V.; Mulkey, Daniel K.

2012-01-01

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) regulate breathing in response to CO2/H+ changes. Their activity is also sensitive to neuromodulatory inputs from multiple respiratory centers, and thus they serve as a key nexus of respiratory control. However, molecular mechanisms that control their activity and susceptibility to neuromodulation are unknown. Here, we show in vitro and in vivo that KCNQ channels are critical determinants of RTN neural activity. In particular, we find that pharmacological block of KCNQ channels (XE991, 10 μM) increased basal activity and CO2-responsivness of RTN neurons in rat brain slices; whereas KCNQ channel activation (retigabine 2–40 μM) silenced these neurons. Interestingly, we also find that KCNQ and apamin sensitive SK channels act synergistically to regulate firing rate of RTN chemoreceptors; simultaneous blockade of both channels led to a increase in CO2-responsivness. Furthermore, we also show that KCNQ channels but not SK channels are downstream effectors of serotonin modulation of RTN activity in vitro. In contrast, inhibition of KCNQ channel did not prevent modulation of RTN activity by Substance P or TRH; previously identified neuromodulators of RTN chemoreception. Importantly, we also show that KCNQ channels are critical for RTN activity in vivo. Inhibition of KCNQ channels lowered the CO2 threshold for phrenic nerve discharge in anesthetized rats and decreased the ventilatory response to serotonin in awake and anesthetized animals. Given that serotonergic dysfunction may contribute to respiratory failure, our findings suggest KCNQ channels as a new therapeutic avenue for respiratory complications associated with multiple neurological disorders. PMID:23175845

Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function

PubMed Central

Saponaro, Andrea; Pauleta, Sofia R.; Cantini, Francesca; Matzapetakis, Manolis; Hammann, Christian; Donadoni, Chiara; Hu, Lei; Thiel, Gerhard; Banci, Lucia; Santoro, Bina; Moroni, Anna

2014-01-01

cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD. PMID:25197093

A Voltage Dependent Non-Inactivating Na+ Channel Activated during Apoptosis in Xenopus Oocytes

PubMed Central

Englund, Ulrika H.; Gertow, Jens; Kågedal, Katarina; Elinder, Fredrik

2014-01-01

Ion channels in the plasma membrane are important for the apoptotic process. Different types of voltage-gated ion channels are up-regulated early in the apoptotic process and block of these channels prevents or delays apoptosis. In the present investigation we examined whether ion channels are up-regulated in oocytes from the frog Xenopus laevis during apoptosis. The two-electrode voltage-clamp technique was used to record endogenous ion currents in the oocytes. During staurosporine-induced apoptosis a voltage-dependent Na+ current increased three-fold. This current was activated at voltages more positive than 0 mV (midpoint of the open-probability curve was +55 mV) and showed almost no sign of inactivation during a 1-s pulse. The current was resistant to the Na+-channel blockers tetrodotoxin (1 µM) and amiloride (10 µM), while the Ca2+-channel blocker verapamil (50 µM) in the bath solution completely blocked the current. The intracellular Na+ concentration increased in staurosporine-treated oocytes, but could be prevented by replacing extracellular Na+ whith either K+ or Choline+. Prevention of this influx of Na+ also prevented the STS-induced up-regulation of the caspase-3 activity, suggesting that the intracellular Na+ increase is required to induce apoptosis. Taken together, we have found that a voltage dependent Na+ channel is up-regulated during apoptosis and that influx of Na+ is a crucial step in the apoptotic process in Xenopus oocytes. PMID:24586320

Klotho Up-regulates Renal Calcium Channel Transient Receptor Potential Vanilloid 5 (TRPV5) by Intra- and Extracellular N-glycosylation-dependent Mechanisms*

PubMed Central

Wolf, Matthias T. F.; An, Sung-Wan; Nie, Mingzhu; Bal, Manjot S.; Huang, Chou-Long

2014-01-01

The anti-aging protein Klotho is a type 1 membrane protein produced predominantly in the distal convoluted tubule. The ectodomain of Klotho is cleaved and secreted into the urine to regulate several ion channels and transporters. Secreted Klotho (sKL) up-regulates the TRPV5 calcium channel from the cell exterior by removing sialic acids from N-glycan of the channel and inhibiting its endocytosis. Because TRPV5 and Klotho coexpress in the distal convoluted tubule, we investigated whether Klotho regulates TRPV5 action from inside the cell. Whole-cell TRPV5-mediated channel activity was recorded in HEK cells coexpressing TRPV5 and sKL or membranous Klotho (mKL). Transfection of sKL, but not mKL, produced detectable Klotho protein in cell culture media. As for sKL, mKL increased TRPV5 current density. The role of sialidase activity of mKL acting inside is supported by findings that mutations of putative sialidase activity sites in sKL and mKL abrogated the regulation of TRPV5 but that the extracellular application of a sialidase inhibitor prevented the regulation of TRPV5 by sKL only. Mechanistically, coexpression with a dominant-negative dynamin II prevented the regulation of TRPV5 by sKL but not by mKL. In contrast, blocking forward trafficking by brefeldin A prevented the effect with mKL but not with sKL. Therefore, Klotho up-regulates TRPV5 from both the inside and outside of cells. The intracellular action of Klotho is likely due to enhanced forward trafficking of channel proteins, whereas the extracellular action is due to inhibition of endocytosis. Both effects involve putative Klotho sialidase activity. These effects of Klotho may play important roles regarding calcium reabsorption in the kidney. PMID:25378396

N-(2-methoxyphenyl) benzenesulfonamide, a novel regulator of neuronal G protein-gated inward rectifier K+ channels.

PubMed

Walsh, Kenneth B; Gay, Elaine A; Blough, Bruce E; Geurkink, David W

2017-11-15

G protein-gated inward rectifier K + (GIRK) channels are members of the super-family of proteins known as inward rectifier K + (Kir) channels and are expressed throughout the peripheral and central nervous systems. Neuronal GIRK channels are the downstream targets of a number of neuromodulators including opioids, somatostatin, dopamine and cannabinoids. Previous studies have demonstrated that the ATP-sensitive K + channel, another member of the Kir channel family, is regulated by sulfonamide drugs. Therefore, to determine if sulfonamides also modulate GIRK channels, we screened a library of arylsulfonamide compounds using a GIRK channel fluorescent assay that utilized pituitary AtT20 cells expressing GIRK channels along with the somatostatin type-2 and -5 receptors. Enhancement of the GIRK channel fluorescent signal by one compound, N-(2-methoxyphenyl) benzenesulfonamide (MPBS), was dependent on the activation of the channel by somatostatin. In whole-cell patch clamp experiments, application of MPBS both shifted the somatostatin concentration-response curve (EC 50 = 3.5nM [control] vs.1.0nM [MPBS]) for GIRK channel activation and increased the maximum GIRK current measured with 100nM somatostatin. However, GIRK channel activation was not observed when MPBS was applied to the cells in the absence of somatostatin. While the MPBS structural analog 4-fluoro-N-(2-methoxyphenyl) benzenesulfonamide also augmented the somatostatin-induced GIRK fluorescent signal, no increase in the signal was observed with the sulfonamides tolbutamide, sulfapyridine and celecoxib. In conclusion, MPBS represents a novel prototypic GPCR-dependent regulator of neuronal GIRK channels. Copyright © 2017 Elsevier B.V. All rights reserved.

Dynamics of receptor-operated Ca(2+) currents through TRPC channels controlled via the PI(4,5)P2-PLC signaling pathway.

PubMed

Mori, Masayuki X; Itsuki, Kyohei; Hase, Hideharu; Sawamura, Seishiro; Kurokawa, Tatsuki; Mori, Yasuo; Inoue, Ryuji

2015-01-01

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that carry receptor-operated Ca(2+) currents (ROCs) triggered by receptor-induced, phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Within the vasculature, TRPC channel ROCs contribute to smooth muscle cell depolarization, vasoconstriction, and vascular remodeling. However, TRPC channel ROCs exhibit a variable response to receptor-stimulation, and the regulatory mechanisms governing TRPC channel activity remain obscure. The variability of ROCs may be explained by their complex regulation by PI(4,5)P2 and its metabolites, which differentially affect TRPC channel activity. To resolve the complex regulation of ROCs, the use of voltage-sensing phosphoinositide phosphatases and model simulation have helped to reveal the time-dependent contribution of PI(4,5)P2 and the possible role of PI(4,5)P2 in the regulation of ROCs. These approaches may provide unprecedented insight into the dynamics of PI(4,5)P2 regulation of TRPC channels and the fundamental mechanisms underlying transmembrane ion flow. Within that context, we summarize the regulation of TRPC channels and their coupling to receptor-mediated signaling, as well as the application of voltage-sensing phosphoinositide phosphatases to this research. We also discuss the controversial bidirectional effects of PI(4,5)P2 using a model simulation that could explain the complicated effects of PI(4,5)P2 on different ROCs.

A Co-operative Regulation of Neuronal Excitability by UNC-7 Innexin and NCA/NALCN Leak Channel

PubMed Central

2011-01-01

Gap junctions mediate the electrical coupling and intercellular communication between neighboring cells. Some gap junction proteins, namely connexins and pannexins in vertebrates, and innexins in invertebrates, may also function as hemichannels. A conserved NCA/Dmα1U/NALCN family cation leak channel regulates the excitability and activity of vertebrate and invertebrate neurons. In the present study, we describe a genetic and functional interaction between the innexin UNC-7 and the cation leak channel NCA in Caenorhabditis elegans neurons. While the loss of the neuronal NCA channel function leads to a reduced evoked postsynaptic current at neuromuscular junctions, a simultaneous loss of the UNC-7 function restores the evoked response. The expression of UNC-7 in neurons reverts the effect of the unc-7 mutation; moreover, the expression of UNC-7 mutant proteins that are predicted to be unable to form gap junctions also reverts this effect, suggesting that UNC-7 innexin regulates neuronal activity, in part, through gap junction-independent functions. We propose that, in addition to gap junction-mediated functions, UNC-7 innexin may also form hemichannels to regulate C. elegans' neuronal activity cooperatively with the NCA family leak channels. PMID:21489288

Masters or slaves? Vesicle release machinery and the regulation of presynaptic calcium channels.

PubMed

Jarvis, Scott E; Zamponi, Gerald W

2005-05-01

Calcium entry through presynaptic voltage-gated calcium channels is essential for neurotransmitter release. The two major types of presynaptic calcium channels contain a synaptic protein interaction site that physically interacts with synaptic vesicle release proteins. This is thought to tighten the coupling between the sources of calcium entry and the neurotransmitter release machinery. Conversely, the binding of synaptic proteins to presynaptic calcium channels regulates calcium channel activity. Hence, presynaptic calcium channels act not only as the masters of the synaptic release process, but also as key targets for feedback inhibition.

The TRPM7 channel kinase regulates store-operated calcium entry.

PubMed

Faouzi, Malika; Kilch, Tatiana; Horgen, F David; Fleig, Andrea; Penner, Reinhold

2017-05-15

Pharmacological and molecular inhibition of transient receptor potential melastatin 7 (TRPM7) reduces store-operated calcium entry (SOCE). Overexpression of TRPM7 in TRPM7 -/- cells restores SOCE. TRPM7 is not a store-operated calcium channel. TRPM7 kinase rather than channel modulates SOCE. TRPM7 channel activity contributes to the maintenance of store Ca 2+ levels at rest. The transient receptor potential melastatin 7 (TRPM7) is a protein that combines an ion channel with an intrinsic kinase domain, enabling it to modulate cellular functions either by conducting ions through the pore or by phosphorylating downstream proteins via its kinase domain. In the present study, we report store-operated calcium entry (SOCE) as a novel target of TRPM7 kinase activity. TRPM7-deficient chicken DT40 B lymphocytes exhibit a strongly impaired SOCE compared to wild-type cells as a result of reduced calcium release activated calcium currents, and independently of potassium channel regulation, membrane potential changes or changes in cell-cycle distribution. Pharmacological blockade of TRPM7 with NS8593 or waixenicin A in wild-type B lymphocytes results in a significant decrease in SOCE, confirming that TRPM7 activity is acutely linked to SOCE, without TRPM7 representing a store-operated channel itself. Using kinase-deficient mutants, we find that TRPM7 regulates SOCE through its kinase domain. Furthermore, Ca 2+ influx through TRPM7 is essential for the maintenance of endoplasmic reticulum Ca 2+ concentration in resting cells, and for the refilling of Ca 2+ stores after a Ca 2+ signalling event. We conclude that the channel kinase TRPM7 and SOCE are synergistic mechanisms regulating intracellular Ca 2+ homeostasis. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

PubMed

Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

2016-04-01

p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. Copyright © 2015 Elsevier Ltd. All rights reserved.

Slack Sodium-activated Potassium Channel Membrane Expression Requires p38 Mitogen-Activated Protein Kinase Phosphorylation

PubMed Central

Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

2016-01-01

p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. PMID:26721627

Redox Regulation of Neuronal Voltage-Gated Calcium Channels

PubMed Central

Jevtovic-Todorovic, Vesna

2014-01-01

Abstract Significance: Voltage-gated calcium channels are ubiquitously expressed in neurons and are key regulators of cellular excitability and synaptic transmitter release. There is accumulating evidence that multiple subtypes of voltage-gated calcium channels may be regulated by oxidation and reduction. However, the redox mechanisms involved in the regulation of channel function are not well understood. Recent Advances: Several studies have established that both T-type and high-voltage-activated subtypes of voltage-gated calcium channel can be redox-regulated. This article reviews different mechanisms that can be involved in redox regulation of calcium channel function and their implication in neuronal function, particularly in pain pathways and thalamic oscillation. Critical Issues: A current critical issue in the field is to decipher precise mechanisms of calcium channel modulation via redox reactions. In this review we discuss covalent post-translational modification via oxidation of cysteine molecules and chelation of trace metals, and reactions involving nitric oxide-related molecules and free radicals. Improved understanding of the roles of redox-based reactions in regulation of voltage-gated calcium channels may lead to improved understanding of novel redox mechanisms in physiological and pathological processes. Future Directions: Identification of redox mechanisms and sites on voltage-gated calcium channel may allow development of novel and specific ion channel therapies for unmet medical needs. Thus, it may be possible to regulate the redox state of these channels in treatment of pathological process such as epilepsy and neuropathic pain. Antioxid. Redox Signal. 21, 880–891. PMID:24161125

Regulation of Neuronal Cav3.1 Channels by Cyclin-Dependent Kinase 5 (Cdk5)

PubMed Central

González-Ramírez, Ricardo; González-Billault, Christian; Felix, Ricardo

2015-01-01

Low voltage-activated (LVA) T-type Ca2+ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca2+ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Cav3.1 to Cav3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca2+ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Cav3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Cav3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Cav3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca2+ channels. PMID:25760945

Regulation of neuronal cav3.1 channels by cyclin-dependent kinase 5 (Cdk5).

PubMed

Calderón-Rivera, Aida; Sandoval, Alejandro; González-Ramírez, Ricardo; González-Billault, Christian; Felix, Ricardo

2015-01-01

Low voltage-activated (LVA) T-type Ca2+ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca2+ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Cav3.1 to Cav3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca2+ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Cav3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Cav3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Cav3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca2+ channels.

Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9*

PubMed Central

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-01-01

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al3+ to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. PMID:25028514

Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9.

PubMed

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-09-12

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Activity-dependent downregulation of M-Type (Kv7) K⁺ channels surface expression requires the activation of iGluRs/Ca²⁺/PKC signaling pathway in hippocampal neuron.

PubMed

Li, Cai; Lu, Qing; Huang, Pengcheng; Fu, Tianli; Li, Changjun; Guo, Lianjun; Xu, Xulin

2015-08-01

M-type (Kv7) K(+) channels, encoded by KCNQ2-KCNQ5 genes, play a pivotal role in controlling neuronal excitability. However, precisely how neuronal activity regulates Kv7 channel translocation has not yet been fully defined. Here we reported activity-dependent changes in Kv7 channel subunits Kv7.2 and Kv7.3 surface expression by glutamate (glu). In the present study, we found that treatment with glutamate rapidly caused a specific decrease in M-current as well as Kv7 channel surface expression in primary cultured hippocampal neurons. The glutamate effects were mimicked by NMDA and AMPA. The glutamate effects on Kv7 channels were partially attenuated by pre-treatment of NMDA receptors antagonist d,l-APV or AMPA-KA receptors antagonist CNQX. The signal required Ca(2+) influx through L-type Ca(2+) channel and intracellular Ca(2+) elevations. PKC activation was involved in the glutamate-induced reduction of Kv7 channel surface expression. Moreover, a significant reduction of Kv7 channel surface expression occurred following glycine-induced "chem"-LTP in vitro and hippocampus-dependent behavioral learning training in vivo. These results demonstrated that activity-dependent reduction of Kv7 channel surface expression through activation of ionotropic glutamate receptors (iGluRs)/Ca(2+)/PKC signaling pathway might be an important molecular mechanism for regulation of neuronal excitability and synaptic plasticity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cholesterol regulates HERG K+ channel activation by increasing phospholipase C β1 expression.

PubMed

Chun, Yoon Sun; Oh, Hyun Geun; Park, Myoung Kyu; Cho, Hana; Chung, Sungkwon

2013-01-01

Human ether-a-go-go-related gene (HERG) K(+) channel underlies the rapidly activating delayed rectifier K(+) conductance (IKr) during normal cardiac repolarization. Also, it may regulate excitability in many neuronal cells. Recently, we showed that enrichment of cell membrane with cholesterol inhibits HERG channels by reducing the levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] due to the activation of phospholipase C (PLC). In this study, we further explored the effect of cholesterol enrichment on HERG channel kinetics. When membrane cholesterol level was mildly increased in human embryonic kidney (HEK) 293 cells expressing HERG channel, the inactivation and deactivation kinetics of HERG current were not affected, but the activation rate was significantly decelerated at all voltages tested. The application of PtdIns(4,5)P2 or inhibitor for PLC prevented the effect of cholesterol enrichment, while the presence of antibody against PtdIns(4,5)P2 in pipette solution mimicked the effect of cholesterol enrichment. These results indicate that the effect of cholesterol enrichment on HERG channel is due to the depletion of PtdIns(4,5)P2. We also found that cholesterol enrichment significantly increases the expression of β1 and β3 isoforms of PLC (PLCβ1, PLCβ3) in the membrane. Since the effects of cholesterol enrichment on HERG channel were prevented by inhibiting transcription or by inhibiting PLCβ1 expression, we conclude that increased PLCβ1 expression leads to the deceleration of HERG channel activation rate via downregulation of PtdIns(4,5)P2. These results confirm a crosstalk between two plasma membrane-enriched lipids, cholesterol and PtdIns(4,5)P2, in the regulation of HERG channels.

CFTR/ENaC dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC.

PubMed

Puga Molina, Lis C; Pinto, Nicolas A; Torres, Nicolás I; Gonzalez-Cota, Ana L; Luque, Guillermina M; Balestrini, Paula A; Romarowski, Ana; Krapf, Dario; Santi, Celia M; Trevino, Claudia L; Darszon, Alberto; Buffone, Mariano G

2018-05-09

To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3--dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (Cystic Fibrosis Transmembrane Conductance Regulator Channel) activity and for the modulation of membrane potential (Em). However, the main HCO3- transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na+/HCO3- cotransporter (NBC) and epithelial Na+ channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3- influx, resulting in lower PKA activity, and that events downstream the cAMP-activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3-  also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

X-ray structure of the mammalian GIRK2-βγ G-protein complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whorton, Matthew R.; MacKinnon, Roderick

2013-07-30

G-protein-gated inward rectifier K + (GIRK) channels allow neurotransmitters, through G-protein-coupled receptor stimulation, to control cellular electrical excitability. In cardiac and neuronal cells this control regulates heart rate and neural circuit activity, respectively. Here we present the 3.5Å resolution crystal structure of the mammalian GIRK2 channel in complex with βγ G-protein subunits, the central signalling complex that links G-protein-coupled receptor stimulation to K + channel activity. Short-range atomic and long-range electrostatic interactions stabilize four βγ G-protein subunits at the interfaces between four K + channel subunits, inducing a pre-open state of the channel. The pre-open state exhibits a conformation thatmore » is intermediate between the closed conformation and the open conformation of the constitutively active mutant. The resultant structural picture is compatible with ‘membrane delimited’ activation of GIRK channels by G proteins and the characteristic burst kinetics of channel gating. The structures also permit a conceptual understanding of how the signalling lipid phosphatidylinositol-4,5-bisphosphate (PIP 2) and intracellular Na + ions participate in multi-ligand regulation of GIRK channels.« less

Regulation of Neurovascular Coupling in Autoimmunity to Water and Ion Channels

PubMed Central

Jukkola, Peter; Gu, Chen

2014-01-01

Much progress has been made in understanding autoimmune channelopathies, but the underlying pathogenic mechanisms are not always clear due to broad expression of some channel proteins. Recent studies show that autoimmune conditions that interfere with neurovascular coupling in the central nervous system (CNS) can lead to neurodegeneration. Cerebral blood flow that meets neuronal activity and metabolic demand is tightly regulated by local neural activity. This process of reciprocal regulation involves coordinated actions of a number of cell types, including neurons, glia, and vascular cells. In particular, astrocytic endfeet cover more than 90% of brain capillaries to assist blood-brain barrier (BBB) function, and wrap around synapses and nodes of Ranvier to communicate with neuronal activity. In this review, we highlight four types of channel proteins that are expressed in astrocytes, regarding their structures, biophysical properties, expression and distribution patterns, and related diseases including autoimmune disorders. Water channel aquaporin 4 (AQP4) and inwardly-rectifying potassium (Kir4.1) channels are concentrated in astrocytic endfeet, whereas some voltage-gated Ca2+ and two-pore-domain K+ channels are expressed throughout the cell body of reactive astrocytes. More channel proteins are found in astrocytes under normal and abnormal conditions. This research field will contribute to a better understanding of pathogenic mechanisms underlying autoimmune disorders. PMID:25462580

Phosphorylation-Dependent Regulation of Ryanodine Receptors

PubMed Central

Marx, Steven O.; Reiken, Steven; Hisamatsu, Yuji; Gaburjakova, Marta; Gaburjakova, Jana; Yang, Yi-Ming; Rosemblit, Nora; Marks, Andrew R.

2001-01-01

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function. PMID:11352932

Regulation of inward rectifier potassium current ionic channel remodeling by AT1 -Calcineurin-NFAT signaling pathway in stretch-induced hypertrophic atrial myocytes.

PubMed

He, Jionghong; Xu, Yanan; Yang, Long; Xia, Guiling; Deng, Na; Yang, Yongyao; Tian, Ye; Fu, Zenan; Huang, Yongqi

2018-05-02

Previous studies have shown that the activation of angiotensin II receptor type I (AT 1 ) is attributed to cardiac remodeling stimulated by increased heart load, and that it is followed by the activation of the calcineurin-nuclear factor of activated T-cells (NFAT) signaling pathway. Additionally, AT 1 has been found to be a regulator of cardiocyte ionic channel remodeling, and calcineurin-NFAT signals participate in the regulation of cardiocyte ionic channel expression. A hypothesis therefore follows that stretch stimulation may regulate cardiocyte ionic channel remodeling by activating the AT 1 -calcineurin-NFAT pathway. Here, we investigated the role of the AT 1 -calcineurin-NFAT pathway in the remodeling of inward rectifier potassium (I k1 ) channel, in addition to its role in changing action potential, in stretch-induced hypertrophic atrial myocytes of neonatal rats. Our results showed that increased stretch significantly led to atrial myocytes hypertrophy; it also increased the activity of calcineurin enzymatic activity, which was subsequently attenuated by telmisartan or cyclosporine-A. The level of NFAT 3 protein in nuclear extracts, the mRNA and protein expression of Kir2.1 in whole cell extracts, and the density of I k1 were noticeably increased in stretched samples. Stretch stimulation significantly shortened the action potential duration (APD) of repolarization at the 50% and 90% level. Telmisartan, cyclosporine-A, and 11R-VIVIT attenuated stretch-induced alterations in the levels of NFAT 3 , mRNA and protein expression of Kir2.1, the density of I k1 , and the APD. Our findings suggest that the AT 1 -calcineurin-NFAT signaling pathway played an important role in regulating I k1 channel remodeling and APD change in stretch-induced hypertrophic atrial myocytes of neonatal rats. This article is protected by copyright. All rights reserved.

Multiple modes of a-type potassium current regulation.

PubMed

Cai, Shi-Qing; Li, Wenchao; Sesti, Federico

2007-01-01

Voltage-dependent potassium (K+) channels (Kv) regulate cell excitability by controlling the movement of K+ ions across the membrane in response to changes in the cell voltage. The Kv family, which includes A-type channels, constitute the largest group of K+ channel genes within the superfamily of Na+, Ca2+ and K+ voltage-gated channels. The name "A-type" stems from the typical profile of these currents that results form the opposing effects of fast activation and inactivation. In neuronal cells, A-type currents (I(A)), determine the interval between two consecutive action potentials during repetitive firing. In cardiac muscle, A-type currents (I(to)), control the initial repolarization of the myocardium. Structurally, A-type channels are tetramers of alpha-subunits each containing six putative transmembrane domains including a voltage-sensor. A-type channels can be modulated by means of protein-protein interactions with so-called beta-subunits that control inactivation voltage sensitivity and other properties, and by post-transcriptional modifications such as phosphorylation or oxidation. Recently a new mode of A-type regulation has been discovered in the form of a class of hybrid beta-subunits that posses their own enzymatic activity. Here, we review the biophysical and physiological properties of these multiple modes of A-type channel regulation.

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+.

PubMed

Li, Mufeng; Silberberg, Shai D; Swartz, Kenton J

2013-09-03

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg(2+). Here we investigated the active forms of ATP and found that the action of MgATP(2-) and ATP(4-) differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP(2-) promotes opening with very low efficacy. In contrast, both free ATP and MgATP(2-) robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg(2+) to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP(2-) and weak regulation by Mg(2+). These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP(2-) and regulation by Mg(2+), and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

PubMed Central

Li, Mufeng; Silberberg, Shai D.; Swartz, Kenton J.

2013-01-01

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg2+. Here we investigated the active forms of ATP and found that the action of MgATP2− and ATP4− differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP2− promotes opening with very low efficacy. In contrast, both free ATP and MgATP2− robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg2+ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP2− and weak regulation by Mg2+. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP2− and regulation by Mg2+, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons. PMID:23959888

The Caenorhabditis elegans Iodotyrosine Deiodinase Ortholog SUP-18 Functions through a Conserved Channel SC-Box to Regulate the Muscle Two-Pore Domain Potassium Channel SUP-9

PubMed Central

de la Cruz, Ignacio Perez; Ma, Long; Horvitz, H. Robert

2014-01-01

Loss-of-function mutations in the Caenorhabditis elegans gene sup-18 suppress the defects in muscle contraction conferred by a gain-of-function mutation in SUP-10, a presumptive regulatory subunit of the SUP-9 two-pore domain K+ channel associated with muscle membranes. We cloned sup-18 and found that it encodes the C. elegans ortholog of mammalian iodotyrosine deiodinase (IYD), an NADH oxidase/flavin reductase that functions in iodine recycling and is important for the biosynthesis of thyroid hormones that regulate metabolism. The FMN-binding site of mammalian IYD is conserved in SUP-18, which appears to require catalytic activity to function. Genetic analyses suggest that SUP-10 can function with SUP-18 to activate SUP-9 through a pathway that is independent of the presumptive SUP-9 regulatory subunit UNC-93. We identified a novel evolutionarily conserved serine-cysteine-rich region in the C-terminal cytoplasmic domain of SUP-9 required for its specific activation by SUP-10 and SUP-18 but not by UNC-93. Since two-pore domain K+ channels regulate the resting membrane potentials of numerous cell types, we suggest that the SUP-18 IYD regulates the activity of the SUP-9 channel using NADH as a coenzyme and thus couples the metabolic state of muscle cells to muscle membrane excitability. PMID:24586202

Heterogeneity in Kv7 channel function in the cerebral and coronary circulation.

PubMed

Lee, Sewon; Yang, Yan; Tanner, Miles A; Li, Min; Hill, Michael A

2015-02-01

Kv7 channels are considered important regulators of vascular smooth muscle contractility. The present study aimed to examine the hypotheses that (i) Kv7 channels are present in mouse cerebral and coronary arteries and regulate vascular reactivity and (ii) regional differences exist in the activity of these channels. PCR confirmed that basilar, Circle of Willis and LAD arteries express predominantly Kv7.1 and 7.4. Western blot analysis, however, showed greater Kv7.4 protein levels in the cerebral vessels. Relaxation to the Kv7 channel activator, retigabine (1-50 μM) was significantly greater in the basilar artery compared to the LAD artery. Similarly, the Kv7 channel inhibitor, linopirdine (10 μM) caused a stronger contraction of the basilar artery. Furthermore, pre-incubation with linopirdine reduced forskolin (cAMP activator)-induced vasorelaxation in basilar while not altering forskolin-induced vasorelaxation of the LAD, suggesting that Kv7 channels play a more prominent role in the cerebral than in the coronary circulation. Consistent with the vessel data, whole cell Kv7 currents in cerebral VSMCs were potentiated by retigabine and inhibited by linopirdine, while these responses were blunted in coronary VSMCs. This study provides evidence that mouse Kv7 channels may contribute differently to regulating the functional properties of cerebral and coronary arteries. Such heterogeneity has important implications for developing novel therapeutics for cardiovascular dysfunction. © 2014 John Wiley & Sons Ltd.

Heterogeneity in Kv7 channel function in the Cerebral and Coronary Circulation

PubMed Central

Tanner, Miles A.; Li, Min; Hill, Michael A.

2014-01-01

Kv7 channels are considered important regulators of vascular smooth muscle contractility. The present study examined the hypotheses that 1. Kv7 channels are present in mouse cerebral and coronary arteries and regulate vascular reactivity, and 2. regional differences exist in the activity of these channels. PCR confirmed that basilar, Circle of Willis and left anterior descending (LAD) arteries express predominantly Kv7.1 and 7.4. Western blot analysis, however, showed greater Kv7.4 protein levels in the cerebral vessels. Relaxation to the Kv7 channel activator, retigabine (1-50μM) was significantly greater in basilar compared to LAD. Similarly, the Kv7 channel inhibitor, linopirdine (10μM) caused stronger contraction of the basilar artery. Furthermore, pre-incubation with linopirdine reduced forskolin (cAMP activator)-induced vasorelaxation in basilar while not altering forskolin-induced vasorelaxation of the LAD, suggesting that Kv7 channels play a more prominent role in the cerebral than coronary circulation. Consistent with the vessel data, whole cell Kv7 currents in cerebral VSMCs were potentiated by retigabine and inhibited by linopirdine, while these responses were blunted in coronary VSMCs. This study provides evidence that mouse Kv7 channels may contribute differently to regulating the functional properties of cerebral and coronary arteries. Such heterogeneity has important implications for developing novel therapeutics for cardiovascular dysfunction. PMID:25476662

Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

PubMed Central

Neely, Alan; Hidalgo, Patricia

2014-01-01

Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels.

PubMed

Meighan, Peter C; Meighan, Starla E; Rich, Elizabeth D; Brown, R Lane; Varnum, Michael D

2012-01-01

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

PubMed Central

Meighan, Peter C.; Meighan, Starla E.; Rich, Elizabeth D.; Brown, R. Lane; Varnum, Michael D.

2012-01-01

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels. PMID:22699690

Gap Junctions

PubMed Central

Nielsen, Morten Schak; Axelsen, Lene Nygaard; Sorgen, Paul L.; Verma, Vandana; Delmar, Mario; Holstein-Rathlou, Niels-Henrik

2013-01-01

Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of gap junction activity is complex. The structure of the various connexins is known to some extent; and structural rearrangements and intramolecular interactions are important for regulation of channel function. Intercellular coupling is further regulated by the number and activity of channels present in gap junctional plaques. The number of connexins in cell-cell channels is regulated by controlling transcription, translation, trafficking, and degradation; and all of these processes are under strict control. Once in the membrane, channel activity is determined by the conductive properties of the connexin involved, which can be regulated by voltage and chemical gating, as well as a large number of posttranslational modifications. The aim of the present article is to review our current knowledge on the structure, regulation, function, and pharmacology of gap junctions. This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease. © 2012 American Physiological Society. Compr Physiol 2:1981-2035, 2012. PMID:23723031

Tyrosine Phosphatases ε and α Perform Specific and Overlapping Functions in Regulation of Voltage-gated Potassium Channels in Schwann Cells

PubMed Central

Tiran, Zohar; Peretz, Asher; Sines, Tal; Shinder, Vera; Sap, Jan; Attali, Bernard

2006-01-01

Tyrosine phosphatases (PTPs) ε and α are closely related and share several molecular functions, such as regulation of Src family kinases and voltage-gated potassium (Kv) channels. Functional interrelationships between PTPε and PTPα and the mechanisms by which they regulate K+ channels and Src were analyzed in vivo in mice lacking either or both PTPs. Lack of either PTP increases Kv channel activity and phosphorylation in Schwann cells, indicating these PTPs inhibit Kv current amplitude in vivo. Open probability and unitary conductance of Kv channels are unchanged, suggesting an effect on channel number or organization. PTPα inhibits Kv channels more strongly than PTPε; this correlates with constitutive association of PTPα with Kv2.1, driven by membranal localization of PTPα. PTPα, but not PTPε, activates Src in sciatic nerve extracts, suggesting Src deregulation is not responsible exclusively for the observed phenotypes and highlighting an unexpected difference between both PTPs. Developmentally, sciatic nerve myelination is reduced transiently in mice lacking either PTP and more so in mice lacking both PTPs, suggesting both PTPs support myelination but are not fully redundant. We conclude that PTPε and PTPα differ significantly in their regulation of Kv channels and Src in the system examined and that similarity between PTPs does not necessarily result in full functional redundancy in vivo. PMID:16870705

Cross-talk between ATP-regulated K+ channels and Na+ transport via cellular metabolism in frog skin principal cells.

PubMed Central

Urbach, V; Van Kerkhove, E; Maguire, D; Harvey, B J

1996-01-01

Isolated frog skin epithelium, mounted in an Ussing chamber and bathed in standard NaCl Ringer solution, recycles K+ across the basolateral membrane of principal cells through an inward-rectifier K+ channel (Kir) operating in parallel with a Na+-K+-ATPase pump. Here we report on the metabolic control of the Kir channel using patch clamping, short-circuit current measurement and enzymatic determination of cellular (ATP (ATPi). 2. The constitutively active Kir channel in the basolateral membrane has the characteristics of an ATP-regulated K+ channel and is now classed as a KATP channel. In excised inside-out patches the open probability (Po) of KATP channels was reduced by ATPi with half-maximum inhibition at an ATPi concentration of 50 microM. 3. ATPi measured (under normal Na+ transport conditions) with luciferin-luciferase was 1.50 +/- 0.23 mM (mean +/- S.E.M.; range, 0.4-3.3 mM n = 11). Thus the KATP channel would be expected to be inactive in intact cells if ATPi was the sole regulator of channel activity. KATP channels which were inactivated by 1 mM ATPi in excised patches could be reactivated by addition of 100 microM ADP on the cytosolic side. When added alone, ADP blocks this channel with half-maximal inhibition at [ADPi] > 5 mM. 4. Sulphonylureas inhibit single KATP channels in cell-attached patches as well as the total basolateral K+ current measured in frog skin epithelia perforated with nystatin on the apical side. 5. Na+-K+-ATPase activity is a major determinant of cytosolic ATP. Blocking the pump activity with ouabain produced a time-dependent increase in ATPi and reduced the open probability of KATP channels in cell-attached membranes. 6. We conclude that the ratio of ATP/ADP is an important metabolic coupling factor between the rate of Na+-K+ pumping and K+ recycling. Images Figure 9 PMID:9011625

AtALMT9 is a malate-activated vacuolar chloride channel required for stomatal opening in Arabidopsis

PubMed Central

De Angeli, Alexis; Zhang, Jingbo; Meyer, Stefan; Martinoia, Enrico

2013-01-01

Water deficit strongly affects crop productivity. Plants control water loss and CO2 uptake by regulating the aperture of the stomatal pores within the leaf epidermis. Stomata aperture is regulated by the two guard cells forming the pore and changing their size in response to ion uptake and release. While our knowledge about potassium and chloride fluxes across the plasma membrane of guard cells is advanced, little is known about fluxes across the vacuolar membrane. Here we present the molecular identification of the long-sought-after vacuolar chloride channel. AtALMT9 is a chloride channel activated by physiological concentrations of cytosolic malate. Single-channel measurements demonstrate that this activation is due to a malate-dependent increase in the channel open probability. Arabidopsis thaliana atalmt9 knockout mutants exhibited impaired stomatal opening and wilt more slowly than the wild type. Our findings show that AtALMT9 is a vacuolar chloride channel having a major role in controlling stomata aperture. PMID:23653216

Distinct pH regulation of slow and rapid anion channels at the plasma membrane of Arabidopsis thaliana hypocotyl cells.

PubMed

Colcombet, Jean; Lelièvre, Françoise; Thomine, Sébastien; Barbier-Brygoo, Hélène; Frachisse, Jean-Marie

2005-07-01

Variations in both intracellular and extracellular pH are known to be involved in a wealth of physiological responses. Using the patch-clamp technique on Arabidopsis hypocotyl cells, it is shown that rapid-type and slow-type anion channels at the plasma membrane are both regulated by pH via distinct mechanisms. Modifications of pH modulate the voltage-dependent gating of the rapid channel. While intracellular alkalinization facilitates channel activation by shifting the voltage gate towards negative potentials, extracellular alkalinization shifts the activation threshold to more positive potentials, away from physiological resting membrane potentials. By contrast, pH modulates slow anion channel activity in a voltage-independent manner. Intracellular acidification and extracellular alkalinization increase slow anion channel currents. The possible role of these distinct modulations in physiological processes involving anion efflux and modulation of extracellular and/or intracellular pH, such as elicitor and ABA signalling, are discussed.

Tyrosine phosphorylation–dependent activation of TRPC6 regulated by PLC-γ1 and nephrin: effect of mutations associated with focal segmental glomerulosclerosis

PubMed Central

Kanda, Shoichiro; Harita, Yutaka; Shibagaki, Yoshio; Sekine, Takashi; Igarashi, Takashi; Inoue, Takafumi; Hattori, Seisuke

2011-01-01

Transient receptor potential canonicals (TRPCs) play important roles in the regulation of intracellular calcium concentration. Mutations in the TRPC6 gene are found in patients with focal segmental glomerulosclerosis (FSGS), a proteinuric disease characterized by dysregulated function of renal glomerular epithelial cells (podocytes). There is as yet no clear picture for the activation mechanism of TRPC6 at the molecular basis, however, and the association between its channel activity and pathogenesis remains unclear. We demonstrate here that tyrosine phosphorylation of TRPC6 induces a complex formation with phospholipase C (PLC)-γ1, which is prerequisite for TRPC6 surface expression. Furthermore, nephrin, an adhesion protein between the foot processes of podocytes, binds to phosphorylated TRPC6 via its cytoplasmic domain, competitively inhibiting TRPC6–PLC-γ1 complex formation, TRPC6 surface localization, and TRPC6 activation. Importantly, FSGS-associated mutations render the mutated TRPC6s insensitive to nephrin suppression, thereby promoting their surface expression and channel activation. These results delineate the mechanism of TRPC6 activation regulated by tyrosine phosphorylation, and imply the cell type–specific regulation, which correlates the FSGS mutations with deregulated TRPC6 channel activity. PMID:21471003

Flavonoid Regulation of HCN2 Channels*

PubMed Central

Carlson, Anne E.; Rosenbaum, Joel C.; Brelidze, Tinatin I.; Klevit, Rachel E.; Zagotta, William N.

2013-01-01

The hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are pacemaker channels whose currents contribute to rhythmic activity in the heart and brain. HCN channels open in response to hyperpolarizing voltages, and the binding of cAMP to their cyclic nucleotide-binding domain (CNBD) facilitates channel opening. Here, we report that, like cAMP, the flavonoid fisetin potentiates HCN2 channel gating. Fisetin sped HCN2 activation and shifted the conductance-voltage relationship to more depolarizing potentials with a half-maximal effective concentration (EC50) of 1.8 μm. When applied together, fisetin and cAMP regulated HCN2 gating in a nonadditive fashion. Fisetin did not potentiate HCN2 channels lacking their CNBD, and two independent fluorescence-based binding assays reported that fisetin bound to the purified CNBD. These data suggest that the CNBD mediates the fisetin potentiation of HCN2 channels. Moreover, binding assays suggest that fisetin and cAMP partially compete for binding to the CNBD. NMR experiments demonstrated that fisetin binds within the cAMP-binding pocket, interacting with some of the same residues as cAMP. Together, these data indicate that fisetin is a partial agonist for HCN2 channels. PMID:24085296

Prolactin receptor in regulation of neuronal excitability and channels

PubMed Central

Patil, Mayur J; Henry, Michael A; Akopian, Armen N

2014-01-01

Prolactin (PRL) activates PRL receptor isoforms to exert regulation of specific neuronal circuitries, and to control numerous physiological and clinically-relevant functions including; maternal behavior, energy balance and food intake, stress and trauma responses, anxiety, neurogenesis, migraine and pain. PRL controls these critical functions by regulating receptor potential thresholds, neuronal excitability and/or neurotransmission efficiency. PRL also influences neuronal functions via activation of certain neurons, resulting in Ca2+ influx and/or electrical firing with subsequent release of neurotransmitters. Although PRL was identified almost a century ago, very little specific information is known about how PRL regulates neuronal functions. Nevertheless, important initial steps have recently been made including the identification of PRL-induced transient signaling pathways in neurons and the modulation of neuronal transient receptor potential (TRP) and Ca2+-dependent K+ channels by PRL. In this review, we summarize current knowledge and recent progress in understanding the regulation of neuronal excitability and channels by PRL. PMID:24758841

Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

PubMed Central

Vocke, Kerstin; Dauner, Kristin; Hahn, Anne; Ulbrich, Anne; Broecker, Jana; Keller, Sandro; Frings, Stephan

2013-01-01

Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca2+/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types. PMID:24081981

Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the SNARE SYP121[W

PubMed Central

Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S.; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R.; Chaumont, François

2012-01-01

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K+ channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K+ channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis. PMID:22942383

Selective regulation of maize plasma membrane aquaporin trafficking and activity by the SNARE SYP121.

PubMed

Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R; Chaumont, François

2012-08-01

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K(+) channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K(+) channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.

Erythrocyte ion channels in regulation of apoptosis.

PubMed

Lang, Florian; Birka, Christina; Myssina, Svetlana; Lang, Karl S; Lang, Philipp A; Tanneur, Valerie; Duranton, Christophe; Wieder, Thomas; Huber, Stephan M

2004-01-01

Erythrocytes lack mitochondria and nuclei, key organelles in the regulation of apoptosis. Until recently, erythrocytes were thus not considered subject to this type of cell death. However, exposure of erythrocytes to the Ca2+ ionophore ionomycin was shown to induce cell shrinkage, cell membrane blebbing and breakdown of phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the cell surface, all typical features of apoptosis. Further studies revealed the participation of ion channels in the regulation of erythrocyte "apoptosis." Osmotic shock, oxidative stress and energy depletion all activate a Ca2(+)-permeable non-selective cation channel in the erythrocyte cell membrane. The subsequent increase of Ca2+ concentration stimulates a scramblase leading to breakdown of cell membrane phosphatidylserine asymmetry and activates Ca2+ sensitive K+ (Gardos) channels leading to KCl loss and (further) cell shrinkage. Phosphatidylserine exposure and cell shrinkage are blunted in the nominal absence of extracellular Ca2+, in the presence of the cation channel inhibitors amiloride or ethylisopropylamiloride, at increased extracellular K+ or in the presence of the Gardos channel inhibitors clotrimazole or charybdotoxin. Thus, increase of cytosolic Ca2+ and cellular loss of K+ participate in the triggering of erythrocyte scramblase. Nevertheless, phosphatidylserine exposure is not completely abrogated in the nominal absence of Ca2+, pointing to additional Ca2(+)-independent pathways. One of those is activation of sphingomyelinase with subsequent formation of ceramide which in turn leads to stimulation of erythrocyte scramblase. The exposure of phosphatidylserine at the extracellular face of the cell membrane stimulates phagocytes to engulf the apoptotic erythrocytes. Thus, sustained activation of the cation channels eventually leads to clearance of affected erythrocytes from peripheral blood. Erythropoietin inhibits the non-selective cation channel and thus interferes with erythrocyte "apoptosis." Susceptibility to scramblase activation is enhanced in thalassemia, sickle cell disease and glucose-6-phosphate dehydrogenase deficiency. Infection with Plasmodium falciparum leads to activation of the cation channel eventually triggering erythrocyte "apoptosis."

BK Channels in the Vascular System.

PubMed

Krishnamoorthy-Natarajan, G; Koide, M

2016-01-01

Autoregulation of blood flow is essential for the preservation of organ function to ensure continuous supply of oxygen and essential nutrients and removal of metabolic waste. This is achieved by controlling the diameter of muscular arteries and arterioles that exhibit a myogenic response to changes in arterial blood pressure, nerve activity and tissue metabolism. Large-conductance voltage and Ca(2+)-dependent K(+) channels (BK channels), expressed exclusively in smooth muscle cells (SMCs) in the vascular wall of healthy arteries, play a critical role in regulating the myogenic response. Activation of BK channels by intracellular, local, and transient ryanodine receptor-mediated "Ca(2+) sparks," provides a hyperpolarizing influence on the SMC membrane potential thereby decreasing the activity of voltage-dependent Ca(2+) channels and limiting Ca(2+) influx to promote SMC relaxation and vasodilation. The BK channel α subunit, a large tetrameric protein with each monomer consisting of seven-transmembrane domains, a long intracellular C-terminal tail and an extracellular N-terminus, associates with the β1 and γ subunits in vascular SMCs. The BK channel is regulated by factors originating within the SMC or from the endothelium, perivascular nerves and circulating blood, that significantly alter channel gating properties, Ca(2+) sensitivity and expression of the α and/or β1 subunit. The BK channel thus serves as a central receiving dock that relays the effects of the changes in several such concomitant autocrine and paracrine factors and influences cardiovascular health. This chapter describes the primary mechanism of regulation of myogenic response by BK channels and the alterations to this mechanism wrought by different vasoactive mediators. © 2016 Elsevier Inc. All rights reserved.

Phosphatidylinositol (4,5)-bisphosphate dynamically regulates the K2P background K+ channel TASK-2.

PubMed

Niemeyer, María Isabel; Cid, L Pablo; Paulais, Marc; Teulon, Jacques; Sepúlveda, Francisco V

2017-03-30

Two-pore domain K 2P K + channels responsible for the background K + conductance and the resting membrane potential, are also finely regulated by a variety of chemical, physical and physiological stimuli. Hormones and transmitters acting through Gq protein-coupled receptors (GqPCRs) modulate the activity of various K 2P channels but the signalling involved has remained elusive, in particular whether dynamic regulation by membrane PI(4,5)P 2 , common among other classes of K + channels, affects K 2P channels is controversial. Here we show that K 2P K + channel TASK-2 requires PI(4,5)P 2 for activity, a dependence that accounts for its run down in the absence of intracellular ATP and its full recovery by addition of exogenous PI(4,5)P 2 , its inhibition by low concentrations of polycation PI scavengers, and inhibition by PI(4,5)P 2 depletion from the membrane. Comprehensive mutagenesis suggests that PI(4,5)P 2 interaction with TASK-2 takes place at C-terminus where three basic aminoacids are identified as being part of a putative binding site.

Two-photon activation of endogenous store-operated calcium channels without optogenetics

NASA Astrophysics Data System (ADS)

Cheng, Pan; Tang, Wanyi; He, Hao

2018-02-01

Store-operated calcium (SOC) channels, regulated by intracellular Ca2+ store, are the essential pathway of calcium signaling and participate in a wide variety of cellular activities such as gene expression, secretion and immune response1. However, our understanding and regulation of SOC channels are mainly based on pharmacological methods. Considering the unique advantages of optical control, optogenetic control of SOC channels has been developed2. However, the process of genetic engineering to express exogenous light-sensitive protein is complicated, which arouses concerns about ethic difficulties in some research of animal and applications in human. In this report, we demonstrate rapid, robust and reproducible two-photon activation of endogenous SOC channels by femtosecond laser without optogenetics. We present that the short-duration two-photon scanning on subcellular microregion induces slow Ca2+ influx from extracellular medium, which can be eliminated by removing extracellular Ca2+. Block of SOC channels using various pharmacological inhibitors or knockdown of SOC channels by RNA interference reduce the probability of two-photon activated Ca2+ influx. On the contrary, overexpression of SOC channels can increase the probability of Ca2+ influx by two-photon scanning. These results collectively indicate Ca2+ influx through two-photon activated SOC channels. Different from classical pathway of SOC entry activated by Ca2+ store depletion, STIM1, the sensor protein of Ca2+ level in endoplasmic reticulum, does not show any aggregation or migration in this two-photon activated Ca2+ influx, which rules out the possibility of intracellular Ca2+ store depletion. Thereby, we propose this all-optical method of two-photon activation of SOC channels is of great potential to be widely applied in the research of cell calcium signaling and related biological research.

Electrophysiological Features of Single Store-Operated Calcium Channels in HEK S4 Cell Line with Stable STIM1 Protein Knockdown.

PubMed

Shalygin, A V; Vigont, V A; Glushankova, L N; Zimina, O A; Kolesnikov, D O; Skopin, A Yu; Kaznacheeva, E V

2017-07-01

An important role in intracellular calcium signaling is played by store-operated channels activated by STIM proteins, calcium sensors of the endoplasmic reticulum. In stable STIM1 knockdown HEK S4 cells, single channels activated by depletion of intracellular calcium stores were detected by cell-attached patch-clamp technique and their electrophysiological parameters were described. Comparison of the properties of single channels in HEK293 and HEK S4 cells revealed no significant differences in their current-voltage curves, while regulation of store-operated calcium channels in these cell lines depended on the level of STIM1 expression. We can conclude that electrophysiological peculiarities of store-regulated calcium entry observed in different cells can be explained by differences in STIM1 expression.

Protein arginine methylation facilitates KCNQ channel-PIP2 interaction leading to seizure suppression

PubMed Central

Kim, Hyun-Ji; Jeong, Myong-Ho; Kim, Kyung-Ran; Jung, Chang-Yun; Lee, Seul-Yi; Kim, Hanna; Koh, Jewoo; Vuong, Tuan Anh; Jung, Seungmoon; Yang, Hyunwoo; Park, Su-Kyung; Choi, Dahee; Kim, Sung Hun; Kang, KyeongJin; Sohn, Jong-Woo; Park, Joo Min; Jeon, Daejong; Koo, Seung-Hoi; Ho, Won-Kyung; Kang, Jong-Sun; Kim, Seong-Tae; Cho, Hana

2016-01-01

KCNQ channels are critical determinants of neuronal excitability, thus emerging as a novel target of anti-epileptic drugs. To date, the mechanisms of KCNQ channel modulation have been mostly characterized to be inhibitory via Gq-coupled receptors, Ca2+/CaM, and protein kinase C. Here we demonstrate that methylation of KCNQ by protein arginine methyltransferase 1 (Prmt1) positively regulates KCNQ channel activity, thereby preventing neuronal hyperexcitability. Prmt1+/- mice exhibit epileptic seizures. Methylation of KCNQ2 channels at 4 arginine residues by Prmt1 enhances PIP2 binding, and Prmt1 depletion lowers PIP2 affinity of KCNQ2 channels and thereby the channel activities. Consistently, exogenous PIP2 addition to Prmt1+/- neurons restores KCNQ currents and neuronal excitability to the WT level. Collectively, we propose that Prmt1-dependent facilitation of KCNQ-PIP2 interaction underlies the positive regulation of KCNQ activity by arginine methylation, which may serve as a key target for prevention of neuronal hyperexcitability and seizures. DOI: http://dx.doi.org/10.7554/eLife.17159.001 PMID:27466704

Modulation of BK channel voltage gating by different auxiliary β subunits

PubMed Central

Contreras, Gustavo F.; Neely, Alan; Alvarez, Osvaldo; Gonzalez, Carlos; Latorre, Ramon

2012-01-01

Calcium- and voltage-activated potassium channels (BK) are regulated by a multiplicity of signals. The prevailing view is that different BK gating mechanisms converge to determine channel opening and that these gating mechanisms are allosterically coupled. In most instances the pore forming α subunit of BK is associated with one of four alternative β subunits that appear to target specific gating mechanisms to regulate the channel activity. In particular, β1 stabilizes the active configuration of the BK voltage sensor having a large effect on BK Ca2+ sensitivity. To determine the extent to which β subunits regulate the BK voltage sensor, we measured gating currents induced by the pore-forming BK α subunit alone and with the different β subunits expressed in Xenopus oocytes (β1, β2IR, β3b, and β4). We found that β1, β2, and β4 stabilize the BK voltage sensor in the active conformation. β3 has no effect on voltage sensor equilibrium. In addition, β4 decreases the apparent number of charges per voltage sensor. The decrease in the charge associated with the voltage sensor in α β4 channels explains most of their biophysical properties. For channels composed of the α subunit alone, gating charge increases slowly with pulse duration as expected if a significant fraction of this charge develops with a time course comparable to that of K+ current activation. In the presence of β1, β2, and β4 this slow component develops in advance of and much more rapidly than ion current activation, suggesting that BK channel opening proceeds in two steps. PMID:23112204

Regulation of ENaC and CFTR expression with K+ channel modulators and effect on fluid absorption across alveolar epithelial cells.

PubMed

Leroy, Claudie; Privé, Anik; Bourret, Jean-Charles; Berthiaume, Yves; Ferraro, Pasquale; Brochiero, Emmanuelle

2006-12-01

In a recent study (Leroy C, Dagenais A, Berthiaume Y, and Brochiero E. Am J Physiol Lung Cell Mol Physiol 286: L1027-L1037, 2004), we identified an ATP-sensitive K(+) (K(ATP)) channel in alveolar epithelial cells, formed by inwardly rectifying K(+) channel Kir6.1/sulfonylurea receptor (SUR)2B subunits. We found that short applications of K(ATP), voltage-dependent K(+) channel KvLQT1, and calcium-activated K(+) (K(Ca)) channel modulators modified Na(+) and Cl(-) currents in alveolar monolayers. In addition, it was shown previously that a K(ATP) opener increased alveolar liquid clearance in human lungs by a mechanism possibly related to epithelial sodium channels (ENaC). We therefore hypothesized that prolonged treatment with K(+) channel modulators could induce a sustained regulation of ENaC activity and/or expression. Alveolar monolayers were treated for 24 h with inhibitors of K(ATP), KvLQT1, and K(Ca) channels identified by PCR. Glibenclamide and clofilium (K(ATP) and KvLQT1 inhibitors) strongly reduced basal transepithelial current, amiloride-sensitive Na(+) current, and forskolin-activated Cl(-) currents, whereas pinacidil, a K(ATP) activator, increased them. Interestingly, K(+) inhibitors or membrane depolarization (induced by valinomycin in high-K(+) medium) decreased alpha-, beta-, and gamma-ENaC and CFTR mRNA. alpha-ENaC and CFTR proteins also declined after glibenclamide or clofilium treatment. Conversely, pinacidil augmented ENaC and CFTR mRNAs and proteins. Since alveolar fluid transport was found to be driven, at least in part, by Na(+) transport through ENaC, we tested the impact of K(+) channel modulators on fluid absorption across alveolar monolayers. We found that glibenclamide and clofilium reduced fluid absorption to a level similar to that seen in the presence of amiloride, whereas pinacidil slightly enhanced it. Long-term regulation of ENaC and CFTR expression by K(+) channel activity could benefit patients with pulmonary diseases affecting ion transport and fluid clearance.

Dendritic calcium channels and their activation by synaptic signals in auditory coincidence detector neurons.

PubMed

Blackmer, Trillium; Kuo, Sidney P; Bender, Kevin J; Apostolides, Pierre F; Trussell, Laurence O

2009-08-01

The avian nucleus laminaris (NL) encodes the azimuthal location of low-frequency sound sources by detecting the coincidence of binaural signals. Accurate coincidence detection requires precise developmental regulation of the lengths of the fine, bitufted dendrites that characterize neurons in NL. Such regulation has been suggested to be driven by local, synaptically mediated, dendritic signals such as Ca(2+). We examined Ca(2+) signaling through patch clamp and ion imaging experiments in slices containing nucleus laminaris from embryonic chicks. Voltage-clamp recordings of neurons located in the NL showed the presence of large Ca(2+) currents of two types, a low voltage-activated, fast inactivating Ni(2+) sensitive channel resembling mammalian T-type channels, and a high voltage-activated, slowly inactivating Cd(2+) sensitive channel. Two-photon Ca(2+) imaging showed that both channel types were concentrated on dendrites, even at their distal tips. Single action potentials triggered synaptically or by somatic current injection immediately elevated Ca(2+) throughout the entire cell. Ca(2+) signals triggered by subthreshold synaptic activity were highly localized. Thus when electrical activity is suprathreshold, Ca(2+) channels ensure that Ca(2+) rises in all dendrites, even those that are synaptically inactive.

New Aspects of the Contribution of ER to SOCE Regulation: TRPC Proteins as a Link Between Plasma Membrane Ion Transport and Intracellular Ca2+ Stores.

PubMed

Bavencoffe, Alexis; Zhu, Michael Xi; Tian, Jin-Bin

2017-01-01

Transient receptor potential canonical (TRPC) proteins were identified as molecular candidates of receptor- and/or store-operated channels because of their close homology to the Drosophila TRP and TRPL. Functional studies have revealed that TRPC channels play an integrated part of phospholipase C-transduced cell signaling, mediating the influx of both Ca 2+ and Na + into cells. As a consequence, the TRPC channels have diverse functional roles in different cell types, including metabotropic receptor-evoked membrane depolarization and intracellular Ca 2+ concentration elevation. Depending on the cellular environment and the protein partners present in the channel complex, the TRPC channels display different biophysical properties and mechanisms of regulation, including but not limited to the Ca 2+ filling state of the endoplasmic reticulum. Despite the overwhelming focus on STIM-regulated Orai channels for store-operated Ca 2+ entry, evidence is growing for STIM-operated TRPC channel activities in various cell types, demonstrating both store-dependent and store-independent mechanisms of TRPC channel gating. The existence of physical and functional interactions between plasma membrane-localized TRPC channels and other proteins involved in sensing and regulating the intracellular Ca 2+ store contents, such as inositol trisphosphate receptors, Junctate, and Homer, further argues for the role of TRPC proteins in linking plasma membrane ion transport with intracellular Ca 2+ stores. The interplay among these proteins will likely define the functional significance of TRPC channel activation in different cellular contexts and under different modes of stimulations.

Cell volume changes regulate slick (Slo2.1), but not slack (Slo2.2) K+ channels.

PubMed

Tejada, Maria A; Stople, Kathleen; Hammami Bomholtz, Sofia; Meinild, Anne-Kristine; Poulsen, Asser Nyander; Klaerke, Dan A

2014-01-01

Slick (Slo2.1) and Slack (Slo2.2) channels belong to the family of high-conductance K+ channels and have been found widely distributed in the CNS. Both channels are activated by Na+ and Cl- and, in addition, Slick channels are regulated by ATP. Therefore, the roles of these channels in regulation of cell excitability as well as ion transport processes, like regulation of cell volume, have been hypothesized. It is the aim of this work to evaluate the sensitivity of Slick and Slack channels to small, fast changes in cell volume and to explore mechanisms, which may explain this type of regulation. For this purpose Slick and Slack channels were co-expressed with aquaporin 1 in Xenopus laevis oocytes and cell volume changes of around 5% were induced by exposure to hypotonic or hypertonic media. Whole-cell currents were measured by two electrode voltage clamp. Our results show that Slick channels are dramatically stimulated (196% of control) by cell swelling and inhibited (57% of control) by a decrease in cell volume. In contrast, Slack channels are totally insensitive to similar cell volume changes. The mechanism underlining the strong volume sensitivity of Slick channels needs to be further explored, however we were able to show that it does not depend on an intact actin cytoskeleton, ATP release or vesicle fusion. In conclusion, Slick channels, in contrast to the similar Slack channels, are the only high-conductance K+ channels strongly sensitive to small changes in cell volume.

Interaction with caveolin-1 modulates vascular ATP-sensitive potassium (KATP) channel activity

PubMed Central

Davies, Lowri M; Purves, Gregor I; Barrett-Jolley, Richard; Dart, Caroline

2010-01-01

ATP-sensitive potassium channels (KATP channels) of arterial smooth muscle are important regulators of arterial tone, and hence blood flow, in response to vasoactive transmitters. Recent biochemical and electron microscopic evidence suggests that these channels localise to small vesicular invaginations of the plasma membrane, known as caveolae, and interact with the caveolae-associated protein, caveolin. Here we report that interaction with caveolin functionally regulates the activity of the vascular subtype of KATP channel, Kir6.1/SUR2B. Pinacidil-evoked recombinant whole-cell Kir6.1/SUR2B currents recorded in HEK293 cells stably expressing caveolin-1 (69.6 ± 8.3 pA pF−1, n= 8) were found to be significantly smaller than currents recorded in caveolin-null cells (179.7 ± 35.9 pA pF−1, n= 6; P < 0.05) indicating that interaction with caveolin may inhibit channel activity. Inclusion in the pipette-filling solution of a peptide corresponding to the scaffolding domain of caveolin-1 had a similar inhibitory effect on whole-cell Kir6.1/SUR2B currents as co-expression with full-length caveolin-1, while a scrambled version of the same peptide had no effect. Interestingly, intracellular dialysis of vascular smooth muscle cells with the caveolin-1 scaffolding domain peptide (SDP) also caused inhibition of pinacidil-evoked native whole-cell KATP currents, indicating that a significant proportion of vascular KATP channels are susceptible to block by exogenously applied SDP. In cell-attached recordings of Kir6.1/SUR2B single channel activity, the presence of caveolin-1 significantly reduced channel open probability (from 0.05 ± 0.01 to 0.005 ± 0.001; P < 0.05) and the amount of time spent in a relatively long-lived open state. These changes in kinetic behaviour can be explained by a caveolin-induced shift in the channel's sensitivity to its physiological regulator MgADP. Our findings thus suggest that interaction with caveolin-1 suppresses vascular-type KATP channel activity. Since caveolin expression is regulated by cellular free cholesterol and plasma levels of low-density lipoprotein (LDL), this interaction may have implications in both the physiological and pathophysiological control of vascular function. PMID:20624795

International Union of Basic and Clinical Pharmacology. C. Nomenclature and Properties of Calcium-Activated and Sodium-Activated Potassium Channels.

PubMed

Kaczmarek, Leonard K; Aldrich, Richard W; Chandy, K George; Grissmer, Stephan; Wei, Aguan D; Wulff, Heike

2017-01-01

A subset of potassium channels is regulated primarily by changes in the cytoplasmic concentration of ions, including calcium, sodium, chloride, and protons. The eight members of this subfamily were originally all designated as calcium-activated channels. More recent studies have clarified the gating mechanisms for these channels and have documented that not all members are sensitive to calcium. This article describes the molecular relationships between these channels and provides an introduction to their functional properties. It also introduces a new nomenclature that differentiates between calcium- and sodium-activated potassium channels. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Distribution and Function of HCN Channels in the Apical Dendritic Tuft of Neocortical Pyramidal Neurons

PubMed Central

Harnett, Mark T.; Magee, Jeffrey C.

2015-01-01

The apical tuft is the most remote area of the dendritic tree of neocortical pyramidal neurons. Despite its distal location, the apical dendritic tuft of layer 5 pyramidal neurons receives substantial excitatory synaptic drive and actively processes corticocortical input during behavior. The properties of the voltage-activated ion channels that regulate synaptic integration in tuft dendrites have, however, not been thoroughly investigated. Here, we use electrophysiological and optical approaches to examine the subcellular distribution and function of hyperpolarization-activated cyclic nucleotide-gated nonselective cation (HCN) channels in rat layer 5B pyramidal neurons. Outside-out patch recordings demonstrated that the amplitude and properties of ensemble HCN channel activity were uniform in patches excised from distal apical dendritic trunk and tuft sites. Simultaneous apical dendritic tuft and trunk whole-cell current-clamp recordings revealed that the pharmacological blockade of HCN channels decreased voltage compartmentalization and enhanced the generation and spread of apical dendritic tuft and trunk regenerative activity. Furthermore, multisite two-photon glutamate uncaging demonstrated that HCN channels control the amplitude and duration of synaptically evoked regenerative activity in the distal apical dendritic tuft. In contrast, at proximal apical dendritic trunk and somatic recording sites, the blockade of HCN channels decreased excitability. Dynamic-clamp experiments revealed that these compartment-specific actions of HCN channels were heavily influenced by the local and distributed impact of the high density of HCN channels in the distal apical dendritic arbor. The properties and subcellular distribution pattern of HCN channels are therefore tuned to regulate the interaction between integration compartments in layer 5B pyramidal neurons. PMID:25609619

Convergent regulation of the lysosomal two-pore channel-2 by Mg2+, NAADP, PI(3,5)P2 and multiple protein kinases

PubMed Central

Jha, Archana; Ahuja, Malini; Patel, Sandip; Brailoiu, Eugen; Muallem, Shmuel

2014-01-01

Lysosomal Ca2+ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca2+ signaling was the discovery of the two-pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated channels regulated by mTORC1, but not by NAADP. Here, we identified Mg2+ and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg2+ specifically inhibited TPC2 outward current, whereas lysosomal Mg2+ partially inhibited both outward and inward currents in a lysosomal lumen pH-dependent manner. Under controlled Mg2+, TPC2 is readily activated by NAADP with channel properties identical to those in response to PI(3,5)P2. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP-mediated Ca2+ release in intact cells is regulated by Mg2+, PI(3,5)P2, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP-mediated Ca2+ signaling and link this pathway to Mg2+ homeostasis and MAP kinases, pointing to roles for lysosomal Ca2+ in cell growth, inflammation and cancer. PMID:24502975

Convergent regulation of the lysosomal two-pore channel-2 by Mg²⁺, NAADP, PI(3,5)P₂ and multiple protein kinases.

PubMed

Jha, Archana; Ahuja, Malini; Patel, Sandip; Brailoiu, Eugen; Muallem, Shmuel

2014-03-03

Lysosomal Ca(2+) homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca(2+) signaling was the discovery of the two-pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated channels regulated by mTORC1, but not by NAADP. Here, we identified Mg(2+) and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg(2+) specifically inhibited TPC2 outward current, whereas lysosomal Mg(2+) partially inhibited both outward and inward currents in a lysosomal lumen pH-dependent manner. Under controlled Mg(2+), TPC2 is readily activated by NAADP with channel properties identical to those in response to PI(3,5)P2. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP-mediated Ca(2+) release in intact cells is regulated by Mg(2+), PI(3,5)P2, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP-mediated Ca(2+) signaling and link this pathway to Mg(2+) homeostasis and MAP kinases, pointing to roles for lysosomal Ca(2+) in cell growth, inflammation and cancer.

cAMP-dependent kinase does not modulate the Slack sodium-activated potassium channel.

PubMed

Nuwer, Megan O; Picchione, Kelly E; Bhattacharjee, Arin

2009-09-01

The Slack gene encodes a Na(+)-activated K(+) channel and is expressed in many different types of neurons. Like the prokaryotic Ca(2+)-gated K(+) channel MthK, Slack contains two 'regulator of K(+) conductance' (RCK) domains within its carboxy terminal, domains likely involved in Na(+) binding and channel gating. It also contains multiple consensus protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites and although regulated by protein kinase C (PKC) phosphorylation, modulation by PKA has not been determined. To test if PKA directly regulates Slack, nystatin-perforated patch whole-cell currents were recorded from a human embryonic kidney (HEK-293) cell line stably expressing Slack. Bath application of forskolin, an adenylate cyclase activator, caused a rapid and complete inhibition of Slack currents however, the inactive homolog of forskolin, 1,9-dideoxyforskolin caused a similar effect. In contrast, bath application of 8-bromo-cAMP did not affect the amplitude nor the activation kinetics of Slack currents. In excised inside-out patch recordings, direct application of the PKA catalytic subunit to patches did not affect the open probability of Slack channels nor was open probability affected by direct application of protein phosphatase 2B. Preincubation of cells with the protein kinase A inhibitor KT5720 also did not change current density. Finally, mutating the consensus phosphorylation site located between RCK domain 1 and domain 2 from serine to glutamate did not affect current activation kinetics. We conclude that unlike PKC, phosphorylation by PKA does not acutely modulate the function and gating activation kinetics of Slack channels.

Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel.

PubMed

Yamada, Toshiki; Krzeminski, Mickael; Bozoky, Zoltan; Forman-Kay, Julie D; Strange, Kevin

2016-11-01

Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple α-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-β-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane α-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structure-function relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Distribution of L-type calcium channels in rat thalamic neurones.

PubMed

Budde, T; Munsch, T; Pape, H C

1998-02-01

One major pathway for calcium entry into neurones is through voltage-activated calcium channels. The distribution of calcium channels over the membrane surface is important for their contribution to neuronal function. Electrophysiological recordings from thalamic cells in situ and after acute isolation demonstrated the presence of high-voltage activated calcium currents. The use of specific L-type calcium channel agonists and antagonists of the dihydropyridine type revealed an about 40% contribution of L-type channels to the total high-voltage-activated calcium current. In order to localize L-type calcium channels in thalamic neurones, fluorescent dihydropyridines were used. They were combined with the fluorescent dye RH414, which allowed the use of a ratio technique and thereby the determination of channel density. The distribution of L-type channels was analysed in the three main thalamic cell types: thalamocortical relay cells, local interneurones and reticular thalamic neurones. While channel density was highest in the soma and decreased significantly in the dendritic region, channels appeared to be clustered differentially in the three types of cells. In thalamocortical cells, L-type channels were clustered in high density around the base of dendrites, while they were more evenly distributed on the soma of interneurones. Reticular thalamic neurones exhibited high density of L-type channels in more central somatic regions. The differential localization of L-type calcium channels found in this study implies their predominate involvement in the regulation of somatic and proximal dendritic calcium-dependent processes, which may be of importance for specific thalamic functions, such as those mediating the transition from rhythmic burst activity during sleep to single spike activity during wakefulness or regulating the relay of visual information.

Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop.

PubMed

Ehrhardt, Annette; Chung, W Joon; Pyle, Louise C; Wang, Wei; Nowotarski, Krzysztof; Mulvihill, Cory M; Ramjeesingh, Mohabir; Hong, Jeong; Velu, Sadanandan E; Lewis, Hal A; Atwell, Shane; Aller, Steve; Bear, Christine E; Lukacs, Gergely L; Kirk, Kevin L; Sorscher, Eric J

2016-01-22

In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Crystal Structure of a Mammalian Voltage-Dependent Shaker Family K+ Channel

NASA Astrophysics Data System (ADS)

Long, Stephen B.; Campbell, Ernest B.; MacKinnon, Roderick

2005-08-01

Voltage-dependent potassium ion (K+) channels (Kv channels) conduct K+ ions across the cell membrane in response to changes in the membrane voltage, thereby regulating neuronal excitability by modulating the shape and frequency of action potentials. Here we report the crystal structure, at a resolution of 2.9 angstroms, of a mammalian Kv channel, Kv1.2, which is a member of the Shaker K+ channel family. This structure is in complex with an oxido-reductase β subunit of the kind that can regulate mammalian Kv channels in their native cell environment. The activation gate of the pore is open. Large side portals communicate between the pore and the cytoplasm. Electrostatic properties of the side portals and positions of the T1 domain and β subunit are consistent with electrophysiological studies of inactivation gating and with the possibility of K+ channel regulation by the β subunit.

Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells

NASA Astrophysics Data System (ADS)

Rich, Devra P.; Anderson, Matthew P.; Gregory, Richard J.; Cheng, Seng H.; Paul, Sucharita; Jefferson, Douglas M.; McCann, John D.; Klinger, Katherine W.; Smith, Alan E.; Welsh, Michael J.

1990-09-01

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (ΔF508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.

Proximal clustering between BK and CaV1.3 channels promotes functional coupling and BK channel activation at low voltage

PubMed Central

Vivas, Oscar; Moreno, Claudia M; Santana, Luis F; Hille, Bertil

2017-01-01

CaV-channel dependent activation of BK channels is critical for feedback control of both calcium influx and cell excitability. Here we addressed the functional and spatial interaction between BK and CaV1.3 channels, unique CaV1 channels that activate at low voltages. We found that when BK and CaV1.3 channels were co-expressed in the same cell, BK channels started activating near −50 mV, ~30 mV more negative than for activation of co-expressed BK and high-voltage activated CaV2.2 channels. In addition, single-molecule localization microscopy revealed striking clusters of CaV1.3 channels surrounding clusters of BK channels and forming a multi-channel complex both in a heterologous system and in rat hippocampal and sympathetic neurons. We propose that this spatial arrangement allows tight tracking between local BK channel activation and the gating of CaV1.3 channels at quite negative membrane potentials, facilitating the regulation of neuronal excitability at voltages close to the threshold to fire action potentials. DOI: http://dx.doi.org/10.7554/eLife.28029.001 PMID:28665272

Modulation of Gardos channel activity by cytokines in sickle erythrocytes.

PubMed

Rivera, Alicia; Jarolim, Petr; Brugnara, Carlo

2002-01-01

It has recently been shown that the Gardos channel activity of mouse erythrocytes can be modified by endothelins, suggesting a functional linkage between endothelin receptors and the Gardos channel. Using (86)Rubidium ((86)Rb) influx, effects were estimated of proinflammatory molecules such as platelet activator factor (PAF), endothelin-1 (ET-1), interleukin-10 (IL-10), and regulated on activation normal T cells expressed and secreted (RANTES) on the Gardos channel activity in human normal and sickle red cells. It was found that PAF (EC(50): 15 +/- 7 nM), RANTES (EC(50), 9 +/- 6 ng/mL [1.2 +/- 0.8 nM]), IL-10 (EC(50), 11 +/- 8 ng/mL [204 +/- 148 nM]), and ET-1 (EC(50), 123 +/- 34 nM) induce a significant increase in Gardos channel activity-between 28% and 84%-over the control. In addition, these agents modify the Gardos channel affinity for internal Ca(++) (K(0.5)) by 2- to 6-fold. Biochemical evidence is provided for the presence of ET receptor subtype B in sickle and normal red cells. Furthermore, it was found that ET-1, PAF, RANTES, and IL-10 induce a significant increase in red cell density (P <.05). These data suggest that activation of the Gardos channel is functionally coupled to receptor motifs such as C-X-C (PAF), C-C (RANTES), and ET receptor subtype B. Thus, cell volume regulation or erythrocyte hydration states might be altered by activation of the Gardos channel by cytokines in vivo. The role of these mediators in promoting sickle cell dehydration in vivo is under investigation.

TRPV1 Channels Are Functionally Coupled with BK(mSlo1) Channels in Rat Dorsal Root Ganglion (DRG) Neurons

PubMed Central

Yan, Zonghe; Kong, Wenjuan; Liu, Beiying; Li, Xia; Yao, Jing; Zhang, Yuexuan; Qin, Feng; Ding, Jiuping

2013-01-01

The transient receptor potential vanilloid receptor 1 (TRPV1) channel is a nonselective cation channel activated by a variety of exogenous and endogenous physical and chemical stimuli, such as temperature (≥42 °C), capsaicin, a pungent compound in hot chili peppers, and allyl isothiocyanate. Large-conductance calcium- and voltage-activated potassium (BK) channels regulate the electric activities and neurotransmitter releases in excitable cells, responding to changes in membrane potentials and elevation of cytosolic calcium ions (Ca2+). However, it is unknown whether the TRPV1 channels are coupled with the BK channels. Using patch-clamp recording combined with an infrared laser device, we found that BK channels could be activated at 0 mV by a Ca2+ influx through TRPV1 channels not the intracellular calcium stores in submilliseconds. The local calcium concentration around BK is estimated over 10 μM. The crosstalk could be affected by 10 mM BAPTA, whereas 5 mM EGTA was ineffectual. Fluorescence and co-immunoprecipitation experiments also showed that BK and TRPV1 were able to form a TRPV1-BK complex. Furthermore, we demonstrated that the TRPV1-BK coupling also occurs in dosal root ganglion (DRG) cells, which plays a critical physiological role in regulating the “pain” signal transduction pathway in the peripheral nervous system. PMID:24147119

Chloride channels in stroke

PubMed Central

Zhang, Ya-ping; Zhang, Hao; Duan, Dayue Darrel

2013-01-01

Vascular remodeling of cerebral arterioles, including proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMCs), is the major cause of changes in the cross-sectional area and diameter of the arteries and sudden interruption of blood flow or hemorrhage in the brain, ie, stroke. Accumulating evidence strongly supports an important role for chloride (Cl−) channels in vascular remodeling and stroke. At least three Cl− channel genes are expressed in VSMCs: 1) the TMEM16A (or Ano1), which may encode the calcium-activated Cl− channels (CACCs); 2) the CLC-3 Cl− channel and Cl−/H+ antiporter, which is closely related to the volume-regulated Cl− channels (VRCCs); and 3) the cystic fibrosis transmembrane conductance regulator (CFTR), which encodes the PKA- and PKC-activated Cl− channels. Activation of the CACCs by agonist-induced increase in intracellular Ca2+ causes membrane depolarization, vasoconstriction, and inhibition of VSMC proliferation. Activation of VRCCs by cell volume increase or membrane stretch promotes the production of reactive oxygen species, induces proliferation and inhibits apoptosis of VSMCs. Activation of CFTR inhibits oxidative stress and may prevent the development of hypertension. In addition, Cl− current mediated by gamma-aminobutyric acid (GABA) receptor has also been implicated a role in ischemic neuron death. This review focuses on the functional roles of Cl− channels in the development of stroke and provides a perspective on the future directions for research and the potential to develop Cl− channels as new targets for the prevention and treatment of stroke. PMID:23103617

Modulation of the olfactory CNG channel by Ptdlns(3,4,5)P3.

PubMed

Zhainazarov, A B; Spehr, M; Wetzel, C H; Hatt, H; Ache, B W

2004-09-01

Recent data suggest that the 3-phosphoinositides can modulate cyclic nucleotide signaling in rat olfactory receptor neurons (ORNs). Given the ability of diverse lipids to modulate ion channels, we asked whether phosphatidylinositol 3,4,5-trisphosphate (PIP3) can regulate the olfactory cyclic nucleotide-gated (CNG) channel as a possible mechanism for this modulation. We show that applying PIP3 to the intracellular side of inside-out patches from rat ORNs inhibits activation of the olfactory CNG channel by cAMP. The effect of PIP3 is immediate and partially reversible, and reflects an increase in the EC50 of cAMP, not a reduction in the single-channel current amplitude. The effect of PIP3 is significantly stronger than that of PIP2; other phospholipids tested have no appreciable effect on channel activity. PIP3 similarly inhibits the recombinant heteromeric (A2/A4) and homomeric (A2) olfactory CNG channel expressed in HEK293 cells, suggesting that PIP3 acts directly on the channel. These findings indicate that 3-phosphoinositides can be functionally important regulators of CNG channels.

Fragile X mental retardation protein controls ion channel expression and activity.

PubMed

Ferron, Laurent

2016-10-15

Fragile X-associated disorders are a family of genetic conditions resulting from the partial or complete loss of fragile X mental retardation protein (FMRP). Among these disorders is fragile X syndrome, the most common cause of inherited intellectual disability and autism. FMRP is an RNA-binding protein involved in the control of local translation, which has pleiotropic effects, in particular on synaptic function. Analysis of the brain FMRP transcriptome has revealed hundreds of potential mRNA targets encoding postsynaptic and presynaptic proteins, including a number of ion channels. FMRP has been confirmed to bind voltage-gated potassium channels (K v 3.1 and K v 4.2) mRNAs and regulates their expression in somatodendritic compartments of neurons. Recent studies have uncovered a number of additional roles for FMRP besides RNA regulation. FMRP was shown to directly interact with, and modulate, a number of ion channel complexes. The sodium-activated potassium (Slack) channel was the first ion channel shown to directly interact with FMRP; this interaction alters the single-channel properties of the Slack channel. FMRP was also shown to interact with the auxiliary β4 subunit of the calcium-activated potassium (BK) channel; this interaction increases calcium-dependent activation of the BK channel. More recently, FMRP was shown to directly interact with the voltage-gated calcium channel, Ca v 2.2, and reduce its trafficking to the plasma membrane. Studies performed on animal models of fragile X syndrome have revealed links between modifications of ion channel activity and changes in neuronal excitability, suggesting that these modifications could contribute to the phenotypes observed in patients with fragile X-associated disorders. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Brain-derived neurotrophic factor (BDNF) induces sustained intracellular Ca2+ elevation through the up-regulation of surface transient receptor potential 3 (TRPC3) channels in rodent microglia.

PubMed

Mizoguchi, Yoshito; Kato, Takahiro A; Seki, Yoshihiro; Ohgidani, Masahiro; Sagata, Noriaki; Horikawa, Hideki; Yamauchi, Yusuke; Sato-Kasai, Mina; Hayakawa, Kohei; Inoue, Ryuji; Kanba, Shigenobu; Monji, Akira

2014-06-27

Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca(2+)]i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca(2+) elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca(2+) elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Tagging of Endogenous BK Channels with a Fluorogen-Activating Peptide Reveals β4-Mediated Control of Channel Clustering in Cerebellum

PubMed Central

Pratt, Christopher P.; Kuljis, Dika A.; Homanics, Gregg E.; He, Jianjun; Kolodieznyi, Dmytro; Dudem, Srikanth; Hollywood, Mark A.; Barth, Alison L.; Bruchez, Marcel P.

2017-01-01

BK channels are critical regulators of neuronal activity, controlling firing, neurotransmitter release, cerebellar function, and BK channel mutations have been linked to seizure disorders. Modulation of BK channel gating is well characterized, regulated by accessory subunit interactions, intracellular signaling pathways, and membrane potential. In contrast, the role of intracellular trafficking mechanisms in controlling BK channel function, especially in live cells, has been less studied. Fluorogen-activating peptides (FAPs) are well-suited for trafficking and physiological studies due to the binding of malachite green (MG)-based dyes with sub-nanomolar affinity to the FAP, resulting in bright, photostable, far-red fluorescence. Cell-excluded MG dyes enable the selective tagging of surface protein and tracking through endocytic pathways. We used CRISPR to insert the FAP at the extracellular N-terminus of BKα in the first exon of its native locus, enabling regulation by the native promoter elements and tag incorporation into multiple splice isoforms. Motor coordination was found to be normal; however, BK channel expression seems to be reduced in some locations. Alternate start site selection or post-translational proteolytic processing resulted in incomplete FAP tagging of the BKα proteins in brain tissues. In Purkinje cell somata, FAP revealed BK channel clustering previously only observed by electron microscopy. Measurement of these clusters in β4+/- and β4-/- mice showed that puncta number and cluster fluorescence intensity on the soma are reduced in β4-/- knockout animals. This novel mouse line provides a versatile fluorescent platform for studying endogenous BK channels in living and fixed tissues. Future studies could apply this line to ex vivo neuronal cultures to study live-cell channel trafficking. PMID:29163049

Modulation of Ionic Channels and Insulin Secretion by Drugs and Hormones in Pancreatic Beta Cells.

PubMed

Velasco, Myrian; Díaz-García, Carlos Manlio; Larqué, Carlos; Hiriart, Marcia

2016-09-01

Pancreatic beta cells, unique cells that secrete insulin in response to an increase in glucose levels, play a significant role in glucose homeostasis. Glucose-stimulated insulin secretion (GSIS) in pancreatic beta cells has been extensively explored. In this mechanism, glucose enters the cells and subsequently the metabolic cycle. During this process, the ATP/ADP ratio increases, leading to ATP-sensitive potassium (KATP) channel closure, which initiates depolarization that is also dependent on the activity of TRP nonselective ion channels. Depolarization leads to the opening of voltage-gated Na(+) channels (Nav) and subsequently voltage-dependent Ca(2+) channels (Cav). The increase in intracellular Ca(2+) triggers the exocytosis of insulin-containing vesicles. Thus, electrical activity of pancreatic beta cells plays a central role in GSIS. Moreover, many growth factors, incretins, neurotransmitters, and hormones can modulate GSIS, and the channels that participate in GSIS are highly regulated. In this review, we focus on the principal ionic channels (KATP, Nav, and Cav channels) involved in GSIS and how classic and new proteins, hormones, and drugs regulate it. Moreover, we also discuss advances on how metabolic disorders such as metabolic syndrome and diabetes mellitus change channel activity leading to changes in insulin secretion. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Regulation of activation and processing of the cystic fibrosis transmembrane conductance regulator (CFTR) by a complex electrostatic interaction between the regulatory domain and cytoplasmic loop 3.

PubMed

Wang, Guangyu; Duan, Dayue Darrel

2012-11-23

NEG2 regulates CFTR gating but the mechanism is unknown. A putative NEG2-CL3 electrostatic attraction, possibly weakened by Arg-764/Arg-766 of the R domain, prohibited CFTR activation. A charge exchange between NEG2 and CL3 caused misprocessing. Electrostatic regulation of CFTR activation and processing may be asymmetric at the CL3-R interface. The CL3-R interface is optimally designed for multiple regulations of CFTR functions. NEG2, a short C-terminal segment (817-838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, Lys-946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of Asp-835, Asp-836, or Glu-838 of NEG2 to prevent the channel activation by PKA. Arg-764 or Arg-766 of the Ser-768 phosphorylation site of the R domain is proposed to promote the channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A, and E838A but also K946A reduced the PKA-dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. However, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity whereas D835R/D836R/E838R/K946D/H950D was fractionally misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing, and Ser-768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2, and the Ser-768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.

The role of transient receptor potential channels in joint diseases.

PubMed

Krupkova, O; Zvick, J; Wuertz-Kozak, K

2017-10-10

Transient receptor potential channels (TRP channels) are cation selective transmembrane receptors with diverse structures, activation mechanisms and physiological functions. TRP channels act as cellular sensors for a plethora of stimuli, including temperature, membrane voltage, oxidative stress, mechanical stimuli, pH and endogenous, as well as, exogenous ligands, thereby illustrating their versatility. As such, TRP channels regulate various functions in both excitable and non-excitable cells, mainly by mediating Ca2+ homeostasis. Dysregulation of TRP channels is implicated in many pathologies, including cardiovascular diseases, muscular dystrophies and hyperalgesia. However, the importance of TRP channel expression, physiological function and regulation in chondrocytes and intervertebral disc (IVD) cells is largely unexplored. Osteoarthritis (OA) and degenerative disc disease (DDD) are chronic age-related disorders that significantly affect the quality of life by causing pain, activity limitation and disability. Furthermore, currently available therapies cannot effectively slow-down or stop progression of these diseases. Both OA and DDD are characterised by reduced tissue cellularity, enhanced inflammatory responses and molecular, structural and mechanical alterations of the extracellular matrix, hence affecting load distribution and reducing joint flexibility. However, knowledge on how chondrocytes and IVD cells sense their microenvironment and respond to its changes is still limited. In this review, we introduced six families of mammalian TRP channels, their mechanisms of activation, as well as, activation-driven cellular consequences. We summarised the current knowledge on TRP channel expression and activity in chondrocytes and IVD cells, as well as, the significance of TRP channels as therapeutic targets for the treatment of OA and DDD.

Structure, function and translational relevance of aquaporin dual water and ion channels.

PubMed

Yool, Andrea J; Campbell, Ewan M

2012-01-01

Aquaporins have been assumed to be selective for water alone, and aquaglyceroporins are accepted as carrying water and small uncharged solutes including glycerol. This review presents an expanded view of aquaporins as channels with more complex mechanisms of regulation and diverse repertoires of substrate permeabilities than were originally appreciated in the early establishment of the field. The role of aquaporins as dual water and gated ion channels is likely to have physiological and potentially translational relevance, and can be evaluated with newly developed molecular and pharmacological tools. Ion channel activity has been shown for Aquaporins -0, -1, and -6, Drosphila Big Brain, and plant Nodulin-26. Although the concept of ion channel function in aquaporins remains controversial, research advances are beginning to define not only the ion channel function but also the detailed molecular mechanisms that govern and mediate the multifunctional capabilities. With regard to physiological relevance, the adaptive benefit of expression of ion channel activity in aquaporins, implied by amino acid sequence conservation of the ion channel gating domains, suggests they provide more than water or glycerol and solute transport. Dual ion and water channels are of interest for understanding the modulation of transmembrane fluid gradients, volume regulation, and possible signal transduction in tissues expressing classes of aquaporins that have the dual function capability. Other aquaporin classes might be found in future work to have ion channel activities, pending identification of the possible signaling pathways that could govern activation. Copyright © 2012 Elsevier Ltd. All rights reserved.

SIGMAR1 Regulates Membrane Electrical Activity in Response to Extracellular Matrix Stimulation to Drive Cancer Cell Invasiveness.

PubMed

Crottès, David; Rapetti-Mauss, Raphael; Alcaraz-Perez, Francisca; Tichet, Mélanie; Gariano, Giuseppina; Martial, Sonia; Guizouarn, Hélène; Pellissier, Bernard; Loubat, Agnès; Popa, Alexandra; Paquet, Agnès; Presta, Marco; Tartare-Deckert, Sophie; Cayuela, Maria Luisa; Martin, Patrick; Borgese, Franck; Soriani, Olivier

2016-02-01

The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K(+) channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/β1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition. ©2015 American Association for Cancer Research.

Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

PubMed

Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T; Scheaffer, Suzanne M; Roswit, William T; Alevy, Yael G; Patel, Anand C; Heier, Richard F; Romero, Arthur G; Nichols, Colin G; Holtzman, Michael J; Brett, Tom J

2012-12-07

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.

Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation*♦

PubMed Central

Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T.; Scheaffer, Suzanne M.; Roswit, William T.; Alevy, Yael G.; Patel, Anand C.; Heier, Richard F.; Romero, Arthur G.; Nichols, Colin G.; Holtzman, Michael J.; Brett, Tom J.

2012-01-01

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface. PMID:23112050

Distribution and function of HCN channels in the apical dendritic tuft of neocortical pyramidal neurons.

PubMed

Harnett, Mark T; Magee, Jeffrey C; Williams, Stephen R

2015-01-21

The apical tuft is the most remote area of the dendritic tree of neocortical pyramidal neurons. Despite its distal location, the apical dendritic tuft of layer 5 pyramidal neurons receives substantial excitatory synaptic drive and actively processes corticocortical input during behavior. The properties of the voltage-activated ion channels that regulate synaptic integration in tuft dendrites have, however, not been thoroughly investigated. Here, we use electrophysiological and optical approaches to examine the subcellular distribution and function of hyperpolarization-activated cyclic nucleotide-gated nonselective cation (HCN) channels in rat layer 5B pyramidal neurons. Outside-out patch recordings demonstrated that the amplitude and properties of ensemble HCN channel activity were uniform in patches excised from distal apical dendritic trunk and tuft sites. Simultaneous apical dendritic tuft and trunk whole-cell current-clamp recordings revealed that the pharmacological blockade of HCN channels decreased voltage compartmentalization and enhanced the generation and spread of apical dendritic tuft and trunk regenerative activity. Furthermore, multisite two-photon glutamate uncaging demonstrated that HCN channels control the amplitude and duration of synaptically evoked regenerative activity in the distal apical dendritic tuft. In contrast, at proximal apical dendritic trunk and somatic recording sites, the blockade of HCN channels decreased excitability. Dynamic-clamp experiments revealed that these compartment-specific actions of HCN channels were heavily influenced by the local and distributed impact of the high density of HCN channels in the distal apical dendritic arbor. The properties and subcellular distribution pattern of HCN channels are therefore tuned to regulate the interaction between integration compartments in layer 5B pyramidal neurons. Copyright © 2015 the authors 0270-6474/15/351024-14$15.00/0.

Functional K(ATP) channels in the rat retinal microvasculature: topographical distribution, redox regulation, spermine modulation and diabetic alteration.

PubMed

Ishizaki, Eisuke; Fukumoto, Masanori; Puro, Donald G

2009-05-15

The essential task of the circulatory system is to match blood flow to local metabolic demand. However, much remains to be learned about this process. To better understand how local perfusion is regulated, we focused on the functional organization of the retinal microvasculature, which is particularly well adapted for the local control of perfusion. Here, we assessed the distribution and regulation of functional K(ATP) channels whose activation mediates the hyperpolarization induced by adenosine. Using microvascular complexes freshly isolated from the rat retina, we found a topographical heterogeneity in the distribution of functional K(ATP) channels; capillaries generate most of the K(ATP) current. The initiation of K(ATP)-induced responses in the capillaries supports the concept that the regulation of retinal perfusion is highly decentralized. Additional study revealed that microvascular K(ATP) channels are redox sensitive, with oxidants increasing their activity. Furthermore, the oxidant-mediated activation of these channels is driven by the polyamine spermine, whose catabolism produces oxidants. In addition, our observation that spermine-dependent oxidation occurs predominately in the capillaries accounts for why they generate most of the K(ATP) current detected in retinal microvascular complexes. Here, we also analysed retinal microvessels of streptozotocin-injected rats. We found that soon after the onset of diabetes, an increase in spermine-dependent oxidation at proximal microvascular sites boosts their K(ATP) current and thereby virtually eliminates the topographical heterogeneity of functional K(ATP) channels. We conclude that spermine-dependent oxidation is a previously unrecognized mechanism by which this polyamine modulates ion channels; in addition to a physiological role, spermine-dependent oxidation may also contribute to microvascular dysfunction in the diabetic retina.

Up-regulation of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 3 (HCN3) by Specific Interaction with K+ Channel Tetramerization Domain-containing Protein 3 (KCTD3)*

PubMed Central

Cao-Ehlker, Xiaochun; Zong, Xiangang; Hammelmann, Verena; Gruner, Christian; Fenske, Stefanie; Michalakis, Stylianos; Wahl-Schott, Christian; Biel, Martin

2013-01-01

Most ion channels consist of the principal ion-permeating core subunit(s) and accessory proteins that are assembled with the channel core. The biological functions of the latter proteins are diverse and include the regulation of the biophysical properties of the ion channel, its connection to signaling pathways and the control of its cell surface expression. There is recent evidence that native hyperpolarization-activated cyclic nucleotide-gated channel complexes (HCN1–4) also contain accessory subunits, among which TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) has been most extensively studied. Here, we identify KCTD3, a so far uncharacterized member of the potassium channel tetramerization-domain containing (KCTD) protein family as an HCN3-interacting protein. KCTD3 is widely expressed in brain and some non-neuronal tissues and colocalizes with HCN3 in specific regions of the brain including hypothalamus. Within the HCN channel family, KCTD3 specifically binds to HCN3 and leads to a profound up-regulation of cell surface expression and current density of this channel. HCN3 can also functionally interact with TRIP8b; however, we found no evidence for channel complexes containing both TRIP8b and KCTD3. The C terminus of HCN3 is crucially required for functional interaction with KCTD3. Replacement of the cytosolic C terminus of HCN2 by the corresponding domain of HCN3 renders HCN2 sensitive to regulation by KCTD3. The C-terminal-half of KCTD3 is sufficient for binding to HCN3. However, the complete protein including the N-terminal tetramerization domain is needed for HCN3 current up-regulation. Together, our experiments indicate that KCTD3 is an accessory subunit of native HCN3 complexes. PMID:23382386

Background and tandem-pore potassium channels in magnocellular neurosecretory cells of the rat supraoptic nucleus

PubMed Central

Han, Jaehee; Gnatenco, Carmen; Sladek, Celia D; Kim, Donghee

2003-01-01

Magnocellular neurosecretory cells (MNCs) were isolated from the supraoptic nucleus of rat hypothalamus, and properties of K+ channels that may regulate the resting membrane potential and the excitability of MNCs were studied. MNCs showed large transient outward currents, typical of vasopressin- and oxytocin-releasing neurons. K+ channels in MNCs were identified by recording K+ channels that were open at rest in cell-attached and inside-out patches in symmetrical 150 mm KCl. Eight different K+ channels were identified and could be distinguished unambiguously by their single-channel kinetics and voltage-dependent rectification. Two K+ channels could be considered functional correlates of TASK-1 and TASK-3, as judged by their single-channel kinetics and high sensitivity to pHo. Three K+ channels showed properties similar to TREK-type tandem-pore K+ channels (TREK-1, TREK-2 and a novel TREK), as judged by their activation by membrane stretch, intracellular acidosis and arachidonic acid. One K+ channel was activated by application of pressure, arachidonic acid and alkaline pHi, and showed single-channel kinetics indistinguishable from those of TRAAK. One K+ channel showed strong inward rectification and single-channel conductance similar to those of a classical inward rectifier, IRK3. Finally, a K+ channel whose cloned counterpart has not yet been identified was highly sensitive to extracellular pH near the physiological range similar to those of TASK channels, and was the most active among all K+ channels. Our results show that in MNCs at rest, eight different types of K+ channels can be found and six of them belong to the tandem-pore K+ channel family. Various physiological and pathophysiological conditions may modulate these K+ channels and regulate the excitability of MNCs. PMID:12562991

Phosphatidylinositol (4,5)-bisphosphate dynamically regulates the K2P background K+ channel TASK-2

PubMed Central

Niemeyer, María Isabel; Cid, L. Pablo; Paulais, Marc; Teulon, Jacques; Sepúlveda, Francisco V.

2017-01-01

Two-pore domain K2P K+ channels responsible for the background K+ conductance and the resting membrane potential, are also finely regulated by a variety of chemical, physical and physiological stimuli. Hormones and transmitters acting through Gq protein-coupled receptors (GqPCRs) modulate the activity of various K2P channels but the signalling involved has remained elusive, in particular whether dynamic regulation by membrane PI(4,5)P2, common among other classes of K+ channels, affects K2P channels is controversial. Here we show that K2P K+ channel TASK-2 requires PI(4,5)P2 for activity, a dependence that accounts for its run down in the absence of intracellular ATP and its full recovery by addition of exogenous PI(4,5)P2, its inhibition by low concentrations of polycation PI scavengers, and inhibition by PI(4,5)P2 depletion from the membrane. Comprehensive mutagenesis suggests that PI(4,5)P2 interaction with TASK-2 takes place at C-terminus where three basic aminoacids are identified as being part of a putative binding site. PMID:28358046

Diadenosine tetraphosphate-gating of cardiac K(ATP) channels requires intact actin cytoskeleton.

PubMed

Jovanović, S; Jovanović, A

2001-09-01

Diadenosine polyphosphates (ApnA) have been recently discovered in the heart, and their levels found to be regulated by ischemia. These signaling molecules are believed to regulate cellular processes that alarm a cell to metabolic stress. In particular, changes in cardiac diadenosine polyphosphates (ApnA) levels may contribute to the regulation of ATP-sensitive K+ (K(ATP)) channel activity, an ion channel that couples the cellular metabolic state with membrane excitability. A feature of myocardial ischemia is the disruption of the actin cytoskeleton which critically regulates the behavior of K(ATP) channels. Whether the integrity of actin microfilaments regulates the interaction of ApnA with K(ATP) channels is not known. The inside-out configuration of the patch-clamp technique was applied to cardiomyocytes isolated from guinea-pig heart. Following patch excision, the prototype dinucleotide, diadenosine tetraphosphate (Ap4A), inhibited K(ATP) channel opening. Treatment of the internal side of membrane patches with either cytochalasin B or DNase I, disrupters of the actin cytoskeleton, prevented Ap4A-induced inhibition of K(ATP) channel opening. Application of purified actin to DNase-treated membrane patches restored the ability of Ap4A to close K(ATP) channels. This study shows that inhibition of cardiac K(ATP) channel by Ap4A, a putative alarmone, requires intact subsarcolemmal actin network. Such interaction between K(ATP) channels, the cardiomyocyte cytoskeleton and intracellular Ap4A could affect different channel-dependent functions.

Characterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology

PubMed Central

Lee, Claire Shuk-Kwan; Tong, Benjamin Chun-Kit; Cheng, Cecily Wing-Hei; Hung, Harry Chun-Hin; Cheung, King-Ho

2016-01-01

Lysosomal calcium (Ca2+) release mediated by NAADP triggers signalling cascades that regulate many cellular processes. The identification of two-pore channel 2 (TPC2) as the NAADP receptor advances our understanding of lysosomal Ca2+ signalling, yet the lysosome is not amenable to traditional patch-clamp electrophysiology. Previous attempts to record TPC2 single-channel activity put TPC2 outside its native environment, which not reflect TPC2’s true physiological properties. To test the feasibility of using nuclear membrane electrophysiology for TPC2 channel characterization, we constructed a stable human TPC2-expressing DT40TKO cell line that lacks endogenous InsP3R and RyR (DT40TKO-hTPC2). Immunostaining revealed hTPC2 expression on the ER and nuclear envelope. Intracellular dialysis of NAADP into Fura-2-loaded DT40TKO-hTPC2 cells elicited cytosolic Ca2+ transients, suggesting that hTPC2 was functionally active. Using nuclear membrane electrophysiology, we detected a ~220 pS single-channel current activated by NAADP with K+ as the permeant ion. The detected single-channel recordings displayed a linear current-voltage relationship, were sensitive to Ned-19 inhibition, were biphasically regulated by NAADP concentration, and regulated by PKA phosphorylation. In summary, we developed a cell model for the characterization of the TPC2 channel and the nuclear membrane patch-clamp technique provided an alternative approach to rigorously investigate the electrophysiological properties of TPC2 with minimal manipulation. PMID:26838264

Activity and Ca2+ regulate the mobility of TRPV1 channels in the plasma membrane of sensory neurons

PubMed Central

Senning, Eric N; Gordon, Sharona E

2015-01-01

TRPV1 channels are gated by a variety of thermal, chemical, and mechanical stimuli. We used optical recording of Ca2+ influx through TRPV1 to measure activity and mobility of single TRPV1 molecules in isolated dorsal root ganglion neurons and cell lines. The opening of single TRPV1 channels produced sparklets, representing localized regions of elevated Ca2+. Unlike sparklets reported for L-type Ca2+ channels, TRPV4 channels, and AchR channels, TRPV1 channels diffused laterally in the plasma membrane as they gated. Mobility was highly variable from channel-to-channel and, to a smaller extent, from cell to cell. Most surprisingly, we found that mobility decreased upon channel activation by capsaicin, but only in the presence of extracellular Ca2+. We propose that decreased mobility of open TRPV1 could act as a diffusion trap to concentrate channels in cell regions with high activity. DOI: http://dx.doi.org/10.7554/eLife.03819.001 PMID:25569155

Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1).

PubMed

De Jesús-Pérez, José J; Cruz-Rangel, Silvia; Espino-Saldaña, Ángeles E; Martínez-Torres, Ataúlfo; Qu, Zhiqiang; Hartzell, H Criss; Corral-Fernandez, Nancy E; Pérez-Cornejo, Patricia; Arreola, Jorge

2018-03-01

The TMEM16A-mediated Ca 2+ -activated Cl - current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca 2+ . On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-β-cyclodextrin M-βCD) or restoration (with M-βCD+cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-βCD alone transiently increases TMEM16A activity and dampens rundown whereas M-βCD+cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-βCD, M-βCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential Protein Kinase C-dependent Modulation of Kv7.4 and Kv7.5 Subunits of Vascular Kv7 Channels*

PubMed Central

Brueggemann, Lioubov I.; Mackie, Alexander R.; Cribbs, Leanne L.; Freda, Jessica; Tripathi, Abhishek; Majetschak, Matthias; Byron, Kenneth L.

2014-01-01

The Kv7 family (Kv7.1–7.5) of voltage-activated potassium channels contributes to the maintenance of resting membrane potential in excitable cells. Previously, we provided pharmacological and electrophysiological evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels and that Kv7 activity is regulated by protein kinase C (PKC) in response to vasoconstrictors in vascular smooth muscle cells. Direct evidence for Kv7.4/7.5 heteromer formation, however, is lacking. Furthermore, it remains to be determined whether both subunits are regulated by PKC. Utilizing proximity ligation assays to visualize single molecule interactions, we now show that Kv7.4/Kv.7.5 heteromers are endogenously expressed in vascular smooth muscle cells. Introduction of dominant-negative Kv7.4 and Kv7.5 subunits in mesenteric artery myocytes reduced endogenous Kv7 currents by 84 and 76%, respectively. Expression of an inducible protein kinase Cα (PKCα) translocation system revealed that PKCα activation is sufficient to suppress endogenous Kv7 currents in A7r5 rat aortic and mesenteric artery smooth muscle cells. Arginine vasopressin (100 and 500 pm) and the PKC activator phorbol 12-myristate 13-acetate (1 nm) each inhibited human (h) Kv7.5 and hKv7.4/7.5, but not hKv7.4 channels expressed in A7r5 cells. A decrease in hKv7.5 and hKv7.4/7.5 current densities was associated with an increase in PKC-dependent phosphorylation of the channel proteins. These findings provide further evidence for a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation and new mechanistic insights into the role of heteromeric subunit assembly for regulation of vascular Kv7 channels. PMID:24297175

CRAC channel activity in C. elegans is mediated by Orai1 and STIM1 homologues and is essential for ovulation and fertility

PubMed Central

Lorin-Nebel, Catherine; Xing, Juan; Yan, Xiaohui; Strange, Kevin

2007-01-01

The Ca2+ release-activated Ca2+ (CRAC) channel is a plasma membrane Ca2+ entry pathway activated by endoplasmic reticulum (ER) Ca2+ store depletion. STIM1 proteins function as ER Ca2+ sensors and regulate CRAC channel activation. Recent studies have demonstrated that CRAC channels are encoded by the human Orai1 gene and a homologous Drosophila gene. C. elegans intestinal cells express a store-operated Ca2+ channel (SOCC) regulated by STIM-1. We cloned a full-length C. elegans cDNA that encodes a 293 amino acid protein, ORAI-1, homologous to human and Drosophila Orai1 proteins. ORAI-1 GFP reporters are co-expressed with STIM-1 in the gonad and intestine. Inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ signalling regulates C. elegans gonad function, fertility and rhythmic posterior body wall muscle contraction (pBoc) required for defecation. RNA interference (RNAi) silencing of orai-1 expression phenocopies stim-1 knockdown and causes sterility and prevents intestinal cell SOCC activation, but has no effect on pBoc or intestinal Ca2+ signalling. Orai-1 RNAi suppresses pBoc defects induced by intestinal expression of a STIM-1 Ca2+-binding mutant, indicating that the proteins function in a common pathway. Co-expression of stim-1 and orai-1 cDNAs in HEK293 cells induces large inwardly rectifying cation currents activated by ER Ca2+ depletion. The properties of this current recapitulate those of the native SOCC current. We conclude that C. elegans expresses bona fide CRAC channels that require the function of Orai1- and STIM1-related proteins. CRAC channels thus arose very early in animal evolution. In C. elegans, CRAC channels do not play obligate roles in all IP3-dependent signalling processes and ER Ca2+ homeostasis. Instead, we suggest that CRAC channels carry out highly specialized and cell-specific signalling roles and that they may function as a failsafe mechanism to prevent Ca2+ store depletion under pathophysiological and stress conditions. PMID:17218360

Molecular basis of activation of the arachidonate-regulated Ca2+ (ARC) channel, a store-independent Orai channel, by plasma membrane STIM1

PubMed Central

Thompson, Jill L; Shuttleworth, Trevor J

2013-01-01

Currently, Orai proteins are known to encode two distinct agonist-activated, highly calcium-selective channels: the store-operated Ca2+ release-activated Ca2+ (CRAC) channels, and the store-independent, arachidonic acid-activated ARC channels. Surprisingly, whilst the trigger for activation of these channels is entirely different, both depend on stromal interacting molecule 1 (STIM1). However, whilst STIM1 in the endoplasmic reticulum membrane is the critical sensor for the depletion of this calcium store that triggers CRAC channel activation, it is the pool of STIM1 constitutively resident in the plasma membrane that is essential for activation of the ARC channels. Here, using a variety of approaches, we show that the key domains within the cytosolic part of STIM1 identified as critical for the activation of CRAC channels are also key for activation of the ARC channels. However, examination of the actual steps involved in such activation reveal marked differences between these two Orai channel types. Specifically, loss of calcium from the EF-hand of STIM1 that forms the key initiation point for activation of the CRAC channels has no effect on ARC channel activity. Secondly, in marked contrast to the dynamic and labile nature of interactions between STIM1 and the CRAC channels, STIM1 in the plasma membrane appears to be constitutively associated with the ARC channels. Finally, specific mutations in STIM1 that induce an extended, constitutively active, conformation for the CRAC channels actually prevent activation of the ARC channels by arachidonic acid. Based on these findings, we propose that the likely role of arachidonic acid lies in inducing the actual gating of the channel. PMID:23690558

Nonreciprocal mechanisms in up- and downregulation of spinal motoneuron excitability by modulators of KCNQ/Kv7 channels.

PubMed

Lombardo, Joseph; Harrington, Melissa A

2016-11-01

KCNQ/K v 7 channels form a slow noninactivating K + current, also known as the M current. They activate in the subthreshold range of membrane potentials and regulate different aspects of excitability in neurons of the central nervous system. In spinal motoneurons (MNs), KCNQ/K v 7 channels have been identified in the somata, axonal initial segment, and nodes of Ranvier, where they generate a slow, noninactivating, K + current sensitive to both muscarinic receptor-mediated inhibition and KCNQ/K v 7 channel blockers. In this study, we thoroughly reevaluated the function of up- and downregulation of KCNQ/K v 7 channels in mouse immature spinal MNs. Using electrophysiological techniques together with specific pharmacological modulators of the activity of KCNQ/K v 7 channels, we show that enhancement of the activity of these channels decreases the excitability of spinal MNs in mouse neonates. This action on MNs results from a combination of hyperpolarization of the resting membrane potential, a decrease in the input resistance, and depolarization of the voltage threshold. On the other hand, the effect of inhibition of KCNQ/K v 7 channels suggested that these channels play a limited role in regulating basal excitability. Computer simulations confirmed that pharmacological enhancement of KCNQ/K v 7 channel activity decreases excitability and also suggested that the effects of inhibition of KCNQ/K v 7 channels on the excitability of spinal MNs do not depend on a direct effect in these neurons but likely on spinal cord synaptic partners. These results indicate that KCNQ/K v 7 channels have a fundamental role in the modulation of the excitability of spinal MNs acting both in these neurons and in their local presynaptic partners. Copyright © 2016 the American Physiological Society.

Nonreciprocal mechanisms in up- and downregulation of spinal motoneuron excitability by modulators of KCNQ/Kv7 channels

PubMed Central

Lombardo, Joseph

2016-01-01

KCNQ/Kv7 channels form a slow noninactivating K+ current, also known as the M current. They activate in the subthreshold range of membrane potentials and regulate different aspects of excitability in neurons of the central nervous system. In spinal motoneurons (MNs), KCNQ/Kv7 channels have been identified in the somata, axonal initial segment, and nodes of Ranvier, where they generate a slow, noninactivating, K+ current sensitive to both muscarinic receptor-mediated inhibition and KCNQ/Kv7 channel blockers. In this study, we thoroughly reevaluated the function of up- and downregulation of KCNQ/Kv7 channels in mouse immature spinal MNs. Using electrophysiological techniques together with specific pharmacological modulators of the activity of KCNQ/Kv7 channels, we show that enhancement of the activity of these channels decreases the excitability of spinal MNs in mouse neonates. This action on MNs results from a combination of hyperpolarization of the resting membrane potential, a decrease in the input resistance, and depolarization of the voltage threshold. On the other hand, the effect of inhibition of KCNQ/Kv7 channels suggested that these channels play a limited role in regulating basal excitability. Computer simulations confirmed that pharmacological enhancement of KCNQ/Kv7 channel activity decreases excitability and also suggested that the effects of inhibition of KCNQ/Kv7 channels on the excitability of spinal MNs do not depend on a direct effect in these neurons but likely on spinal cord synaptic partners. These results indicate that KCNQ/Kv7 channels have a fundamental role in the modulation of the excitability of spinal MNs acting both in these neurons and in their local presynaptic partners. PMID:27512022

Ion channel signaling influences cellular proliferation and phagocyte activity during axolotl tail regeneration.

PubMed

Franklin, Brandon M; Voss, S Randal; Osborn, Jeffrey L

2017-08-01

Little is known about the potential for ion channels to regulate cellular behaviors during tissue regeneration. Here, we utilized an amphibian tail regeneration assay coupled with a chemical genetic screen to identify ion channel antagonists that altered critical cellular processes during regeneration. Inhibition of multiple ion channels either partially (anoctamin1/Tmem16a, anoctamin2/Tmem16b, K V 2.1, K V 2.2, L-type Ca V channels and H/K ATPases) or completely (GlyR, GABA A R, K V 1.5 and SERCA pumps) inhibited tail regeneration. Partial inhibition of tail regeneration by blocking the calcium activated chloride channels, anoctamin1&2, was associated with a reduction of cellular proliferation in tail muscle and mesenchymal regions. Inhibition of anoctamin 1/2 also altered the post-amputation transcriptional response of p44/42 MAPK signaling pathway genes, including decreased expression of erk1/erk2. We also found that complete inhibition via voltage gated K + channel blockade was associated with diminished phagocyte recruitment to the amputation site. The identification of H + pumps as required for axolotl tail regeneration supports findings in Xenopus and Planaria models, and more generally, the conservation of ion channels as regulators of tissue regeneration. This study provides a preliminary framework for an in-depth investigation of the mechanistic role of ion channels and their potential involvement in regulating cellular proliferation and other processes essential to wound healing, appendage regeneration, and tissue repair. Copyright © 2017 Elsevier B.V. All rights reserved.

Roles of Ion Channels in the Environmental Responses of Plants

NASA Astrophysics Data System (ADS)

Furuichi, Takuya; Kawano, Tomonori; Tatsumi, Hitoshi; Sokabe, Masahiro

When plant cells are exposed to environmental stresses or perceive internal signal molecules involved in growth and development, ion channels are transiently activated to convert these stimuli into intracellular signals. Among the ions taken up by plant cells, Ca2+ plays an essential role as an intracellular second messenger in plants; the cytoplasmic free Ca2+ concentration ([Ca2+]c) is therefore strictly regulated. Signal transduction pathways mediated by changes in [Ca2+]c - termed Ca2+ signaling - are initiated by the activation of Ca2+-permeable channels in many cases. To date, a large body of electrophysiological and recent molecular biological studies have revealed that plants possess Ca2+ channels belonging to distinct types with different gating mechanisms, and a variety of genes for Ca2+-permeable channels have been isolated and functionally characterized. Topics in this chapter focus on long-distance signal translocation in plants and the characteristics of a variety of plant Ca2+-permeable channels including voltage-dependent Ca2+-permeable channels, cyclic nucleotide-gated cation channels, ionotropic glutamate receptors and mechanosensitive channels. We discuss their roles in environmental responses and in the regulation of growth and development.

Non-basic amino acids in the ROMK1 channels via an appropriate distance modulate PIP2 regulated pHi-gating.

PubMed

Lee, Chien-Hsing; Huang, Po-Tsang; Liou, Horng-Huei; Lin, Mei-Ying; Lou, Kuo-Long; Chen, Chung-Yi

2016-04-22

The ROMK1 (Kir1.1) channel activity is predominantly regulated by intracellular pH (pHi) and phosphatidylinositol 4,5-bisphosphate (PIP2). Although several residues were reported to be involved in the regulation of pHi associated with PIP2 interaction, the detailed molecular mechanism remains unclear. We perform experiments in ROMK1 pHi-gating with electrophysiology combined with mutational and structural analysis. In the present study, non basic residues of C-terminal region (S219, N215, I192, L216 and L220) in ROMK1 channels have been found to mediate channel-PIP2 interaction and pHi gating. Further, our structural results show these residues with an appropriate distance to interact with membrane PIP2. Meanwhile, a cluster of basic residues (R188, R217 and K218), which was previously discovered regarding the interaction with PIP2, exists in this appropriate distance to discriminate the regulation of channel-PIP2 interaction and pHi-gating. This appropriate distance can be observed with high conservation in the Kir channel family. Our results provide insight that an appropriate distance cooperates with the electrostatics interaction of channel-PIP2 to regulate pHi-gating. Copyright © 2016 Elsevier Inc. All rights reserved.

PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels

PubMed Central

Itsuki, Kyohei; Imai, Yuko; Hase, Hideharu; Okamura, Yasushi; Inoue, Ryuji

2014-01-01

Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC). PI(4,5)P2 depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P2 reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P2 or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to Gq and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P2 reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C–insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P2. We determined the functional dissociation constant of PI(4,5)P2 to TRPC channels using FRET of the PLCδ Pleckstrin homology domain (PHd), which binds PI(4,5)P2, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P2 and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P2 regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P2 depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total current. In conclusion, our studies demonstrate a fundamental role for PI(4,5)P2 in regulating TRPC6 and TRPC7 activity triggered by PLC-coupled receptor stimulation. PMID:24470487

Anchored PDE4 regulates chloride conductance in wild-type and ΔF508-CFTR human airway epithelia

PubMed Central

Blanchard, Elise; Zlock, Lorna; Lao, Anna; Mika, Delphine; Namkung, Wan; Xie, Moses; Scheitrum, Colleen; Gruenert, Dieter C.; Verkman, Alan S.; Finkbeiner, Walter E.; Conti, Marco; Richter, Wito

2014-01-01

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that impair its expression and/or chloride channel function. Here, we provide evidence that type 4 cyclic nucleotide phosphodiesterases (PDE4s) are critical regulators of the cAMP/PKA-dependent activation of CFTR in primary human bronchial epithelial cells. In non-CF cells, PDE4 inhibition increased CFTR activity under basal conditions (ΔISC 7.1 μA/cm2) and after isoproterenol stimulation (increased ΔISC from 13.9 to 21.0 μA/cm2) and slowed the return of stimulated CFTR activity to basal levels by >3-fold. In cells homozygous for ΔF508-CFTR, the most common mutation found in CF, PDE4 inhibition alone produced minimal channel activation. However, PDE4 inhibition strongly amplified the effects of CFTR correctors, drugs that increase expression and membrane localization of CFTR, and/or CFTR potentiators, drugs that increase channel gating, to reach ∼25% of the chloride conductance observed in non-CF cells. Biochemical studies indicate that PDE4s are anchored to CFTR and mediate a local regulation of channel function. Taken together, our results implicate PDE4 as an important determinant of CFTR activity in airway epithelia, and support the use of PDE4 inhibitors to potentiate the therapeutic benefits of CFTR correctors and potentiators.—Blanchard, E., Zlock, L., Lao, A., Mika, D., Namkung, W., Xie, M., Scheitrum, C., Gruenert, D.C., Verkman, A.S., Finkbeiner, W.E., Conti, M., Richter, W. Anchored PDE4 regulates chloride conductance in wild type and ΔF508-CFTR human airway epithelia. PMID:24200884

Functional Characterization of Cnidarian HCN Channels Points to an Early Evolution of Ih.

PubMed

Baker, Emma C; Layden, Michael J; van Rossum, Damian B; Kamel, Bishoy; Medina, Monica; Simpson, Eboni; Jegla, Timothy

2015-01-01

HCN channels play a unique role in bilaterian physiology as the only hyperpolarization-gated cation channels. Their voltage-gating is regulated by cyclic nucleotides and phosphatidylinositol 4,5-bisphosphate (PIP2). Activation of HCN channels provides the depolarizing current in response to hyperpolarization that is critical for intrinsic rhythmicity in neurons and the sinoatrial node. Additionally, HCN channels regulate dendritic excitability in a wide variety of neurons. Little is known about the early functional evolution of HCN channels, but the presence of HCN sequences in basal metazoan phyla and choanoflagellates, a protozoan sister group to the metazoans, indicate that the gene family predates metazoan emergence. We functionally characterized two HCN channel orthologs from Nematostella vectensis (Cnidaria, Anthozoa) to determine which properties of HCN channels were established prior to the emergence of bilaterians. We find Nematostella HCN channels share all the major functional features of bilaterian HCNs, including reversed voltage-dependence, activation by cAMP and PIP2, and block by extracellular Cs+. Thus bilaterian-like HCN channels were already present in the common parahoxozoan ancestor of bilaterians and cnidarians, at a time when the functional diversity of voltage-gated K+ channels was rapidly expanding. NvHCN1 and NvHCN2 are expressed broadly in planulae and in both the endoderm and ectoderm of juvenile polyps.

Physical and functional interaction between integrins and hERG potassium channels.

PubMed

Arcangeli, A; Becchetti, A; Cherubini, A; Crociani, O; Defilippi, P; Guasti, L; Hofmann, G; Pillozzi, S; Olivotto, M; Wanke, E

2004-11-01

Integrins are adhesion receptors capable of transmitting intracellular signals that regulate many different cellular functions. Among integrin-mediated signals, the activation of ion channels can be included. We demonstrated that a long-lasting activation of hERG (human ether-a-go-go-related gene) potassium channels occurs in both human neuroblastoma and leukaemia cells after the activation of the beta1 integrin subunit. This activation is apparently a determining factor inducing neurite extension and osteoclastic differentiation in both the cell types. More recently, we provided evidences that beta1 integrins and hERG channels co-precipitate in both the cell types. Preliminary results suggest that a macromolecular signalling complex indeed occurs between integrins and the hERG1 protein and that hERG channel activity can modulate integrin downstream signalling.

Threading the biophysics of mammalian Slo1 channels onto structures of an invertebrate Slo1 channel

PubMed Central

2017-01-01

For those interested in the machinery of ion channel gating, the Ca2+ and voltage-activated BK K+ channel provides a compelling topic for investigation, by virtue of its dual allosteric regulation by both voltage and intracellular Ca2+ and because its large-single channel conductance facilitates detailed kinetic analysis. Over the years, biophysical analyses have illuminated details of the allosteric regulation of BK channels and revealed insights into the mechanism of BK gating, e.g., inner cavity size and accessibility and voltage sensor-pore coupling. Now the publication of two structures of an Aplysia californica BK channel—one liganded and one metal free—promises to reinvigorate functional studies and interpretation of biophysical results. The new structures confirm some of the previous functional inferences but also suggest new perspectives regarding cooperativity between Ca2+-binding sites and the relationship between voltage- and Ca2+-dependent gating. Here we consider the extent to which the two structures explain previous functional data on pore-domain properties, voltage-sensor motions, and divalent cation binding and activation of the channel. PMID:29025867

The Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels: from Biophysics to Pharmacology of a Unique Family of Ion Channels.

PubMed

Sartiani, Laura; Mannaioni, Guido; Masi, Alessio; Novella Romanelli, Maria; Cerbai, Elisabetta

2017-10-01

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are important members of the voltage-gated pore loop channels family. They show unique features: they open at hyperpolarizing potential, carry a mixed Na/K current, and are regulated by cyclic nucleotides. Four different isoforms have been cloned (HCN1-4) that can assemble to form homo- or heterotetramers, characterized by different biophysical properties. These proteins are widely distributed throughout the body and involved in different physiologic processes, the most important being the generation of spontaneous electrical activity in the heart and the regulation of synaptic transmission in the brain. Their role in heart rate, neuronal pacemaking, dendritic integration, learning and memory, and visual and pain perceptions has been extensively studied; these channels have been found also in some peripheral tissues, where their functions still need to be fully elucidated. Genetic defects and altered expression of HCN channels are linked to several pathologies, which makes these proteins attractive targets for translational research; at the moment only one drug (ivabradine), which specifically blocks the hyperpolarization-activated current, is clinically available. This review discusses current knowledge about HCN channels, starting from their biophysical properties, origin, and developmental features, to (patho)physiologic role in different tissues and pharmacological modulation, ending with their present and future relevance as drug targets. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Regulatory crosstalk by protein kinases on CFTR trafficking and activity

NASA Astrophysics Data System (ADS)

Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

2016-01-01

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function.

PubMed

Concepcion, Axel R; Vaeth, Martin; Wagner, Larry E; Eckstein, Miriam; Hecht, Lee; Yang, Jun; Crottes, David; Seidl, Maximilian; Shin, Hyosup P; Weidinger, Carl; Cameron, Scott; Turvey, Stuart E; Issekutz, Thomas; Meyts, Isabelle; Lacruz, Rodrigo S; Cuk, Mario; Yule, David I; Feske, Stefan

2016-11-01

Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release-activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel-deficient patients and mice with ectodermal tissue-specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice.

Dendritic small conductance calcium-activated potassium channels activated by action potentials suppress EPSPs and gate spike-timing dependent synaptic plasticity.

PubMed

Jones, Scott L; To, Minh-Son; Stuart, Greg J

2017-10-23

Small conductance calcium-activated potassium channels (SK channels) are present in spines and can be activated by backpropagating action potentials (APs). This suggests they may play a critical role in spike-timing dependent synaptic plasticity (STDP). Consistent with this idea, EPSPs in both cortical and hippocampal pyramidal neurons were suppressed by preceding APs in an SK-dependent manner. In cortical pyramidal neurons EPSP suppression by preceding APs depended on their precise timing as well as the distance of activated synapses from the soma, was dendritic in origin, and involved SK-dependent suppression of NMDA receptor activation. As a result SK channel activation by backpropagating APs gated STDP induction during low-frequency AP-EPSP pairing, with both LTP and LTD absent under control conditions but present after SK channel block. These findings indicate that activation of SK channels in spines by backpropagating APs plays a key role in regulating both EPSP amplitude and STDP induction.

Central Regulation of Glucose Production May Be Impaired in Type 2 Diabetes

PubMed Central

Esterson, Yonah B.; Carey, Michelle; Boucai, Laura; Goyal, Akankasha; Raghavan, Pooja; Zhang, Kehao; Mehta, Deeksha; Feng, Daorong; Wu, Licheng; Kehlenbrink, Sylvia; Koppaka, Sudha; Kishore, Preeti

2016-01-01

The challenges of achieving optimal glycemic control in type 2 diabetes highlight the need for new therapies. Inappropriately elevated endogenous glucose production (EGP) is the main source of hyperglycemia in type 2 diabetes. Because activation of central ATP-sensitive potassium (KATP) channels suppresses EGP in nondiabetic rodents and humans, this study examined whether type 2 diabetic humans and rodents retain central regulation of EGP. The KATP channel activator diazoxide was administered in a randomized, placebo-controlled crossover design to eight type 2 diabetic subjects and seven age- and BMI-matched healthy control subjects. Comprehensive measures of glucose turnover and insulin sensitivity were performed during euglycemic pancreatic clamp studies following diazoxide and placebo administration. Complementary rodent clamp studies were performed in Zucker Diabetic Fatty rats. In type 2 diabetic subjects, extrapancreatic KATP channel activation with diazoxide under fixed hormonal conditions failed to suppress EGP, whereas matched control subjects demonstrated a 27% reduction in EGP (P = 0.002) with diazoxide. Diazoxide also failed to suppress EGP in diabetic rats. These results suggest that suppression of EGP by central KATP channel activation may be lost in type 2 diabetes. Restoration of central regulation of glucose metabolism could be a promising therapeutic target to reduce hyperglycemia in type 2 diabetes. PMID:27207526

The Kv7 Channel and Cardiovascular Risk Factors.

PubMed

Fosmo, Andreas L; Skraastad, Øyvind B

2017-01-01

Potassium channels play a pivotal role in the regulation of excitability in cells such as neurons, cardiac myocytes, and vascular smooth muscle cells. The KCNQ (Kv7) family of voltage-activated K + channels hyperpolarizes the cell and stabilizes the membrane potential. Here, we outline how Kv7 channel activity may contribute to the development of the cardiovascular risk factors such as hypertension, diabetes, and obesity. Questions and hypotheses regarding previous and future research have been raised. Alterations in the Kv7 channel may contribute to the development of cardiovascular disease (CVD). Pharmacological modification of Kv7 channels may represent a possible treatment for CVD in the future.

The Kv7 Channel and Cardiovascular Risk Factors

PubMed Central

Fosmo, Andreas L.; Skraastad, Øyvind B.

2017-01-01

Potassium channels play a pivotal role in the regulation of excitability in cells such as neurons, cardiac myocytes, and vascular smooth muscle cells. The KCNQ (Kv7) family of voltage-activated K+ channels hyperpolarizes the cell and stabilizes the membrane potential. Here, we outline how Kv7 channel activity may contribute to the development of the cardiovascular risk factors such as hypertension, diabetes, and obesity. Questions and hypotheses regarding previous and future research have been raised. Alterations in the Kv7 channel may contribute to the development of cardiovascular disease (CVD). Pharmacological modification of Kv7 channels may represent a possible treatment for CVD in the future. PMID:29259974

I(A) channels encoded by Kv1.4 and Kv4.2 regulate neuronal firing in the suprachiasmatic nucleus and circadian rhythms in locomotor activity.

PubMed

Granados-Fuentes, Daniel; Norris, Aaron J; Carrasquillo, Yarimar; Nerbonne, Jeanne M; Herzog, Erik D

2012-07-18

Neurons in the suprachiasmatic nucleus (SCN) display coordinated circadian changes in electrical activity that are critical for daily rhythms in physiology, metabolism, and behavior. SCN neurons depolarize spontaneously and fire repetitively during the day and hyperpolarize, drastically reducing firing rates, at night. To explore the hypothesis that rapidly activating and inactivating A-type (I(A)) voltage-gated K(+) (Kv) channels, which are also active at subthreshold membrane potentials, are critical regulators of the excitability of SCN neurons, we examined locomotor activity and SCN firing in mice lacking Kv1.4 (Kv1.4(-/-)), Kv4.2 (Kv4.2(-/-)), or Kv4.3 (Kv4.3(-/-)), the pore-forming (α) subunits of I(A) channels. Mice lacking either Kv1.4 or Kv4.2 α subunits have markedly shorter (0.5 h) periods of locomotor activity than wild-type (WT) mice. In vitro extracellular multi-electrode recordings revealed that Kv1.4(-/-) and Kv4.2(-/-) SCN neurons display circadian rhythms in repetitive firing, but with shorter periods (0.5 h) than WT cells. In contrast, the periods of wheel-running activity in Kv4.3(-/-) mice and firing in Kv4.3(-/-) SCN neurons were indistinguishable from WT animals and neurons. Quantitative real-time PCR revealed that the transcripts encoding all three Kv channel α subunits, Kv1.4, Kv4.2, and Kv4.3, are expressed constitutively throughout the day and night in the SCN. Together, these results demonstrate that Kv1.4- and Kv4.2-encoded I(A) channels regulate the intrinsic excitability of SCN neurons during the day and night and determine the period and amplitude of circadian rhythms in SCN neuron firing and locomotor behavior.

HCN2 channels in the ventral tegmental area regulate behavioral responses to chronic stress

PubMed Central

Zhong, Peng; Vickstrom, Casey R; Liu, Xiaojie; Hu, Ying; Yu, Laikang; Yu, Han-Gang

2018-01-01

Dopamine neurons in the ventral tegmental area (VTA) are powerful regulators of depression-related behavior. Dopamine neuron activity is altered in chronic stress-based models of depression, but the underlying mechanisms remain incompletely understood. Here, we show that mice subject to chronic mild unpredictable stress (CMS) exhibit anxiety- and depressive-like behavior, which was associated with decreased VTA dopamine neuron firing in vivo and ex vivo. Dopamine neuron firing is governed by voltage-gated ion channels, in particular hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Following CMS, HCN-mediated currents were decreased in nucleus accumbens-projecting VTA dopamine neurons. Furthermore, shRNA-mediated HCN2 knockdown in the VTA was sufficient to recapitulate CMS-induced depressive- and anxiety-like behavior in stress-naïve mice, whereas VTA HCN2 overexpression largely prevented CMS-induced behavioral deficits. Together, these results reveal a critical role for HCN2 in regulating VTA dopamine neuronal activity and depressive-related behaviors. PMID:29256865

Small-Conductance Ca2+-Activated Potassium Channels Negatively Regulate Aldosterone Secretion in Human Adrenocortical Cells.

PubMed

Yang, Tingting; Zhang, Hai-Liang; Liang, Qingnan; Shi, Yingtang; Mei, Yan-Ai; Barrett, Paula Q; Hu, Changlong

2016-09-01

Aldosterone, which plays a key role in maintaining water and electrolyte balance, is produced by zona glomerulosa cells of the adrenal cortex. Autonomous overproduction of aldosterone from zona glomerulosa cells causes primary hyperaldosteronism. Recent clinical studies have highlighted the pathological role of the KCNJ5 potassium channel in primary hyperaldosteronism. Our objective was to determine whether small-conductance Ca(2+)-activated potassium (SK) channels may also regulate aldosterone secretion in human adrenocortical cells. We found that apamin, the prototypic inhibitor of SK channels, decreased membrane voltage, raised intracellular Ca(2+) and dose dependently increased aldosterone secretion from human adrenocortical H295R cells. By contrast, 1-Ethyl-2-benzimidazolinone, an agonist of SK channels, antagonized apamin's action and decreased aldosterone secretion. Commensurate with an increase in aldosterone production, apamin increased mRNA expression of steroidogenic acute regulatory protein and aldosterone synthase that control the early and late rate-limiting steps in aldosterone biosynthesis, respectively. In addition, apamin increased angiotensin II-stimulated aldosterone secretion, whereas 1-Ethyl-2-benzimidazolinone suppressed both angiotensin II- and high K(+)-stimulated production of aldosterone in H295R cells. These findings were supported by apamin-modulation of basal and angiotensin II-stimulated aldosterone secretion from acutely prepared slices of human adrenals. We conclude that SK channel activity negatively regulates aldosterone secretion in human adrenocortical cells. Genetic association studies are necessary to determine whether mutations in SK channel subtype 2 genes may also drive aldosterone excess in primary hyperaldosteronism. © 2016 American Heart Association, Inc.

The regulation of transient receptor potential canonical 4 (TRPC4) channel by phosphodiesterase 5 inhibitor via the cyclic guanosine 3'5'-monophosphate.

PubMed

Wie, Jinhong; Jeong, SeungJoo; Kwak, Misun; Myeong, Jongyun; Chae, MeeRee; Park, Jong Kwan; Lee, Sung Won; So, Insuk

2017-06-01

The transient receptor potential (TRP) protein superfamily consists of a diverse group of cation channels that bear structural similarities to the fruit fly Drosophila TRP. The TRP superfamily is distinct from other groups of ion channels in displaying a large diversity in ion selectivity, modes of activation, and physiological functions. Classical TRP (transient receptor potential canonical (TRPC)) channels are activated by stimulation of Gq-PLC-coupled receptors and modulated by phosphorylation. The cyclic guanosine monophosphate (cGMP)-PKG pathway is involved in the regulation of TRPC3 and TRPC6 channels. Phosphodiesterase (PDE) 5 inhibitor induced muscle relaxation in corporal smooth muscle cells and was used to treat erectile dysfunction by inhibiting cGMP degradation. Here, we report the functional relationship between TRPC4 and cGMP. In human embryonic kidney (HEK) 293 cells overexpressing TRPC4, cGMP selectively activated TRPC4 channels and increased cytosolic calcium level through TRPC4 channel. We investigated phosphorylation sites in TRPC4 channels and identified S688 as an important phosphorylation site for the cGMP-PKG pathway. Cyclic GMP also activated TRPC4-like current with doubly rectifying current-voltage relationship in prostate smooth muscle cell lines. Taken together, these results show that TRPC4 is phosphorylated by the cGMP-PKG pathway and might be an important target for modulating prostate function by PDE5 inhibitors.

Biophysics of BK Channel Gating.

PubMed

Pantazis, A; Olcese, R

2016-01-01

BK channels are universal regulators of cell excitability, given their exceptional unitary conductance selective for K(+), joint activation mechanism by membrane depolarization and intracellular [Ca(2+)] elevation, and broad expression pattern. In this chapter, we discuss the structural basis and operational principles of their activation, or gating, by membrane potential and calcium. We also discuss how the two activation mechanisms interact to culminate in channel opening. As members of the voltage-gated potassium channel superfamily, BK channels are discussed in the context of archetypal family members, in terms of similarities that help us understand their function, but also seminal structural and biophysical differences that confer unique functional properties. © 2016 Elsevier Inc. All rights reserved.

Taurine activates delayed rectifier KV channels via a metabotropic pathway in retinal neurons

PubMed Central

Bulley, Simon; Liu, Yufei; Ripps, Harris; Shen, Wen

2013-01-01

Taurine is one of the most abundant amino acids in the retina, throughout the CNS, and in heart and muscle cells. In keeping with its broad tissue distribution, taurine serves as a modulator of numerous basic processes, such as enzyme activity, cell development, myocardial function and cytoprotection. Despite this multitude of functional roles, the precise mechanism underlying taurine's actions has not yet been identified. In this study we report findings that indicate a novel role for taurine in the regulation of voltage-gated delayed rectifier potassium (KV) channels in retinal neurons by means of a metabotropic receptor pathway. The metabotropic taurine response was insensitive to the Cl− channel blockers, picrotoxin and strychnine, but it was inhibited by a specific serotonin 5-HT2A receptor antagonist, MDL11939. Moreover, we found that taurine enhanced KV channels via intracellular protein kinase C-mediated pathways. When 5-HT2A receptors were expressed in human embryonic kidney cells, taurine and AL34662, a non-specific 5-HT2 receptor activator, produced a similar regulation of KIR channels. In sum, this study provides new evidence that taurine activates a serotonin system, apparently via 5-HT2A receptors and related intracellular pathways. PMID:23045337

Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells

NASA Technical Reports Server (NTRS)

Duncan, R. L.; Kizer, N.; Barry, E. L.; Friedman, P. A.; Hruska, K. A.

1996-01-01

By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.

CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes

PubMed Central

Nagel, G; Barbry, P; Chabot, H; Brochiero, E; Hartung, K; Grygorczyk, R

2005-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR–ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain. PMID:15746174

A highly active ATP-insensitive K+ import pathway in plant mitochondria.

PubMed

Ruy, Fernando; Vercesi, Anibal E; Andrade, Paula B M; Bianconi, M Lucia; Chaimovich, Hernan; Kowaltowski, Alicia J

2004-04-01

We describe here a regulated and highly active K+ uptake pathway in potato (Solanum tuberosum), tomato (Lycopersicon esculentum), and maize (Zea mays) mitochondria. K+ transport was not inhibited by ATP, NADH, or thiol reagents, which regulate ATP-sensitive K+ channels previously described in plant and mammalian mitochondria. However, K+ uptake was completely prevented by quinine, a broad spectrum K+ channel inhibitor. Increased K+ uptake in plants leads to mitochondrial swelling, respiratory stimulation, heat release, and the prevention of reactive oxygen species formation. This newly described ATP-insensitive K+ import pathway is potentially involved in metabolism regulation and prevention of oxidative stress.

Managing the complexity of communication: regulation of gap junctions by post-translational modification

PubMed Central

Axelsen, Lene N.; Calloe, Kirstine; Holstein-Rathlou, Niels-Henrik; Nielsen, Morten S.

2013-01-01

Gap junctions are comprised of connexins that form cell-to-cell channels which couple neighboring cells to accommodate the exchange of information. The need for communication does, however, change over time and therefore must be tightly controlled. Although the regulation of connexin protein expression by transcription and translation is of great importance, the trafficking, channel activity and degradation are also under tight control. The function of connexins can be regulated by several post translational modifications, which affect numerous parameters; including number of channels, open probability, single channel conductance or selectivity. The most extensively investigated post translational modifications are phosphorylations, which have been documented in all mammalian connexins. Besides phosphorylations, some connexins are known to be ubiquitinated, SUMOylated, nitrosylated, hydroxylated, acetylated, methylated, and γ-carboxyglutamated. The aim of the present review is to summarize our current knowledge of post translational regulation of the connexin family of proteins. PMID:24155720

Heme Regulates Allosteric Activation of the Slo1 BK Channel

PubMed Central

Horrigan, Frank T.; Heinemann, Stefan H.; Hoshi, Toshinori

2005-01-01

Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531–535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G–V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G–V simulated by weakening the coupling of both Ca2+ binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca2+- and voltage-dependent gating as well as intrinsic stability of the open state. PMID:15955873

Exploring the biophysical evidence that mammalian two‐pore channels are NAADP‐activated calcium‐permeable channels

PubMed Central

Reilly‐O'Donnell, Benedict; Sitsapesan, Rebecca

2016-01-01

Abstract Nicotinic acid adenine dinucleotide phosphate (NAADP) potently releases Ca2+ from acidic intracellular endolysosomal Ca2+ stores. It is widely accepted that two types of two‐pore channels, termed TPC1 and TPC2, are responsible for the NAADP‐mediated Ca2+ release but the underlying mechanisms regulating their gating appear to be different. For example, although both TPC1 and TPC2 are activated by NAADP, TPC1 appears to be additionally regulated by cytosolic Ca2+. Ion conduction and permeability also differ markedly. TPC1 and TPC2 are permeable to a range of cations although biophysical experiments suggest that TPC2 is slightly more selective for Ca2+ over K+ than TPC1 and hence capable of releasing greater quantities of Ca2+ from acidic stores. TPC1 is also permeable to H+ and therefore may play a role in regulating lysosomal and cytosolic pH, possibly creating localised acidic domains. The significantly different gating and ion conducting properties of TPC1 and TPC2 suggest that these two ion channels may play complementary physiological roles as Ca2+‐release channels of the endolysosomal system. PMID:26872338

The Interaction between Logjams, Channel Evolution, and Sports Fisheries on a Dam Regulated Low Gradient River.

NASA Astrophysics Data System (ADS)

Schenk, E.; Hupp, C. R.; Moulin, B.

2014-12-01

The purpose of our study was to determine the interaction between in-stream large wood (LW), bank erosion, and sports fisheries in the 210 river kilometer (km) Coastal Plain segment of the dam-regulated Roanoke River, North Carolina. Methods included collecting background geomorphic data including a 200 km channel geometry survey and measurements from 701 bank erosion pins at 36 cross-sections over 132 km. LW concentrations were evaluated over a 177 km reach using georeferenced aerial video taken during regulated low flow (56 m3/s). LW transport was measured using 290 radio tagged LW pieces (mean diameter = 35.0 cm, length = 9.3 m) installed between 2008 and 2010. Largemouth bass (Micropterus salmoides) were surveyed in 2010 at 29 sites using a boat mounted electroshock unit. The abundance of LW in logjams was 59 pieces/km and these were concentrated (21.5 logjams/km) in an actively eroding reach with relatively high sinuosity, high local LW production rates, and narrow channel widths. Most jams (70%) are available nearly year round as aquatic habitat, positioned either on the lower bank or submerged at low-water flows. The actively eroding reach is adjusting to upstream dam regulation by channel widening. The channel upstream of this reach has widened and stabilized while the channel downstream of the eroding reach is still relatively narrow but with lower bank erosion rates. Repeat surveys of radio tagged LW determined that transport was common throughout the study area despite dam regulation and a low channel gradient (0.0016). The mean distance travelled by a radio tagged piece of LW was 11.9 km with a maximum of 101 km (84 tags moved, 96 stationary, 110 not found). Radio tagged LW that moved during the study was found at low flow either in logjams (44%), as individual LW (43%), or submerged mid-channel (14%). Largemouth bass biomass density (g/hr effort) was highest in the actively eroding reach where logjams were most common. Our results support the hypothesis that channel evolution processes control bank stability and complexity that in turn control logjam frequency. Areas with higher concentrations of logjams have larger and more largemouth bass, a valued sports fish.

Activation of TRPV1 channels inhibits mechanosensitive Piezo channel activity by depleting membrane phosphoinositides

PubMed Central

Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

2015-01-01

Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor PI(4)P from the plasma membrane through Ca2+-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 or PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin. PMID:25670203

Effects of unsaturated fatty acids on the kinetics of voltage‐gated proton channels heterologously expressed in cultured cells

PubMed Central

Kawanabe, Akira

2016-01-01

Key points Arachidonic acid (AA) greatly enhances the activity of the voltage‐gated proton (Hv) channel, although its mechanism of action and physiological function remain unclear.In the present study, we analysed the effects of AA on proton currents through Hv channels heterologously expressed in HEK293T cells.The dramatic increase in proton current amplitude elicited by AA was accompanied by accelerated activation kinetics and a leftward shift in the voltage‐dependence of activation.Mutagenesis studies suggest the two aforementioned effects of AA reflect two distinct structural mechanisms.Application of phospholipase A2, which liberates AA from phospholipids in the membrane, also enhances Hv channel activity, supporting the idea that AA modulates Hv channel activity within physiological contexts. Abstract Unsaturated fatty acids are key components of the biological membranes of all cells, and precursors of mediators for cell signalling. Arachidonic acid (AA) is an unsaturated fatty acid known to modulate the activities of various ion channels, including the voltage‐gated proton (Hv) channel, which supports the rapid production of reactive oxygen species (ROS) in phagocytes through regulation of pH and membrane potential. However, the molecular mechanisms and physiological functions of the effects of AA on Hv channels remain unclear. In the present study, we report an electrophysiological analysis of the effects of AA on the mouse Hv channel (mHv1) heterologously expressed in HEK293T cells. Application of AA to excised inside‐out patch membranes rapidly induced a robust increase in the amplitude of the proton current through mHv1. The current increase was accompanied by accelerated activation kinetics and a small leftward shift of the current–voltage relationship. In monomeric channels lacking the coiled‐coil region of the channel protein, the shift in the current–voltage relationship was diminished but activation and deactivation remained accelerated. Studies with several AA derivatives showed that double bonds and hydrophilic head groups are essential for the effect of AA, although charge was not important. The application of phospholipase A2 (PLA2), which generates AA from cell membrane phospholipids, stimulated mHv1 activity to a similar extent as direct application of ∼20 μm AA, suggesting that endogenous AA may regulate Hv channel activity. PMID:26563684

BAD and KATP channels regulate neuron excitability and epileptiform activity

PubMed Central

Fernández-Agüera, María Carmen; Nathwani, Nidhi; Lahmann, Carolina; Burnham, Veronica L

2018-01-01

Brain metabolism can profoundly influence neuronal excitability. Mice with genetic deletion or alteration of Bad (BCL-2 agonist of cell death) exhibit altered brain-cell fuel metabolism, accompanied by resistance to acutely induced epileptic seizures; this seizure protection is mediated by ATP-sensitive potassium (KATP) channels. Here we investigated the effect of BAD manipulation on KATP channel activity and excitability in acute brain slices. We found that BAD’s influence on neuronal KATP channels was cell-autonomous and directly affected dentate granule neuron (DGN) excitability. To investigate the role of neuronal KATP channels in the anticonvulsant effects of BAD, we imaged calcium during picrotoxin-induced epileptiform activity in entorhinal-hippocampal slices. BAD knockout reduced epileptiform activity, and this effect was lost upon knockout or pharmacological inhibition of KATP channels. Targeted BAD knockout in DGNs alone was sufficient for the antiseizure effect in slices, consistent with a ‘dentate gate’ function that is reinforced by increased KATP channel activity. PMID:29368690

International Union of Basic and Clinical Pharmacology. LXXVI. Current Progress in the Mammalian TRP Ion Channel Family

PubMed Central

Wu, Long-Jun; Sweet, Tara-Beth

2010-01-01

Transient receptor potential (TRP) channels are a large family of ion channel proteins, surpassed in number in mammals only by voltage-gated potassium channels. TRP channels are activated and regulated through strikingly diverse mechanisms, making them suitable candidates for cellular sensors. They respond to environmental stimuli such as temperature, pH, osmolarity, pheromones, taste, and plant compounds, and intracellular stimuli such as Ca2+ and phosphatidylinositol signal transduction pathways. However, it is still largely unknown how TRP channels are activated in vivo. Despite the uncertainties, emerging evidence using TRP channel knockout mice indicates that these channels have broad function in physiology. Here we review the recent progress on the physiology, pharmacology and pathophysiological function of mammalian TRP channels. PMID:20716668

Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels

PubMed Central

Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry

2016-01-01

Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

Sildenafil prevents the up-regulation of transient receptor potential canonical channels in the development of cardiomyocyte hypertrophy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kiso, Hironori; Ohba, Takayoshi; Iino, Kenji

2013-07-05

Highlights: •Transient receptor potential canonical (TRPC1, 3 and 6) are up-regulated by ET-1. •Sildenafil inhibited hypertrophic responses (BNP, Ca entry, NFAT activation). •Sildenafil suppressed TRPC1, 3 and 6 expression. -- Abstract: Background: Transient receptor potential canonical (TRPCs) channels are up-regulated in the development of cardiac hypertrophy. Sildenafil inhibits TRPC6 activation and expression, leading to the prevention of cardiac hypertrophy. However, the effects of sildenafil on the expression of other TRPCs remain unknown. We hypothesized that in addition to its effects of TRPC6, sildenafil blocks the up-regulation of other TRPC channels to suppress cardiomyocyte hypertrophy. Methods and results: In cultured neonatalmore » rat cardiomyocytes, a 48 h treatment with 10 nM endothelin (ET)-1 induced hypertrophic responses characterized by nuclear factor of activated T cells activation and enhancement of brain natriuretic peptide expression and cell surface area. Co-treatment with sildenafil (1 μM, 48 h) inhibited these ET-1-induced hypertrophic responses. Although ET-1 enhanced the gene expression of TRPCs, sildenafil inhibited the enhanced gene expression of TRPC1, C3 and C6. Moreover, co-treatment with sildenafil abolished the augmentation of SOCE in the hypertrophied cardiomyocytes. Conclusions: These results suggest that sildenafil inhibits cardiomyocyte hypertrophy by suppressing the up-regulation of TRPC expression.« less

CFTR and lung homeostasis

PubMed Central

Matalon, Sadis

2014-01-01

CFTR is a cAMP-activated chloride and bicarbonate channel that is critical for lung homeostasis. Decreases in CFTR expression have dire consequences in cystic fibrosis (CF) and have been suggested to be a component of the lung pathology in chronic obstructive pulmonary disease. Decreases or loss of channel function often lead to mucus stasis, chronic bacterial infections, and the accompanying chronic inflammatory responses that promote progressive lung destruction, and, eventually in CF, lung failure. Here we discuss CFTR's functional role airway surface liquid hydration and pH, in regulation of other channels such as the epithelial sodium channel, and in regulating inflammatory responses in the lung. PMID:25381027

KV7 channels in the human detrusor: channel modulator effects and gene and protein expression.

PubMed

Bientinesi, Riccardo; Mancuso, Cesare; Martire, Maria; Bassi, Pier Francesco; Sacco, Emilio; Currò, Diego

2017-02-01

Voltage-gated type 7 K + (K V 7 or KCNQ) channels regulate the contractility of various smooth muscles. With this study, we aimed to assess the role of K V 7 channels in the regulation of human detrusor contractility, as well as the gene and protein expression of K V 7 channels in this tissue. For these purposes, the isolated organ technique, RT-qPCR, and Western blot were used, respectively. XE-991, a selective K V 7 channel blocker, concentration-dependently contracted the human detrusor; mean EC 50 and E max of XE-991-induced concentration-response curve were 14.1 μM and 28.8 % of the maximal bethanechol-induced contraction, respectively. Flupirtine and retigabine, selective K V 7.2-7.5 channel activators, induced concentration-dependent relaxations of bethanechol-precontracted strips, with maximal relaxations of 51.6 and 51.8 % of the precontraction, respectively. XE-991 blocked the relaxations induced by flupirtine and retigabine. All five KCNQ genes were found to be expressed in the detrusor with KCNQ4 being the most expressed among them. Different bands, having sizes similar to some of reported K V 7.1, 7.4, and 7.5 channel subunit isoforms, were detected in the detrusor by Western blot with the K V 7.4 band being the most intense among them. In conclusion, K V 7 channels contribute to set the basal tone of the human detrusor. In addition, K V 7 channel activators significantly relax the detrusor. The K V 7.4 channels are probably the most important K V 7 channels expressed in the human detrusor. These data suggest that selective K V 7.4 channel activators might represent new pharmacological tools for inducing therapeutic relaxation of the detrusor.

Two-pore channels: Regulation by NAADP and customized roles in triggering calcium signals

PubMed Central

Patel, Sandip; Marchant, Jonathan; Brailoiu, Eugen

2010-01-01

NAADP is a potent regulator of cytosolic calcium levels. Much evidence suggests that NAADP activates a novel channel located on an acidic (lysosomal-like) calcium store, the mobilisation of which results in further calcium release from the endoplasmic reticulum. Here, we discuss the recent identification of a family of poorly characterized ion channels (the two-pore channels) as endo-lysosomal NAADP receptors. The generation of calcium signals by these channels is likened to those evoked by depolarisation during excitation-contraction coupling in muscle. We discuss the idea that two pore-channels can mediate a trigger release of calcium which is then amplified by calcium-induced calcium release from the endoplasmic reticulum. This is similar to the activation of voltage-sensitive calcium channels and subsequent mobilisation of sarcoplasmic reticulum calcium stores in cardiac tissue. We suggest that two-pore channels may physically interact with ryanodine receptors to account for more direct release of calcium from the endoplasmic reticulum in analogy with the conformational coupling of voltage-sensitive calcium channels and ryanodine receptors in skeletal muscle. Interaction of two-pore channels with other calcium release channels likely occurs between stores “trans-chatter” and possibly within the same store “cis-chatter”. We also speculate that trafficking of two-pore channels through the endolysosomal system facilitates interactions with calcium entry channels. Strategic placing of two-pore channels thus provides a versatile means of generating spatiotemporally complex cellular calcium signals. PMID:20621760

Gating of human ClC-2 chloride channels and regulation by carboxy-terminal domains

PubMed Central

Garcia-Olivares, Jennie; Alekov, Alexi; Boroumand, Mohammad Reza; Begemann, Birgit; Hidalgo, Patricia; Fahlke, Christoph

2008-01-01

Eukaryotic ClC channels are dimeric proteins with each subunit forming an individual protopore. Single protopores are gated by a fast gate, whereas the slow gate is assumed to control both protopores through a cooperative movement of the two carboxy-terminal domains. We here study the role of the carboxy-terminal domain in modulating fast and slow gating of human ClC-2 channels, a ubiquitously expressed ClC-type chloride channel involved in transepithelial solute transport and in neuronal chloride homeostasis. Partial truncation of the carboxy-terminus abolishes function of ClC-2 by locking the channel in a closed position. However, unlike other isoforms, its complete removal preserves function of ClC-2. ClC-2 channels without the carboxy-terminus exhibit fast and slow gates that activate and deactivate significantly faster than in WT channels. In contrast to the prevalent view, a single carboxy-terminus suffices for normal slow gating, whereas both domains regulate fast gating of individual protopores. Our findings demonstrate that the carboxy-terminus is not strictly required for slow gating and that the cooperative gating resides in other regions of the channel protein. ClC-2 is expressed in neurons and believed to open at negative potentials and increased internal chloride concentrations after intense synaptic activity. We propose that the function of the ClC-2 carboxy-terminus is to slow down the time course of channel activation in order to stabilize neuronal excitability PMID:18801843

Gating of human ClC-2 chloride channels and regulation by carboxy-terminal domains.

PubMed

Garcia-Olivares, Jennie; Alekov, Alexi; Boroumand, Mohammad Reza; Begemann, Birgit; Hidalgo, Patricia; Fahlke, Christoph

2008-11-15

Eukaryotic ClC channels are dimeric proteins with each subunit forming an individual protopore. Single protopores are gated by a fast gate, whereas the slow gate is assumed to control both protopores through a cooperative movement of the two carboxy-terminal domains. We here study the role of the carboxy-terminal domain in modulating fast and slow gating of human ClC-2 channels, a ubiquitously expressed ClC-type chloride channel involved in transepithelial solute transport and in neuronal chloride homeostasis. Partial truncation of the carboxy-terminus abolishes function of ClC-2 by locking the channel in a closed position. However, unlike other isoforms, its complete removal preserves function of ClC-2. ClC-2 channels without the carboxy-terminus exhibit fast and slow gates that activate and deactivate significantly faster than in WT channels. In contrast to the prevalent view, a single carboxy-terminus suffices for normal slow gating, whereas both domains regulate fast gating of individual protopores. Our findings demonstrate that the carboxy-terminus is not strictly required for slow gating and that the cooperative gating resides in other regions of the channel protein. ClC-2 is expressed in neurons and believed to open at negative potentials and increased internal chloride concentrations after intense synaptic activity. We propose that the function of the ClC-2 carboxy-terminus is to slow down the time course of channel activation in order to stabilize neuronal excitability.

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

PubMed Central

Scholl, Elizabeth Storer; Pirone, Antonella; Cox, Daniel H; Duncan, R Keith; Jacob, Michele H

2014-01-01

Small conductance Ca2+-sensitive potassium (SK2) channels are voltage-independent, Ca2+-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca2+ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca2+ and Ca2+-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca2+ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses. PMID:24394769

Myogenic tone is impaired at low arterial pressure in mice deficient in the low-voltage-activated CaV 3.1 T-type Ca(2+) channel.

PubMed

Björling, K; Morita, H; Olsen, M F; Prodan, A; Hansen, P B; Lory, P; Holstein-Rathlou, N-H; Jensen, L J

2013-04-01

Using mice deficient in the CaV 3.1 T-type Ca(2+) channel, the aim of the present study was to elucidate the molecular identity of non-L-type channels involved in vascular tone regulation in mesenteric arteries and arterioles. We used immunofluorescence microscopy to localize CaV 3.1 channels, patch clamp electrophysiology to test the effects of a putative T-type channel blocker NNC 55-0396 on whole-cell Ca(2+) currents, pressure myography and Ca(2+) imaging to test diameter and Ca(2+) responses of the applied vasoconstrictors, and Q-PCR to check mRNA expression levels of several Ca(2+) handling proteins in wild-type and CaV 3.1(-/-) mice. Our data indicated that CaV 3.1 channels are important for the maintenance of myogenic tone at low pressures (40-80 mm Hg), whereas they are not involved in high-voltage-activated Ca(2+) currents, Ca(2+) entry or vasoconstriction to high KCl in mesenteric arteries and arterioles. Furthermore, we show that NNC 55-0396 is not a specific T-type channel inhibitor, as it potently blocks L-type and non-L-type high-voltage-activated Ca(2+) currents in mouse mesenteric vascular smooth muscle cell. Our data using mice deficient in the CaV 3.1 T-type channel represent new evidence for the involvement of non-L-type channels in arteriolar tone regulation. We showed that CaV 3.1 channels are important for the myogenic tone at low arterial pressure, which is potentially relevant under resting conditions in vivo. Moreover, CaV 3.1 channels are not involved in Ca(2+) entry and vasoconstriction to large depolarization with, for example, high KCl. Finally, we caution against using NNC 55-0396 as a specific T-type channel blocker in native cells expressing high-voltage-activated Ca(2+) channels. Acta Physiologica © 2013 Scandinavian Physiological Society.

IGF-1 activates hEAG K(+) channels through an Akt-dependent signaling pathway in breast cancer cells: role in cell proliferation.

PubMed

Borowiec, Anne-Sophie; Hague, Frédéric; Harir, Noria; Guénin, Stéphanie; Guerineau, François; Gouilleux, Fabrice; Roudbaraki, Morad; Lassoued, Kaiss; Ouadid-Ahidouch, Halima

2007-09-01

Previous work from our laboratory has shown that human ether à go-go (hEAG) K(+) channels are crucial for breast cancer cell proliferation and cell cycle progression. In this study, we investigated the regulation of hEAG channels by an insulin-like growth factor-1 (IGF-1), which is known to stimulate cell proliferation. Acute applications of IGF-1 increased K(+) current-density and hyperpolarized MCF-7 cells. The effects of IGF-1 were inhibited by hEAG inhibitors. Moreover, IGF-1 increased mRNA expression of hEAG in a time-dependent manner in parallel with an enhancement of cell proliferation. The MCF-7 cell proliferation induced by IGF-1 is inhibited pharmacologically by Astemizole or Quinidine or more specifically using siRNA against hEAG channel. Either mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K) are known to mediate IGF-1 cell proliferative signals through the activation of extracellular signal-regulated kinase 1/2 (Erk 1/2) and Akt, respectively. In MCF-7 cells, IGF-1 rapidly stimulated Akt phosphorylation, whereas IGF-1 had little stimulating effect on Erk 1/2 which seems to be constitutively activated. The application of wortmannin was found to block the effects of IGF-1 on K(+) current. Moreover, the inhibition of Akt phosphorylation by the application of wortmannin or by a specific reduction of Akt kinase activity reduced the hEAG mRNA levels. Taken together, our results show, for the first time, that IGF-1 increases both the activity and the expression of hEAG channels through an Akt-dependent pathway. Since a hEAG channel is necessary for cell proliferation, its regulation by IGF-1 may thus play an important role in IGF-1 signaling to promote a mitogenic effect in breast cancer cells.

Cyclic AMP-dependent regulation of P-type calcium channels expressed in Xenopus oocytes.

PubMed

Fournier, F; Bourinet, E; Nargeot, J; Charnet, P

1993-05-01

Xenopus oocytes injected with rat cerebellum mRNA, express voltage-dependent calcium channels (VDCC). These were identified as P-type Ca2+ channels by their insensitivity to dihydropyridines and omega-conotoxin and by their blockade by Agelenopsis aperta venom (containing the funnel-web spider toxins: FTX and omega-Aga-IV-A). Coinjection of cerebellar mRNA and antisense oligonucleotide complementary to the dihydropyridine-resistant brain Ca2+ channel, named BI [Mori Y. et al. (1991) Nature 350:398-402] or rbA [Starr T. V. B. et al. (1991) Proc Natl Acad Sci USA 88:5621-5625], strongly reduced the expressed Ba2+ current suggesting that these clones encode a P-type VDCC. The macroscopic Ca2+ channel activity was increased by direct intraoocyte injection of cAMP. This increase in current amplitude was concomitant with a slowing of current inactivation, and was attributed to activation of protein kinase A, since it could be antagonized by a peptidic inhibitor of this enzyme. Positive regulation of P-type VDCC could be of importance in Purkinje neurons and motor nerve terminals where this channel is predominant.

TRPC5-eNOS Axis Negatively Regulates ATP-Induced Cardiomyocyte Hypertrophy.

PubMed

Sunggip, Caroline; Shimoda, Kakeru; Oda, Sayaka; Tanaka, Tomohiro; Nishiyama, Kazuhiro; Mangmool, Supachoke; Nishimura, Akiyuki; Numaga-Tomita, Takuro; Nishida, Motohiro

2018-01-01

Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca 2+ -dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 5'-triphosphate (ATP), is also known as most potent Ca 2+ -mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca 2+ signaling requires inositol 1,4,5-trisphosphate (IP 3 ) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca 2+ /NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP 3 -mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation.

Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function

PubMed Central

Hristov, Kiril L.; Smith, Amy C.; Parajuli, Shankar P.; Malysz, John; Rovner, Eric S.

2016-01-01

Transient receptor potential melastatin 4 (TRPM4) channels are Ca2+-activated nonselective cation channels that have been recently identified as regulators of detrusor smooth muscle (DSM) function in rodents. However, their expression and function in human DSM remain unexplored. We provide insights into the functional role of TRPM4 channels in human DSM under physiological conditions. We used a multidisciplinary experimental approach, including RT-PCR, Western blotting, immunohistochemistry and immunocytochemistry, patch-clamp electrophysiology, and functional studies of DSM contractility. DSM samples were obtained from patients without preoperative overactive bladder symptoms. RT-PCR detected mRNA transcripts for TRPM4 channels in human DSM whole tissue and freshly isolated single cells. Western blotting and immunohistochemistry with confocal microscopy revealed TRPM4 protein expression in human DSM. Immunocytochemistry further detected TRPM4 protein expression in DSM single cells. Patch-clamp experiments showed that 9-phenanthrol, a selective TRPM4 channel inhibitor, significantly decreased the transient inward cation currents and voltage step-induced whole cell currents in freshly isolated human DSM cells. In current-clamp mode, 9-phenanthrol hyperpolarized the human DSM cell membrane potential. Furthermore, 9-phenanthrol attenuated the spontaneous phasic, carbachol-induced and nerve-evoked contractions in human DSM isolated strips. Significant species-related differences in TRPM4 channel activity between human, rat, and guinea pig DSM were revealed, suggesting a more prominent physiological role for the TRPM4 channel in the regulation of DSM function in humans than in rodents. In conclusion, TRPM4 channels regulate human DSM excitability and contractility and are critical determinants of human urinary bladder function. Thus, TRPM4 channels could represent promising novel targets for the pharmacological or genetic control of overactive bladder. PMID:26791488

Impact of the F508del mutation on ovine CFTR, a Cl− channel with enhanced conductance and ATP-dependent gating

PubMed Central

Cai, Zhiwei; Palmai-Pallag, Timea; Khuituan, Pissared; Mutolo, Michael J; Boinot, Clément; Liu, Beihui; Scott-Ward, Toby S; Callebaut, Isabelle; Harris, Ann; Sheppard, David N

2015-01-01

Cross-species comparative studies are a powerful approach to understanding the epithelial Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl− channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl− currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl− channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del. Key points Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function. Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del. PMID:25763566

Ryanodine receptors regulate arterial diameter and wall [Ca2+] in cerebral arteries of rat via Ca2+-dependent K+ channels

PubMed Central

Knot, Harm J; Standen, Nicholas B; Nelson, Mark T

1998-01-01

The effects of inhibitors of ryanodine-sensitive calcium release (RyR) channels in the sarcoplasmic reticulum (SR) and Ca2+-dependent potassium (KCa) channels on the membrane potential, intracellular [Ca2+], and diameters of small pressurized (60 mmHg) cerebral arteries (100–200 μm) were studied using digital fluorescence video imaging of arterial diameter and wall [Ca2+], combined with microelectrode measurements of arterial membrane potential. Ryanodine (10 μm), an inhibitor of RyR channels, depolarized by 9 mV, increased intracellular [Ca2+] by 46 nm and constricted pressurized (to 60 mmHg) arteries with myogenic tone by 44 μm (∼22 %). Iberiotoxin (100 nm), a blocker of KCa channels, under the same conditions, depolarized the arteries by 10 mV, increased arterial wall calcium by 51 nm, and constricted by 37 μm (∼19 %). The effects of ryanodine and iberiotoxin were not additive and were blocked by inhibitors of voltage-dependent Ca2+ channels. Caffeine (10 mm), an activator of RyR channels, transiently increased arterial wall [Ca2+] by 136 ± 9 nm in control arteries and by 158 ± 12 nm in the presence of iberiotoxin. Caffeine was relatively ineffective in the presence of ryanodine, increasing [calcium] by 18 ± 5 nm. In the presence of blockers of voltage-dependent Ca2+ channels (nimodipine, diltiazem), ryanodine and inhibitors of the SR calcium ATPase (thapsigargin, cyclopiazonic acid) were without effect on arterial wall [Ca2+] and diameter. These results suggest that local Ca2+ release originating from RyR channels (Ca2+ sparks) in the SR of arterial smooth muscle regulates myogenic tone in cerebral arteries solely through activation of KCa channels, which regulate membrane potential through tonic hyperpolarization, thus limiting Ca2+ entry through L-type voltage-dependent Ca2+ channels. KCa channels therefore act as a negative feedback control element regulating arterial diameter through a reduction in global intracellular free [Ca2+]. PMID:9490841

Cholesterol up-regulates neuronal G protein-gated inwardly rectifying potassium (GIRK) channel activity in the hippocampus

PubMed Central

Bukiya, Anna N.; Noskov, Sergei; Rosenhouse-Dantsker, Avia

2017-01-01

Hypercholesterolemia is a well known risk factor for the development of neurodegenerative disease. However, the underlying mechanisms are mostly unknown. In recent years, it has become increasingly evident that cholesterol-driven effects on physiology and pathophysiology derive from its ability to alter the function of a variety of membrane proteins including ion channels. Yet, the effect of cholesterol on G protein-gated inwardly rectifying potassium (GIRK) channels expressed in the brain is unknown. GIRK channels mediate the actions of inhibitory brain neurotransmitters. As a result, loss of GIRK function can enhance neuron excitability, whereas gain of GIRK function can reduce neuronal activity. Here we show that in rats on a high-cholesterol diet, cholesterol levels in hippocampal neurons are increased. We also demonstrate that cholesterol plays a critical role in modulating neuronal GIRK currents. Specifically, cholesterol enrichment of rat hippocampal neurons resulted in enhanced channel activity. In accordance, elevated currents upon cholesterol enrichment were also observed in Xenopus oocytes expressing GIRK2 channels, the primary GIRK subunit expressed in the brain. Furthermore, using planar lipid bilayers, we show that although cholesterol did not affect the unitary conductance of GIRK2, it significantly enhanced the frequency of channel openings. Last, combining computational and functional approaches, we identified two putative cholesterol-binding sites in the transmembrane domain of GIRK2. These findings establish that cholesterol plays a critical role in modulating GIRK activity in the brain. Because up-regulation of GIRK function can reduce neuronal activity, our findings may lead to novel approaches for prevention and therapy of cholesterol-driven neurodegenerative disease. PMID:28213520

Targeting G-Protein Signaling for the Therapeutics of Prostate Tumor Bone Metastases and the Associated Chronic Bone Pain

DTIC Science & Technology

2015-09-01

results in increased activity/expression of key pain-sensing receptor channels, such as TRPV1 , such that the channels are constitutively activated...Keywords: Prostate Cancer Bone Metastasis, Bone Cancer Pain, Heterotrimeric G protein betagamma subunits, G protein coupled receptors (GPCRs), TRPV1 ...vitro, as well as mediating GPCR-regulated TRPV1 channel function in cultured mouse sensory neurons (Aim 1) Major Goal/Objective 1: Determine the

Targeting G-Protein Signaling for the Therapeutics of Prostate Tumor Bone Metastases and the Associated Chronic Bone Pain

DTIC Science & Technology

2013-07-01

results in increased activity/expression of key pain-sensing receptor channels, such as TRPV1 , such that the channels are constitutively activated...Keywords: Prostate Cancer Bone Metastasis, Bone Cancer Pain, Heterotrimeric G protein betagamma subunits, G protein coupled receptors (GPCRs), TRPV1 ...cell growth, migration and invasion in vitro, as well as mediating GPCR-regulated TRPV1 channel function in cultured mouse sensory neurons (Aim 1

Activation of the Ca2+-sensing receptors increases currents through inward rectifier K+ channels via activation of phosphatidylinositol 4-kinase.

PubMed

Liu, Chung-Hung; Chang, Hsueh-Kai; Lee, Sue-Ping; Shieh, Ru-Chi

2016-11-01

Inward rectifier K + channels are important for maintaining normal electrical function in many cell types. The proper function of these channels requires the presence of membrane phosphoinositide 4,5-bisphosphate (PIP 2 ). Stimulation of the Ca 2+ -sensing receptor CaR, a pleiotropic G protein-coupled receptor, activates both G q/11 , which decreases PIP 2 , and phosphatidylinositol 4-kinase (PI-4-K), which, conversely, increases PIP 2 . How membrane PIP 2 levels are regulated by CaR activation and whether these changes modulate inward rectifier K + are unknown. In this study, we found that activation of CaR by the allosteric agonist, NPSR568, increased inward rectifier K + current (I K1 ) in guinea pig ventricular myocytes and currents mediated by Kir2.1 channels exogenously expressed in HEK293T cells with a similar sensitivity. Moreover, using the fluorescent PIP 2 reporter tubby-R332H-cYFP to monitor PIP 2 levels, we found that CaR activation in HEK293T cells increased membrane PIP 2 concentrations. Pharmacological studies showed that both phospholipase C (PLC) and PI-4-K are activated by CaR stimulation with the latter played a dominant role in regulating membrane PIP 2 and, thus, Kir currents. These results provide the first direct evidence that CaR activation upregulates currents through inward rectifier K + channels by accelerating PIP 2 synthesis. The regulation of I K1 plays a critical role in the stability of the electrical properties of many excitable cells, including cardiac myocytes and neurons. Further, synthetic allosteric modulators that increase CaR activity have been used to treat hyperparathyroidism, and negative CaR modulators are of potential importance in the treatment of osteoporosis. Thus, our results provide further insight into the roles played by CaR in the cardiovascular system and are potentially valuable for heart disease treatment and drug safety.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiator VX-770 (Ivacaftor) Opens the Defective Channel Gate of Mutant CFTR in a Phosphorylation-dependent but ATP-independent Manner* ♦

PubMed Central

Eckford, Paul D. W.; Li, Canhui; Ramjeesingh, Mohabir; Bear, Christine E.

2012-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) acts as a channel on the apical membrane of epithelia. Disease-causing mutations in the cystic fibrosis gene can lead to CFTR protein misfolding as in the case of the F508del mutation and/or channel dysfunction. Recently, a small molecule, VX-770 (ivacaftor), has shown efficacy in restoring lung function in patients bearing the G551D mutation, and this has been linked to repair of its channel gating defect. However, these studies did not reveal the mechanism of action of VX-770 in detail. Normally, CFTR channel activity is regulated by phosphorylation, ATP binding, and hydrolysis. Hence, it has been hypothesized that VX-770 modifies one or more of these metabolic events. In this study, we examined VX-770 activity using a reconstitution system for purified CFTR protein, a system that enables control of known regulatory factors. We studied the consequences of VX-770 interaction with CFTR incorporated in planar lipid bilayers and in proteoliposomes, using a novel flux-based assay. We found that purified and phosphorylated CFTR was potentiated in the presence of Mg-ATP, suggesting that VX-770 bound directly to the CFTR protein, rather than associated kinases or phosphatases. Interestingly, we also found that VX-770 enhanced the channel activity of purified and mutant CFTR in the nominal absence of Mg-ATP. These findings suggest that VX-770 can cause CFTR channel opening through a nonconventional ATP-independent mechanism. This work sets the stage for future studies of the structural properties that mediate CFTR gating using VX-770 as a probe. PMID:22942289

Cystic fibrosis transmembrane conductance regulator (CFTR) potentiator VX-770 (ivacaftor) opens the defective channel gate of mutant CFTR in a phosphorylation-dependent but ATP-independent manner.

PubMed

Eckford, Paul D W; Li, Canhui; Ramjeesingh, Mohabir; Bear, Christine E

2012-10-26

The cystic fibrosis transmembrane conductance regulator (CFTR) acts as a channel on the apical membrane of epithelia. Disease-causing mutations in the cystic fibrosis gene can lead to CFTR protein misfolding as in the case of the F508del mutation and/or channel dysfunction. Recently, a small molecule, VX-770 (ivacaftor), has shown efficacy in restoring lung function in patients bearing the G551D mutation, and this has been linked to repair of its channel gating defect. However, these studies did not reveal the mechanism of action of VX-770 in detail. Normally, CFTR channel activity is regulated by phosphorylation, ATP binding, and hydrolysis. Hence, it has been hypothesized that VX-770 modifies one or more of these metabolic events. In this study, we examined VX-770 activity using a reconstitution system for purified CFTR protein, a system that enables control of known regulatory factors. We studied the consequences of VX-770 interaction with CFTR incorporated in planar lipid bilayers and in proteoliposomes, using a novel flux-based assay. We found that purified and phosphorylated CFTR was potentiated in the presence of Mg-ATP, suggesting that VX-770 bound directly to the CFTR protein, rather than associated kinases or phosphatases. Interestingly, we also found that VX-770 enhanced the channel activity of purified and mutant CFTR in the nominal absence of Mg-ATP. These findings suggest that VX-770 can cause CFTR channel opening through a nonconventional ATP-independent mechanism. This work sets the stage for future studies of the structural properties that mediate CFTR gating using VX-770 as a probe.

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca2+- Permeable Channels and Stomatal Closure

PubMed Central

Munemasa, Shintaro; Wang, Yong-Fei; Andreoli, Shannon; Tiriac, Hervé; Alonso, Jose M; Harper, Jeffery F; Ecker, Joseph R; Kwak, June M; Schroeder, Julian I

2006-01-01

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca2+ in guard cell ion channel regulation. However, genetic mutants in Ca2+ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca2+-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca2+ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca2+-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca2+-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca2+ oscillation experiments revealed that Ca2+-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca2+-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca2+-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling. PMID:17032064

Loss of Transient Receptor Potential Ankyrin 1 Channel Deregulates Emotion, Learning and Memory, Cognition, and Social Behavior in Mice.

PubMed

Lee, Kuan-I; Lin, Hui-Ching; Lee, Hsueh-Te; Tsai, Feng-Chuan; Lee, Tzong-Shyuan

2017-07-01

The transient receptor potential ankyrin 1 (TRPA1) channel is a non-selective cation channel that helps regulate inflammatory pain sensation and nociception and the development of inflammatory diseases. However, the potential role of the TRPA1 channel and the underlying mechanism in brain functions are not fully resolved. In this study, we demonstrated that genetic deletion of the TRPA1 channel in mice or pharmacological inhibition of its activity increased neurite outgrowth. In vivo study in mice provided evidence of the TRPA1 channel as a negative regulator in hippocampal functions; functional ablation of the TRPA1 channel in mice enhanced hippocampal functions, as evidenced by less anxiety-like behavior, and enhanced fear-related or spatial learning and memory, and novel location recognition as well as social interactions. However, the TRPA1 channel appears to be a prerequisite for motor function; functional loss of the TRPA1 channel in mice led to axonal bundle fragmentation, downregulation of myelin basic protein, and decreased mature oligodendrocyte population in the brain, for impaired motor function. The TRPA1 channel may play a crucial role in neuronal development and oligodendrocyte maturation and be a potential regulator in emotion, cognition, learning and memory, and social behavior.

Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

PubMed

Thuleau, P; Ward, J M; Ranjeva, R; Schroeder, J I

1994-07-01

Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.

ABA signaling in guard cells entails a dynamic protein-protein interaction relay from the PYL-RCAR family receptors to ion channels.

PubMed

Lee, Sung Chul; Lim, Chae Woo; Lan, Wenzhi; He, Kai; Luan, Sheng

2013-03-01

Plant hormone abscisic acid (ABA) serves as an integrator of environmental stresses such as drought to trigger stomatal closure by regulating specific ion channels in guard cells. We previously reported that SLAC1, an outward anion channel required for stomatal closure, was regulated via reversible protein phosphorylation events involving ABA signaling components, including protein phosphatase 2C members and a SnRK2-type kinase (OST1). In this study, we reconstituted the ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors, to the PP2C-SnRK2 phosphatase-kinase pairs, to the ion channel SLAC1. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure. Both yeast two-hybrid and bimolecular fluorescence complementation assays were used to verify the interactions among the components in the pathway. These biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners. The SLAC1 channel activity was used as an endpoint readout for the strength of the signaling pathway, depending on the presence of different combinations of signaling components. Further study using transgenic plants overexpressing one of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity.

Transient Receptor Potential Canonical (TRPC)/Orai1-dependent Store-operated Ca2+ Channels: NEW TARGETS OF ALDOSTERONE IN CARDIOMYOCYTES.

PubMed

Sabourin, Jessica; Bartoli, Fiona; Antigny, Fabrice; Gomez, Ana Maria; Benitah, Jean-Pierre

2016-06-17

Store-operated Ca(2+) entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca(2+) influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum- and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca(2+)]i, which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum- and glucocorticoid-regulated kinase 1 pathway. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Transient Receptor Potential Canonical (TRPC)/Orai1-dependent Store-operated Ca2+ Channels

PubMed Central

Sabourin, Jessica; Bartoli, Fiona; Antigny, Fabrice; Gomez, Ana Maria; Benitah, Jean-Pierre

2016-01-01

Store-operated Ca2+ entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca2+ influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum- and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca2+]i, which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum- and glucocorticoid-regulated kinase 1 pathway. PMID:27129253

Impacts of channel deposition on the risk of flooding in a watershed

NASA Astrophysics Data System (ADS)

Ting-Yue, Hong; Chia-Ling, Chang

2017-04-01

Taiwan is located in East Asian where is always hit by typhoons. Typhoons usually bring huge amounts of rainfall and result in the problems of channel deposition. Deposition influences the functions of channel and increases the risk of flooding. The Luliao Reservoir Watershed is the case area in this study. It is the major water source for agricultural activity and domestic use. The objective of this study is to assess the possible impacts of channel deposition on the watershed environment. This study applies the Storm Water Management Model (SWMM) to predict the hydrologic responses and evaluate the risk of flooding. The results show that the decrease of cross section induced by deposition in a channel may increase the risk of flooding and impact the safety of watershed environment. Therefore, canal desilting is important in channel regulation. The discussion and analysis can be useful references for channel regulation.

Up-Regulatory Effects of Curcumin on Large Conductance Ca2+-Activated K+ Channels

PubMed Central

Hei, Hongya; Li, Fangping; Wang, Yunman; Peng, Wen; Zhang, Xuemei

2015-01-01

Large conductance Ca2+-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms. PMID:26672753

A novel type of ATP block on a Ca(2+)-activated K(+) channel from bullfrog erythrocytes.

PubMed

Shindo, M; Imai, Y; Sohma, Y

2000-07-01

Using the patch-clamp technique, we have identified an intermediate conductance Ca(2+)-activated K(+) channel from bullfrog (Rana catesbeiana) erythrocytes and have investigated the regulation of channel activity by cytosolic ATP. The channel was highly selective for K(+) over Na(+), gave a linear I-V relationship with symmetrical 117.5 mM K(+) solutions and had a single-channel conductance of 60 pS. Channel activity was dependent on Ca(2+) concentration (K(1/2) = 600 nM) but voltage-independent. These basic characteristics are similar to those of human and frog erythrocyte Ca(2+)-activated K(+) (Gardos) channels previously reported. However, cytoplasmic application of ATP reduced channel activity with block exhibiting a novel bell-shaped concentration dependence. The channel was inhibited most by approximately 10 microM ATP (P(0) reduced to 5% of control) but less blocked by lower and higher concentrations of ATP. Moreover, the novel type of ATP block did not require Mg(2+), was independent of PKA or PKC, and was mimicked by a nonhydrolyzable ATP analog, AMP-PNP. This suggests that ATP exerts its effect by direct binding to sites on the channel or associated regulatory proteins, but not by phosphorylation of either of these components.

G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity.

PubMed

Stott, Jennifer B; Povstyan, Oleksandr V; Carr, Georgina; Barrese, Vincenzo; Greenwood, Iain A

2015-05-19

Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein βγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K(+) currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gβγ subunits (2-250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein-coupled receptors. Gallein, an inhibitor of Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein-coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone.

G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity

PubMed Central

Stott, Jennifer B.; Povstyan, Oleksandr V.; Carr, Georgina; Barrese, Vincenzo; Greenwood, Iain A.

2015-01-01

Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein βγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K+ currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gβγ subunits (2–250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein–coupled receptors. Gallein, an inhibitor of Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein–coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone. PMID:25941381

Cholinergic and ghrelinergic receptors and KCNQ channels in the medial PFC regulate the expression of palatability.

PubMed

Parent, Marc A; Amarante, Linda M; Swanson, Kyra; Laubach, Mark

2015-01-01

The medial prefrontal cortex (mPFC) is a key brain region for the control of consummatory behavior. Neuronal activity in this area is modulated when rats initiate consummatory licking and reversible inactivations eliminate reward contrast effects and reduce a measure of palatability, the duration of licking bouts. Together, these data suggest the hypothesis that rhythmic neuronal activity in the mPFC is crucial for the control of consummatory behavior. The muscarinic cholinergic system is known to regulate membrane excitability and control low-frequency rhythmic activity in the mPFC. Muscarinic receptors (mAChRs) act through KCNQ (Kv7) potassium channels, which have recently been linked to the orexigenic peptide ghrelin. To understand if drugs that act on KCNQ channels within the mPFC have effects on consummatory behavior, we made infusions of several muscarinic drugs (scopolamine, oxotremorine, physostigmine), the KCNQ channel blocker XE-991, and ghrelin into the mPFC and evaluated their effects on consummatory behavior. A consistent finding across all drugs was an effect on the duration of licking bouts when animals consume solutions with a relatively high concentration of sucrose. The muscarinic antagonist scopolamine reduced bout durations, both systemically and intra-cortically. By contrast, the muscarinic agonist oxotremorine, the cholinesterase inhibitor physostigmine, the KCNQ channel blocker XE-991, and ghrelin all increased the durations of licking bouts when infused into the mPFC. Our findings suggest that cholinergic and ghrelinergic signaling in the mPFC, acting through KCNQ channels, regulates the expression of palatability.

PI3-kinase promotes TRPV2 activity independently of channel translocation to the plasma membrane.

PubMed

Penna, Aubin; Juvin, Véronique; Chemin, Jean; Compan, Vincent; Monet, Michael; Rassendren, François-A

2006-06-01

Cellular or chemical activators for most transient receptor potential channels of the vanilloid subfamily (TRPV) have been identified in recent years. A remarkable exception to this is TRPV2, for which cellular events leading to channel activation are still a matter of debate. Diverse stimuli such as extreme heat or phosphatidylinositol-3 kinase (PI3-kinase) regulated membrane insertion have been shown to promote TRPV2 channel activity. However, some of these results have proved difficult to reproduce and may underlie different gating mechanisms depending on the cell type in which TRPV2 channels are expressed. Here, we show that expression of recombinant TRPV2 can induce cytotoxicity that is directly related to channel activity since it can be prevented by introducing a charge substitution in the pore-forming domain of the channel, or by reducing extracellular calcium. In stably transfected cells, TRPV2 expression results in an outwardly rectifying current that can be recorded at all potentials, and in an increase of resting intracellular calcium concentration that can be partly prevented by serum starvation. Using cytotoxicity as a read-out of channel activity and direct measurements of cell surface expression of TRPV2, we show that inhibition of the PI3-kinase decreases TRPV2 channel activity but does not affect the trafficking of the channel to the plasma membrane. It is concluded that PI3-kinase induces or modulates the activity of recombinant TRPV2 channels; in contrast to the previously proposed mechanism, activation of TRPV2 channels by PI3-kinase is not due to channel translocation to the plasma membrane.

Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.

PubMed

Fang, Xiao-Qian; Qiao, Haifa; Groveman, Bradley R; Feng, Shuang; Pflueger, Melissa; Xin, Wen-Kuan; Ali, Mohammad K; Lin, Shuang-Xiu; Xu, Jindong; Duclot, Florian; Kabbaj, Mohamed; Wang, Wei; Ding, Xin-Sheng; Santiago-Sim, Teresa; Jiang, Xing-Hong; Salter, Michael W; Yu, Xian-Min

2015-11-19

Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor internalization-induced changes in neuronal functions of the CNS.

Phosphatidylinositol (4,5)Bisphosphate Inhibits K+-Efflux Channel Activity in NT1 Tobacco Cultured Cells1[W][OA

PubMed Central

Ma, Xiaohong; Shor, Oded; Diminshtein, Sofia; Yu, Ling; Im, Yang Ju; Perera, Imara; Lomax, Aaron; Boss, Wendy F.; Moran, Nava

2009-01-01

In the animal world, the regulation of ion channels by phosphoinositides (PIs) has been investigated extensively, demonstrating a wide range of channels controlled by phosphatidylinositol (4,5)bisphosphate (PtdInsP2). To understand PI regulation of plant ion channels, we examined the in planta effect of PtdInsP2 on the K+-efflux channel of tobacco (Nicotiana tabacum), NtORK (outward-rectifying K channel). We applied a patch clamp in the whole-cell configuration (with fixed “cytosolic” Ca2+ concentration and pH) to protoplasts isolated from cultured tobacco cells with genetically manipulated plasma membrane levels of PtdInsP2 and cellular inositol (1,4,5)trisphosphate: “Low PIs” had depressed levels of these PIs, and “High PIs” had elevated levels relative to controls. In all of these cells, K channel activity, reflected in the net, steady-state outward K+ currents (IK), was inversely related to the plasma membrane PtdInsP2 level. Consistent with this, short-term manipulations decreasing PtdInsP2 levels in the High PIs, such as pretreatment with the phytohormone abscisic acid (25 μm) or neutralizing the bath solution from pH 5.6 to pH 7, increased IK (i.e. NtORK activity). Moreover, increasing PtdInsP2 levels in controls or in abscisic acid-treated high-PI cells, using the specific PI-phospholipase C inhibitor U73122 (2.5–4 μm), decreased NtORK activity. In all cases, IK decreases stemmed largely from decreased maximum attainable NtORK channel conductance and partly from shifted voltage dependence of channel gating to more positive potentials, making it more difficult to activate the channels. These results are consistent with NtORK inhibition by the negatively charged PtdInsP2 in the internal plasma membrane leaflet. Such effects are likely to underlie PI signaling in intact plant cells. PMID:19052153

Phosphatidylinositol (4,5)bisphosphate inhibits K+-efflux channel activity in NT1 tobacco cultured cells.

PubMed

Ma, Xiaohong; Shor, Oded; Diminshtein, Sofia; Yu, Ling; Im, Yang Ju; Perera, Imara; Lomax, Aaron; Boss, Wendy F; Moran, Nava

2009-02-01

In the animal world, the regulation of ion channels by phosphoinositides (PIs) has been investigated extensively, demonstrating a wide range of channels controlled by phosphatidylinositol (4,5)bisphosphate (PtdInsP2). To understand PI regulation of plant ion channels, we examined the in planta effect of PtdInsP2 on the K+-efflux channel of tobacco (Nicotiana tabacum), NtORK (outward-rectifying K channel). We applied a patch clamp in the whole-cell configuration (with fixed "cytosolic" Ca2+ concentration and pH) to protoplasts isolated from cultured tobacco cells with genetically manipulated plasma membrane levels of PtdInsP2 and cellular inositol (1,4,5)trisphosphate: "Low PIs" had depressed levels of these PIs, and "High PIs" had elevated levels relative to controls. In all of these cells, K channel activity, reflected in the net, steady-state outward K+ currents (IK), was inversely related to the plasma membrane PtdInsP2 level. Consistent with this, short-term manipulations decreasing PtdInsP2 levels in the High PIs, such as pretreatment with the phytohormone abscisic acid (25 microM) or neutralizing the bath solution from pH 5.6 to pH 7, increased IK (i.e. NtORK activity). Moreover, increasing PtdInsP2 levels in controls or in abscisic acid-treated high-PI cells, using the specific PI-phospholipase C inhibitor U73122 (2.5-4 microM), decreased NtORK activity. In all cases, IK decreases stemmed largely from decreased maximum attainable NtORK channel conductance and partly from shifted voltage dependence of channel gating to more positive potentials, making it more difficult to activate the channels. These results are consistent with NtORK inhibition by the negatively charged PtdInsP2 in the internal plasma membrane leaflet. Such effects are likely to underlie PI signaling in intact plant cells.

Single channel recording of a mitochondrial calcium uniporter.

PubMed

Wu, Guangyan; Li, Shunjin; Zong, Guangning; Liu, Xiaofen; Fei, Shuang; Shen, Linda; Guan, Xiangchen; Yang, Xue; Shen, Yuequan

2018-01-29

Mitochondrial calcium uniporter (MCU) is the pore-forming subunit of the entire uniporter complex and plays an important role in mitochondrial calcium uptake. However, the single channel recording of MCU remains controversial. Here, we expressed and purified different MCU proteins and then reconstituted them into planar lipid bilayers for single channel recording. We showed that MCU alone from Pyronema omphalodes (pMCU) is active with prominent single channel Ca 2+ currents. In sharp contrast, MCU alone from Homo sapiens (hMCU) is inactive. The essential MCU regulator (EMRE) activates hMCU, and therefore, the complex (hMCU-hEMRE) shows prominent single channel Ca 2+ currents. These single channel currents are sensitive to the specific MCU inhibitor Ruthenium Red. Our results clearly demonstrate that active MCU can conduct large amounts of calcium into the mitochondria. Copyright © 2018 Elsevier Inc. All rights reserved.

Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents.

PubMed

Mani, Bharath K; Robakowski, Christina; Brueggemann, Lyubov I; Cribbs, Leanne L; Tripathi, Abhishek; Majetschak, Matthias; Byron, Kenneth L

2016-03-01

Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth muscle cell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K(+) currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth muscle cells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled β-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 > Kv7.4/Kv7.5 > Kv7.4. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents

PubMed Central

Mani, Bharath K.; Robakowski, Christina; Brueggemann, Lyubov I.; Cribbs, Leanne L.; Tripathi, Abhishek; Majetschak, Matthias

2016-01-01

Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth muscle cell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K+ currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth muscle cells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled β-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4. PMID:26700561

[G-protein potentiates the activation of TNF-alpha on calcium-activated potassium channel in ECV304].

PubMed

Lin, L; Zheng, Y; Qu, J; Bao, G

2000-06-01

Observe the effect of tumor necrosis factor-alpha (TNF-alpha) on calcium-activated potassium channel in ECV304 and the possible involvement of G-protein mediation in the action of TNF-alpha. Using the cell-attached configuration of patch clamp technique. (1) the activity of high-conductance calcium-activated potassium channel (BKca) was recorded. Its conductance is (202.54 +/- 16.62) pS; (2) the activity of BKca was potentiated by 200 U/ml TNF-alpha; (3) G-protein would intensify this TNF-alpha activation. TNF-alpha acted on vascular endothelial cell ECV304 could rapidly activate the activity of BKca. Opening of BKca resulted in membrane hyper-polarization which could increase electro-chemical gradient for the resting Ca2+ influx and open leakage calcium channel, thus resting cytoplasmic free Ca2+ concentration could be elevated. G-protein may exert an important regulation in this process.

Carbachol-induced colonic mucus formation requires transport via NKCC1, K+ channels and CFTR

PubMed Central

Lindén, Sara K.; Alwan, Ala H.; Scholte, Bob J.; Hansson, Gunnar C.; Sjövall, Henrik

2016-01-01

The colonic mucosa protects itself from the luminal content by secreting mucus that keeps the bacteria at a distance from the epithelium. For this barrier to be effective, the mucus has to be constantly replenished which involves exocytosis and expansion of the secreted mucins. Mechanisms involved in regulation of mucus exocytosis and expansion are poorly understood, and the aim of this study was to investigate whether epithelial anion secretion regulates mucus formation in the colon. The muscarinic agonist carbachol was used to induce parallel secretion of anions and mucus, and by using established inhibitors of ion transport, we studied how inhibition of epithelial transport affected mucus formation in mouse colon. Anion secretion and mucin exocytosis were measured by changes in membrane current and epithelial capacitance, respectively. Mucus thickness measurements were used to determine the carbachol effect on mucus growth. The results showed that the carbachol-induced increase in membrane current was dependent on NKCC1 co-transport, basolateral K+ channels and Cftr activity. In contrast, the carbachol-induced increase in capacitance was partially dependent on NKCC1 and K+ channel activity, but did not require Cftr activity. Carbachol also induced an increase in mucus thickness that was inhibited by the NKCC1 blocker bumetanide. However, mice that lacked a functional Cftr channel did not respond to carbachol with an increase in mucus thickness, suggesting that carbachol-induced mucin expansion requires Cftr channel activity. In conclusion, these findings suggest that colonic epithelial transport regulates mucus formation by affecting both exocytosis and expansion of the mucin molecules. PMID:25139191

Carbachol-induced colonic mucus formation requires transport via NKCC1, K⁺ channels and CFTR.

PubMed

Gustafsson, Jenny K; Lindén, Sara K; Alwan, Ala H; Scholte, Bob J; Hansson, Gunnar C; Sjövall, Henrik

2015-07-01

The colonic mucosa protects itself from the luminal content by secreting mucus that keeps the bacteria at a distance from the epithelium. For this barrier to be effective, the mucus has to be constantly replenished which involves exocytosis and expansion of the secreted mucins. Mechanisms involved in regulation of mucus exocytosis and expansion are poorly understood, and the aim of this study was to investigate whether epithelial anion secretion regulates mucus formation in the colon. The muscarinic agonist carbachol was used to induce parallel secretion of anions and mucus, and by using established inhibitors of ion transport, we studied how inhibition of epithelial transport affected mucus formation in mouse colon. Anion secretion and mucin exocytosis were measured by changes in membrane current and epithelial capacitance, respectively. Mucus thickness measurements were used to determine the carbachol effect on mucus growth. The results showed that the carbachol-induced increase in membrane current was dependent on NKCC1 co-transport, basolateral K(+) channels and Cftr activity. In contrast, the carbachol-induced increase in capacitance was partially dependent on NKCC1 and K(+) channel activity, but did not require Cftr activity. Carbachol also induced an increase in mucus thickness that was inhibited by the NKCC1 blocker bumetanide. However, mice that lacked a functional Cftr channel did not respond to carbachol with an increase in mucus thickness, suggesting that carbachol-induced mucin expansion requires Cftr channel activity. In conclusion, these findings suggest that colonic epithelial transport regulates mucus formation by affecting both exocytosis and expansion of the mucin molecules.

Mechanosensitive channels are activated by stress in the actin stress fibres, and could be involved in gravity sensing in plants.

PubMed

Tatsumi, H; Furuichi, T; Nakano, M; Toyota, M; Hayakawa, K; Sokabe, M; Iida, H

2014-01-01

Mechanosensitive (MS) channels are expressed in a variety of cells. The molecular and biophysical mechanism involved in the regulation of MS channel activities is a central interest in basic biology. MS channels are thought to play crucial roles in gravity sensing in plant cells. To date, two mechanisms have been proposed for MS channel activation. One is that tension development in the lipid bilayer directly activates MS channels. The second mechanism proposes that the cytoskeleton is involved in the channel activation, because MS channel activities are modulated by pharmacological treatments that affect the cytoskeleton. We tested whether tension in the cytoskeleton activates MS channels. Mammalian endothelial cells were microinjected with phalloidin-conjugated beads, which bound to stress fibres, and a traction force to the actin cytoskeleton was applied by dragging the beads with optical tweezers. MS channels were activated when the force was applied, demonstrating that a sub-pN force to the actin filaments activates a single MS channel. Plants may use a similar molecular mechanism in gravity sensing, since the cytoplasmic Ca(2+) concentration increase induced by changes in the gravity vector was attenuated by potential MS channel inhibitors, and by actin-disrupting drugs. These results support the idea that the tension increase in actin filaments by gravity-dependent sedimentation of amyloplasts activates MS Ca(2+) -permeable channels, which can be the molecular mechanism of a Ca(2+) concentration increase through gravistimulation. We review recent progress in the study of tension sensing by actin filaments and MS channels using advanced biophysical methods, and discuss their possible roles in gravisensing. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells.

PubMed

Engbers, Jordan D T; Anderson, Dustin; Asmara, Hadhimulya; Rehak, Renata; Mehaffey, W Hamish; Hameed, Shahid; McKay, Bruce E; Kruskic, Mirna; Zamponi, Gerald W; Turner, Ray W

2012-02-14

Encoding sensory input requires the expression of postsynaptic ion channels to transform key features of afferent input to an appropriate pattern of spike output. Although Ca(2+)-activated K(+) channels are known to control spike frequency in central neurons, Ca(2+)-activated K(+) channels of intermediate conductance (KCa3.1) are believed to be restricted to peripheral neurons. We now report that cerebellar Purkinje cells express KCa3.1 channels, as evidenced through single-cell RT-PCR, immunocytochemistry, pharmacology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-activated Cav3.2 Ca(2+) channels at the nanodomain level to support a previously undescribed transient voltage- and Ca(2+)-dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca(2+) influx to trigger a KCa3.1-mediated regulation of the EPSP and subsequent after-hyperpolarization. The Cav3-KCa3.1 complex provides powerful control over temporal summation of EPSPs, effectively suppressing low frequencies of parallel fiber input. KCa3.1 channels thus contribute to a high-pass filter that allows Purkinje cells to respond preferentially to high-frequency parallel fiber bursts characteristic of sensory input.

The role of endogenous molecules in modulating pain through transient receptor potential vanilloid 1 (TRPV1)

PubMed Central

Morales-Lázaro, Sara L; Simon, Sidney A; Rosenbaum, Tamara

2013-01-01

Pain is a physiological response to a noxious stimulus that decreases the quality of life of those sufferring from it. Research aimed at finding new therapeutic targets for the treatment of several maladies, including pain, has led to the discovery of numerous molecular regulators of ion channels in primary afferent nociceptive neurons. Among these receptors is TRPV1 (transient receptor potential vanilloid 1), a member of the TRP family of ion channels. TRPV1 is a calcium-permeable channel, which is activated or modulated by diverse exogenous noxious stimuli such as high temperatures, changes in pH, and irritant and pungent compounds, and by selected molecules released during tissue damage and inflammatory processes. During the last decade the number of endogenous regulators of TRPV1's activity has increased to include lipids that can negatively regulate TRPV1, as is the case for cholesterol and PIP2 (phosphatidylinositol 4,5-biphosphate) while, in contrast, other lipids produced in response to tissue injury and ischaemic processes are known to positively regulate TRPV1. Among the latter, lysophosphatidic acid activates TRPV1 while amines such as N-acyl-ethanolamines and N-acyl-dopamines can sensitize or directly activate TRPV1. It has also been found that nucleotides such as ATP act as mediators of chemically induced nociception and pain and gases, such as hydrogen sulphide and nitric oxide, lead to TRPV1 activation. Finally, the products of lipoxygenases and omega-3 fatty acids among other molecules, such as divalent cations, have also been shown to endogenously regulate TRPV1 activity. Here we provide a comprehensive review of endogenous small molecules that regulate the function of TRPV1. Acting through mechanisms that lead to sensitization and desensitization of TRPV1, these molecules regulate pathways involved in pain and nociception. Understanding how these compounds modify TRPV1 activity will allow us to comprehend how some pathologies are associated with its deregulation. PMID:23613529

The role of endogenous molecules in modulating pain through transient receptor potential vanilloid 1 (TRPV1).

PubMed

Morales-Lázaro, Sara L; Simon, Sidney A; Rosenbaum, Tamara

2013-07-01

Pain is a physiological response to a noxious stimulus that decreases the quality of life of those sufferring from it. Research aimed at finding new therapeutic targets for the treatment of several maladies, including pain, has led to the discovery of numerous molecular regulators of ion channels in primary afferent nociceptive neurons. Among these receptors is TRPV1 (transient receptor potential vanilloid 1), a member of the TRP family of ion channels. TRPV1 is a calcium-permeable channel, which is activated or modulated by diverse exogenous noxious stimuli such as high temperatures, changes in pH, and irritant and pungent compounds, and by selected molecules released during tissue damage and inflammatory processes. During the last decade the number of endogenous regulators of TRPV1's activity has increased to include lipids that can negatively regulate TRPV1, as is the case for cholesterol and PIP2 (phosphatidylinositol 4,5-biphosphate) while, in contrast, other lipids produced in response to tissue injury and ischaemic processes are known to positively regulate TRPV1. Among the latter, lysophosphatidic acid activates TRPV1 while amines such as N-acyl-ethanolamines and N-acyl-dopamines can sensitize or directly activate TRPV1. It has also been found that nucleotides such as ATP act as mediators of chemically induced nociception and pain and gases, such as hydrogen sulphide and nitric oxide, lead to TRPV1 activation. Finally, the products of lipoxygenases and omega-3 fatty acids among other molecules, such as divalent cations, have also been shown to endogenously regulate TRPV1 activity. Here we provide a comprehensive review of endogenous small molecules that regulate the function of TRPV1. Acting through mechanisms that lead to sensitization and desensitization of TRPV1, these molecules regulate pathways involved in pain and nociception. Understanding how these compounds modify TRPV1 activity will allow us to comprehend how some pathologies are associated with its deregulation.

Regulation of the desensitization and ion selectivity of ATP-gated P2X2 channels by phosphoinositides

PubMed Central

Fujiwara, Yuichiro; Kubo, Yoshihiro

2006-01-01

Phosphoinositides (PIPns) are known to regulate the activity of some ion channels. Here we determined that ATP-gated P2X2 channels also are regulated by PIPns, and investigated the structural background and the unique features of this regulation. We initially used two-electrode voltage clamp to analyse the electrophysiological properties of P2X2 channels expressed in Xenopus oocytes, and observed that preincubation with wortmannin or LY294002, two PI3K inhibitors, accelerated channel desensitization. K365Q or K369Q mutation of the conserved, positively charged, amino acid residues in the proximal region of the cytoplasmic C-terminal domain also accelerated desensitization, whereas a K365R or K369R mutation did not. We observed that the permeability of the channel to N-methyl-d-glucamine (NMDG) transiently increased and then decreased after ATP application, and that the speed of the decrease was accelerated by K365Q or K369Q mutation or PI3K inhibition. Using GST-tagged recombinant proteins spanning the proximal C-terminal region, we then analysed their binding of the P2X2 cytoplasmic domain to anionic lipids using PIPns-coated nitrocellulose membranes. We found that the recombinant proteins that included the positively charged region bound to PIPs and PIP2s, and that this binding was eliminated by the K365Q and K369Q mutations. We also used a fluorescence assay to confirm that fusion proteins comprising the proximal C-terminal region of P2X2 with EGFP expressed in COS-7 cells closely associated with the membrane. Taken together, these results show that membrane-bound PIPns play a key role in maintaining channel activity and regulating pore dilation through electrostatic interaction with the proximal region of the P2X2 cytoplasmic C-terminal domain. PMID:16857707

Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

NASA Astrophysics Data System (ADS)

House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

2015-06-01

Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

The β1 Subunit Enhances Oxidative Regulation of Large-Conductance Calcium-activated K+ Channels

PubMed Central

Santarelli, Lindsey Ciali; Chen, Jianguo; Heinemann, Stefan H.; Hoshi, Toshinori

2004-01-01

Oxidative stress may alter the functions of many proteins including the Slo1 large conductance calcium-activated potassium channel (BKCa). Previous results demonstrated that in the virtual absence of Ca2+, the oxidant chloramine-T (Ch-T), without the involvement of cysteine oxidation, increases the open probability and slows the deactivation of BKCa channels formed by human Slo1 (hSlo1) α subunits alone. Because native BKCa channel complexes may include the auxiliary subunit β1, we investigated whether β1 influences the oxidative regulation of hSlo1. Oxidation by Ch-T with β1 present shifted the half-activation voltage much further in the hyperpolarizing direction (−75 mV) as compared with that with α alone (−30 mV). This shift was eliminated in the presence of high [Ca2+]i, but the increase in open probability in the virtual absence of Ca2+ remained significant at physiologically relevant voltages. Furthermore, the slowing of channel deactivation after oxidation was even more dramatic in the presence of β1. Oxidation of cysteine and methionine residues within β1 was not involved in these potentiated effects because expression of mutant β1 subunits lacking cysteine or methionine residues produced results similar to those with wild-type β1. Unlike the results with α alone, oxidation by Ch-T caused a significant acceleration of channel activation only when β1 was present. The β1 M177 mutation disrupted normal channel activation and prevented the Ch-T–induced acceleration of activation. Overall, the functional effects of oxidation of the hSlo1 pore-forming α subunit are greatly amplified by the presence of β1, which leads to the additional increase in channel open probability and the slowing of deactivation. Furthermore, M177 within β1 is a critical structural determinant of channel activation and oxidative sensitivity. Together, the oxidized BKCa channel complex with β1 has a considerable chance of being open within the physiological voltage range even at low [Ca2+]i. PMID:15452197

Calcium channels in chicken sperm regulate motility and the acrosome reaction.

PubMed

Nguyen, Thi Mong Diep; Duittoz, Anne; Praud, Christophe; Combarnous, Yves; Blesbois, Elisabeth

2016-05-01

Intracellular cytoplasmic calcium ([Ca(2+) ]i ) has an important regulatory role in gamete functions. However, the biochemical components involved in Ca(2+) transport are still unknown in birds, an animal class that has lost functional sperm-specific CatSper channels. Here, we provide evidence for the presence and expression of various Ca(2+) channels in chicken sperm, including high voltage-activated channels (L and R types), the store-operated Ca(2+) channel (SOC) component Orai1, the transient receptor potential channel (TRPC1) and inositol-1,4,5-trisphosphate receptors (IP3 R1). L- and R-type channels were mainly localized in the acrosome and the midpiece, and T-type channels were not detected in chicken sperm. Orai1 was found in all compartments, but with a weak, diffuse signal in the flagellum. TRCP1 was mainly localized in the acrosome and the midpiece, but a weak diffuse signal was also observed in the nucleus and the flagellum. IP3 R1 was mainly detected in the nucleus. The L-type channel inhibitor nifedipine, the R-type channel inhibitor SNX-482 and the SOC inhibitors MRS-1845, 2-APB and YM-58483 decreased [Ca(2+) ]i sperm motility and acrosome reaction capability, with the SOC inhibitors inhibiting these functions most efficiently. Furthermore, we showed that Ca(2+) -mediated induction of AMP-activated protein kinase (AMPK) phosphorylation was blocked by SOC inhibition. Our identification of important regulators of Ca(2+) signaling in avian sperm suggests that SOCs play a predominant role in gamete function, whereas T-type channels may not be involved. In addition, Ca(2+) entry via SOCs appears to be the most likely pathway for AMPK activation and energy-requiring sperm functions such as motility and the acrosome reaction. © 2016 Federation of European Biochemical Societies.

Volume-activated trimethylamine oxide efflux in red blood cells of spiny dogfish (Squalus acanthias).

PubMed

Koomoa, D L; Musch, M W; MacLean, A V; Goldstein, L

2001-09-01

The aims of this study were to determine the pathway of swelling-activated trimethylamine oxide (TMAO) efflux and its regulation in spiny dogfish (Squalus acanthias) red blood cells and compare the characteristics of this efflux pathway with the volume-activated osmolyte (taurine) channel present in erythrocytes of fishes. The characteristics of the TMAO efflux pathway were similar to those of the taurine efflux pathway. The swelling-activated effluxes of both TMAO and taurine were significantly inhibited by known anion transport inhibitors (DIDS and niflumic acid) and by the general channel inhibitor quinine. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea activated both TMAO and taurine effluxes similarly. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea also stimulated the activity of tyrosine kinases p72syk and p56lyn, although the stimulations by the latter two treatments were less than by hypotonicity. The volume activations of both TMAO and taurine effluxes were inhibited by tyrosine kinase inhibitors, suggesting that activation of tyrosine kinases may play a role in activating the osmolyte effluxes. These results indicate that the volume-activated TMAO efflux occurs via the organic osmolyte (taurine) channel and may be regulated by the volume activation of tyrosine kinases.

Active Dendrites and Differential Distribution of Calcium Channels Enable Functional Compartmentalization of Golgi Cells.

PubMed

Rudolph, Stephanie; Hull, Court; Regehr, Wade G

2015-11-25

Interneurons are essential to controlling excitability, timing, and synaptic integration in neuronal networks. Golgi cells (GoCs) serve these roles at the input layer of the cerebellar cortex by releasing GABA to inhibit granule cells (grcs). GoCs are excited by mossy fibers (MFs) and grcs and provide feedforward and feedback inhibition to grcs. Here we investigate two important aspects of GoC physiology: the properties of GoC dendrites and the role of calcium signaling in regulating GoC spontaneous activity. Although GoC dendrites are extensive, previous studies concluded they are devoid of voltage-gated ion channels. Hence, the current view holds that somatic voltage signals decay passively within GoC dendrites, and grc synapses onto distal dendrites are not amplified and are therefore ineffective at firing GoCs because of strong passive attenuation. Using whole-cell recording and calcium imaging in rat slices, we find that dendritic voltage-gated sodium channels allow somatic action potentials to activate voltage-gated calcium channels (VGCCs) along the entire dendritic length, with R-type and T-type VGCCs preferentially located distally. We show that R- and T-type VGCCs located in the dendrites can boost distal synaptic inputs and promote burst firing. Active dendrites are thus critical to the regulation of GoC activity, and consequently, to the processing of input to the cerebellar cortex. In contrast, we find that N-type channels are preferentially located near the soma, and control the frequency and pattern of spontaneous firing through their close association with calcium-activated potassium (KCa) channels. Thus, VGCC types are differentially distributed and serve specialized functions within GoCs. Interneurons are essential to neural processing because they modulate excitability, timing, and synaptic integration within circuits. At the input layer of the cerebellar cortex, a single type of interneuron, the Golgi cell (GoC), carries these functions. The extent of inhibition depends on both spontaneous activity of GoCs and the excitatory synaptic input they receive. In this study, we find that different types of calcium channels are differentially distributed, with dendritic calcium channels being activated by somatic activity, boosting synaptic inputs and enabling bursting, and somatic calcium cannels promoting regular firing. We therefore challenge the current view that GoC dendrites are passive and identify the mechanisms that contribute to GoCs regulating the flow of sensory information in the cerebellar cortex. Copyright © 2015 the authors 0270-6474/15/3515492-13$15.00/0.

Light-induced modification of plant plasma membrane ion transport.

PubMed

Marten, I; Deeken, R; Hedrich, R; Roelfsema, M R G

2010-09-01

Light is not only the driving force for electron and ion transport in the thylakoid membrane, but also regulates ion transport in various other membranes of plant cells. Light-dependent changes in ion transport at the plasma membrane and associated membrane potential changes have been studied intensively over the last century. These studies, with various species and cell types, revealed that apart from regulation by chloroplasts, plasma membrane transport can be controlled by phytochromes, phototropins or channel rhodopsins. In this review, we compare light-dependent plasma membrane responses of unicellular algae (Eremosphaera and Chlamydomonas), with those of a multicellular alga (Chara), liverworts (Conocephalum), mosses (Physcomitrella) and several angiosperm cell types. Light-dependent plasma membrane responses of Eremosphaera and Chara are characterised by the dominant role of K(+) channels during membrane potential changes. In most other species, the Ca(2+)-dependent activation of plasma membrane anion channels represents a general light-triggered event. Cell type-specific responses are likely to have evolved by modification of this general response or through the development of additional light-dependent signalling pathways. Future research to elucidate these light-activated signalling chains is likely to benefit from the recent identification of S-type anion channel genes and proteins capable of regulating these channels.

Transient Receptor Potential Channels in the Vasculature

PubMed Central

Earley, Scott; Brayden, Joseph E.

2015-01-01

The mammalian genome encodes 28 distinct members of the transient receptor potential (TRP) superfamily of cation channels, which exhibit varying degrees of selectivity for different ionic species. Multiple TRP channels are present in all cells and are involved in diverse aspects of cellular function, including sensory perception and signal transduction. Notably, TRP channels are involved in regulating vascular function and pathophysiology, the focus of this review. TRP channels in vascular smooth muscle cells participate in regulating contractility and proliferation, whereas endothelial TRP channel activity is an important contributor to endothelium-dependent vasodilation, vascular wall permeability, and angiogenesis. TRP channels are also present in perivascular sensory neurons and astrocytic endfeet proximal to cerebral arterioles, where they participate in the regulation of vascular tone. Almost all of these functions are mediated by changes in global intracellular Ca2+ levels or subcellular Ca2+ signaling events. In addition to directly mediating Ca2+ entry, TRP channels influence intracellular Ca2+ dynamics through membrane depolarization associated with the influx of cations or through receptor- or store-operated mechanisms. Dysregulation of TRP channels is associated with vascular-related pathologies, including hypertension, neointimal injury, ischemia-reperfusion injury, pulmonary edema, and neurogenic inflammation. In this review, we briefly consider general aspects of TRP channel biology and provide an in-depth discussion of the functions of TRP channels in vascular smooth muscle cells, endothelial cells, and perivascular cells under normal and pathophysiological conditions. PMID:25834234

BAD and KATP channels regulate neuron excitability and epileptiform activity.

PubMed

Martínez-François, Juan Ramón; Fernández-Agüera, María Carmen; Nathwani, Nidhi; Lahmann, Carolina; Burnham, Veronica L; Danial, Nika N; Yellen, Gary

2018-01-25

Brain metabolism can profoundly influence neuronal excitability. Mice with genetic deletion or alteration of Bad ( B CL-2 a gonist of cell d eath) exhibit altered brain-cell fuel metabolism, accompanied by resistance to acutely induced epileptic seizures; this seizure protection is mediated by ATP-sensitive potassium (K ATP ) channels. Here we investigated the effect of BAD manipulation on K ATP channel activity and excitability in acute brain slices. We found that BAD's influence on neuronal K ATP channels was cell-autonomous and directly affected dentate granule neuron (DGN) excitability. To investigate the role of neuronal K ATP channels in the anticonvulsant effects of BAD, we imaged calcium during picrotoxin-induced epileptiform activity in entorhinal-hippocampal slices. BAD knockout reduced epileptiform activity, and this effect was lost upon knockout or pharmacological inhibition of K ATP channels. Targeted BAD knockout in DGNs alone was sufficient for the antiseizure effect in slices, consistent with a 'dentate gate' function that is reinforced by increased K ATP channel activity. © 2018, Martínez-François et al.

Structural basis of dual Ca2+/pH regulation of the endolysosomal TRPML1 channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Minghui; Zhang, Wei K.; Benvin, Nicole M.

The activities of organellar ion channels are often regulated by Ca2+ and H+, which are present in high concentrations in many organelles. Here we report a structural element critical for dual Ca2+/pH regulation of TRPML1, a Ca2+-release channel crucial for endolysosomal function. TRPML1 mutations cause mucolipidosis type IV (MLIV), a severe lysosomal storage disorder characterized by neurodegeneration, mental retardation and blindness. We obtained crystal structures of the 213-residue luminal domain of human TRPML1 containing three missense MLIV-causing mutations. This domain forms a tetramer with a highly electronegative central pore formed by a novel luminal pore loop. Cysteine cross-linking and cryo-EMmore » analyses confirmed that this architecture occurs in the full-length channel. Structure–function studies demonstrated that Ca2+ and H+ interact with the luminal pore and exert physiologically important regulation. The MLIV-causing mutations disrupt the luminal-domain structure and cause TRPML1 mislocalization. Our study reveals the structural underpinnings of TRPML1's regulation, assembly and pathogenesis.« less

Voltage-Gated K+ Channel, Kv3.3 Is Involved in Hemin-Induced K562 Differentiation

PubMed Central

Song, Min Seok; Choi, Seon Young; Ryu, Pan Dong; Lee, So Yeong

2016-01-01

Voltage-gated K+ (Kv) channels are well known to be involved in cell proliferation. However, even though cell proliferation is closely related to cell differentiation, the relationship between Kv channels and cell differentiation remains poorly investigated. This study demonstrates that Kv3.3 is involved in K562 cell erythroid differentiation. Down-regulation of Kv3.3 using siRNA-Kv3.3 increased hemin-induced K562 erythroid differentiation through decreased activation of signal molecules such as p38, cAMP response element-binding protein, and c-fos. Down-regulation of Kv3.3 also enhanced cell adhesion by increasing integrin β3 and this effect was amplified when the cells were cultured with fibronectin. The Kv channels, or at least Kv3.3, appear to be associated with cell differentiation; therefore, understanding the mechanisms of Kv channel regulation of cell differentiation would provide important information regarding vital cellular processes. PMID:26849432

Control of the neurovascular coupling by nitric oxide-dependent regulation of astrocytic Ca2+ signaling

PubMed Central

Muñoz, Manuel F.; Puebla, Mariela; Figueroa, Xavier F.

2015-01-01

Neuronal activity must be tightly coordinated with blood flow to keep proper brain function, which is achieved by a mechanism known as neurovascular coupling. Then, an increase in synaptic activity leads to a dilation of local parenchymal arterioles that matches the enhanced metabolic demand. Neurovascular coupling is orchestrated by astrocytes. These glial cells are located between neurons and the microvasculature, with the astrocytic endfeet ensheathing the vessels, which allows fine intercellular communication. The neurotransmitters released during neuronal activity reach astrocytic receptors and trigger a Ca2+ signaling that propagates to the endfeet, activating the release of vasoactive factors and arteriolar dilation. The astrocyte Ca2+ signaling is coordinated by gap junction channels and hemichannels formed by connexins (Cx43 and Cx30) and channels formed by pannexins (Panx-1). The neuronal activity-initiated Ca2+ waves are propagated among neighboring astrocytes directly via gap junctions or through ATP release via connexin hemichannels or pannexin channels. In addition, Ca2+ entry via connexin hemichannels or pannexin channels may participate in the regulation of the astrocyte signaling-mediated neurovascular coupling. Interestingly, nitric oxide (NO) can activate connexin hemichannel by S-nitrosylation and the Ca2+-dependent NO-synthesizing enzymes endothelial NO synthase (eNOS) and neuronal NOS (nNOS) are expressed in astrocytes. Therefore, the astrocytic Ca2+ signaling triggered in neurovascular coupling may activate NO production, which, in turn, may lead to Ca2+ influx through hemichannel activation. Furthermore, NO release from the hemichannels located at astrocytic endfeet may contribute to the vasodilation of parenchymal arterioles. In this review, we discuss the mechanisms involved in the regulation of the astrocytic Ca2+ signaling that mediates neurovascular coupling, with a special emphasis in the possible participation of NO in this process. PMID:25805969

SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake.

PubMed

Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M

2017-05-01

Mitochondrial calcium ([Ca 2+ ] m ) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca 2+ ] m uptake upon SK channel activation as detected by time lapse mitochondrial Ca 2+ measurements with the Ca 2+ -binding mitochondria-targeted aequorin and FRET-based [Ca 2+ ] m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca 2+ ] m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death.

SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake

PubMed Central

Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M

2017-01-01

Mitochondrial calcium ([Ca2+]m) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca2+]m uptake upon SK channel activation as detected by time lapse mitochondrial Ca2+ measurements with the Ca2+-binding mitochondria-targeted aequorin and FRET-based [Ca2+]m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca2+]m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death. PMID:28282037

SLO-2 potassium channel is an important regulator of neurotransmitter release in Caenorhabditis elegans

PubMed Central

Liu, Ping; Chen, Bojun; Wang, Zhao-Wen

2014-01-01

Slo2 channels are prominent K+ channels in mammalian neurons but their physiological functions are not well understood. Here we investigate physiological functions and regulation of the C. elegans homologue SLO-2 in motor neurons through electrophysiological analyses of wild-type and mutant worms. We find that SLO-2 is the primary K+ channel conducting delayed outward current in cholinergic motor neurons, and one of two K+ channels with this function in GABAergic motor neurons. Loss-of-function mutation of slo-2 increases the duration and charge transfer rate of spontaneous postsynaptic current bursts at the neuromuscular junction, which are physiological signals used by motor neurons to control muscle cells, without altering postsynaptic receptor sensitivity. SLO-2 activity in motor neurons depends on Ca2+ entry through EGL-19, an L-type voltage-gated Ca2+ channel (CaV1), but not on other proteins implicated in either Ca2+ entry or intracellular Ca2+ release. Thus, SLO-2 is functionally coupled with CaV1 and regulates neurotransmitter release. PMID:25300429

The Transcription Factors Islet and Lim3 Combinatorially Regulate Ion Channel Gene Expression

PubMed Central

Wolfram, Verena; Southall, Tony D.; Günay, Cengiz; Prinz, Astrid A.; Brand, Andrea H.

2014-01-01

Expression of appropriate ion channels is essential to allow developing neurons to form functional networks. Our previous studies have identified LIM-homeodomain (HD) transcription factors (TFs), expressed by developing neurons, that are specifically able to regulate ion channel gene expression. In this study, we use the technique of DNA adenine methyltransferase identification (DamID) to identify putative gene targets of four such TFs that are differentially expressed in Drosophila motoneurons. Analysis of targets for Islet (Isl), Lim3, Hb9, and Even-skipped (Eve) identifies both ion channel genes and genes predicted to regulate aspects of dendritic and axonal morphology. Significantly, some ion channel genes are bound by more than one TF, consistent with the possibility of combinatorial regulation. One such gene is Shaker (Sh), which encodes a voltage-dependent fast K+ channel (Kv1.1). DamID reveals that Sh is bound by both Isl and Lim3. We used body wall muscle as a test tissue because in conditions of low Ca2+, the fast K+ current is carried solely by Sh channels (unlike neurons in which a second fast K+ current, Shal, also contributes). Ectopic expression of isl, but not Lim3, is sufficient to reduce both Sh transcript and Sh current level. By contrast, coexpression of both TFs is additive, resulting in a significantly greater reduction in both Sh transcript and current compared with isl expression alone. These observations provide evidence for combinatorial activity of Isl and Lim3 in regulating ion channel gene expression. PMID:24523544

The N-terminal domain of Slack determines the formation and trafficking of Slick/Slack heteromeric sodium-activated potassium channels.

PubMed

Chen, Haijun; Kronengold, Jack; Yan, Yangyang; Gazula, Valeswara-Rao; Brown, Maile R; Ma, Liqun; Ferreira, Gonzalo; Yang, Youshan; Bhattacharjee, Arin; Sigworth, Fred J; Salkoff, Larry; Kaczmarek, Leonard K

2009-04-29

Potassium channels activated by intracellular Na(+) ions (K(Na)) play several distinct roles in regulating the firing patterns of neurons, and, at the single channel level, their properties are quite diverse. Two known genes, Slick and Slack, encode K(Na) channels. We have now found that Slick and Slack subunits coassemble to form heteromeric channels that differ from the homomers in their unitary conductance, kinetic behavior, subcellular localization, and response to activation of protein kinase C. Heteromer formation requires the N-terminal domain of Slack-B, one of the alternative splice variants of the Slack channel. This cytoplasmic N-terminal domain of Slack-B also facilitates the localization of heteromeric K(Na) channels to the plasma membrane. Immunocytochemical studies indicate that Slick and Slack-B subunits are coexpressed in many central neurons. Our findings provide a molecular explanation for some of the diversity in reported properties of neuronal K(Na) channels.

Caenorhabditis elegans TRPV Channels Function in a Modality-Specific Pathway to Regulate Response to Aberrant Sensory Signaling

PubMed Central

Ezak , Meredith J.; Hong , Elizabeth; Chaparro-Garcia , Angela; Ferkey , Denise M.

2010-01-01

Olfaction and some forms of taste (including bitter) are mediated by G protein-coupled signal transduction pathways. Olfactory and gustatory ligands bind to chemosensory G protein-coupled receptors (GPCRs) in specialized sensory cells to activate intracellular signal transduction cascades. G protein-coupled receptor kinases (GRKs) are negative regulators of signaling that specifically phosphorylate activated GPCRs to terminate signaling. Although loss of GRK function usually results in enhanced cellular signaling, Caenorhabditis elegans lacking GRK-2 function are not hypersensitive to chemosensory stimuli. Instead, grk-2 mutant animals do not chemotax toward attractive olfactory stimuli or avoid aversive tastes and smells. We show here that loss-of-function mutations in the transient receptor potential vanilloid (TRPV) channels OSM-9 and OCR-2 selectively restore grk-2 behavioral avoidance of bitter tastants, revealing modality-specific mechanisms for TRPV channel function in the regulation of C. elegans chemosensation. Additionally, a single amino acid point mutation in OCR-2 that disrupts TRPV channel-mediated gene expression, but does not decrease channel function in chemosensory primary signal transduction, also restores grk-2 bitter taste avoidance. Thus, loss of GRK-2 function may lead to changes in gene expression, via OSM-9/OCR-2, to selectively alter the levels of signaling components that transduce or regulate bitter taste responses. Our results suggest a novel mechanism and multiple modality-specific pathways that sensory cells employ in response to aberrant signal transduction. PMID:20176974

C-Terminal β9-Strand of the Cyclic Nucleotide-Binding Homology Domain Stabilizes Activated States of Kv11.1 Channels

PubMed Central

Ng, Chai Ann; Ke, Ying; Perry, Matthew D.; Tan, Peter S.; Hill, Adam P.; Vandenberg, Jamie I.

2013-01-01

Kv11.1 potassium channels are important for regulation of the normal rhythm of the heartbeat. Reduced activity of Kv11.1 channels causes long QT syndrome type 2, a disorder that increases the risk of cardiac arrhythmias and sudden cardiac arrest. Kv11.1 channels are members of the KCNH subfamily of voltage-gated K+ channels. However, they also share many similarities with the cyclic nucleotide gated ion channel family, including having a cyclic nucleotide-binding homology (cNBH) domain. Kv11.1 channels, however, are not directly regulated by cyclic nucleotides. Recently, crystal structures of the cNBH domain from mEAG and zELK channels, both members of the KCNH family of voltage-gated potassium channels, revealed that a C-terminal β9-strand in the cNBH domain occupied the putative cyclic nucleotide-binding site thereby precluding binding of cyclic nucleotides. Here we show that mutations to residues in the β9-strand affect the stability of the open state relative to the closed state of Kv11.1 channels. We also show that disrupting the structure of the β9-strand reduces the stability of the inactivated state relative to the open state. Clinical mutations located in this β9-strand result in reduced trafficking efficiency, which suggests that binding of the C-terminal β9-strand to the putative cyclic nucleotide-binding pocket is also important for assembly and trafficking of Kv11.1 channels. PMID:24204727

G-protein mediated gating of inward-rectifier K+ channels.

PubMed

Mark, M D; Herlitze, S

2000-10-01

G-protein regulated inward-rectifier potassium channels (GIRK) are part of a superfamily of inward-rectifier K+ channels which includes seven family members. To date four GIRK subunits, designated GIRK1-4 (also designated Kir3.1-4), have been identified in mammals, and GIRK5 has been found in Xenopus oocytes. GIRK channels exist in vivo both as homotetramers and heterotetramers. In contrast to the other mammalian GIRK family members, GIRK1 can not form functional channels by itself and has to assemble with GIRK2, 3 or 4. As the name implies, GIRK channels are modulated by G-proteins; they are also modulated by phosphatidylinositol 4,5-bisphosphate, intracellular sodium, ethanol and mechanical stretch. Recently a family of GTPase activating proteins known as regulators of G-protein signaling were shown to be the missing link for the fast deactivation kinetics of GIRK channels in native cells, which contrast with the slow kinetics observed in heterologously expressed channels. GIRK1, 2 and 3 are highly abundant in brain, while GIRK4 has limited distribution. Here, GIRK1/2 seems to be the predominant heterotetramer. In general, neuronal GIRK channels are involved in the regulation of the excitability of neurons and may contribute to the resting potential. Interestingly, only the GIRK1 and 4 subunits are distributed in the atrial and sinoatrial node cells of the heart and are involved in the regulation of cardiac rate. Our main objective of this review is to assess the current understanding of the G-protein modulation of GIRK channels and their physiological importance in mammals.

KCa2 and KCa3 Channels in Learning and Memory Processes, and Neurodegeneration

PubMed Central

Kuiper, Els F. E.; Nelemans, Ad; Luiten, Paul; Nijholt, Ingrid; Dolga, Amalia; Eisel, Uli

2012-01-01

Calcium-activated potassium (KCa) channels are present throughout the central nervous system as well as many peripheral tissues. Activation of KCa channels contribute to maintenance of the neuronal membrane potential and was shown to underlie the afterhyperpolarization (AHP) that regulates action potential firing and limits the firing frequency of repetitive action potentials. Different subtypes of KCa channels were anticipated on the basis of their physiological and pharmacological profiles, and cloning revealed two well defined but phylogenetic distantly related groups of channels. The group subject of this review includes both the small conductance KCa2 channels (KCa2.1, KCa2.2, and KCa2.3) and the intermediate-conductance (KCa3.1) channel. These channels are activated by submicromolar intracellular Ca2+ concentrations and are voltage independent. Of all KCa channels only the KCa2 channels can be potently but differentially blocked by the bee-venom apamin. In the past few years modulation of KCa channel activation revealed new roles for KCa2 channels in controlling dendritic excitability, synaptic functioning, and synaptic plasticity. Furthermore, KCa2 channels appeared to be involved in neurodegeneration, and learning and memory processes. In this review, we focus on the role of KCa2 and KCa3 channels in these latter mechanisms with emphasis on learning and memory, Alzheimer’s disease and on the interplay between neuroinflammation and different neurotransmitters/neuromodulators, their signaling components and KCa channel activation. PMID:22701424

TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

PubMed Central

Lemonnier, Loic; Shapovalov, George; Gordienko, Dmitri; Poux, Céline; Bernardini, Michela; Bokhobza, Alexandre; Bidaux, Gabriel; Degerny, Cindy; Verreman, Kathye; Guarmit, Basma; Benahmed, Mohamed; de Launoit, Yvan; Bindels, Rene J.M.; Pla, Alessandra Fiorio; Prevarskaya, Natalia

2015-01-01

TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor–activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named “TRP channel–associated factors” (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity. PMID:25559186

TRPV2 enhances axon outgrowth through its activation by membrane stretch in developing sensory and motor neurons.

PubMed

Shibasaki, Koji; Murayama, Namie; Ono, Katsuhiko; Ishizaki, Yasuki; Tominaga, Makoto

2010-03-31

Thermosensitive TRP (thermo TRP) channels are well recognized for their contributions to sensory transduction, responding to a wide variety of stimuli including temperature, nociceptive stimuli, touch, and osmolarity. However, the precise roles for the thermo TRP channels during development have not been determined. To explore the functional importance of thermo TRP channels during neural development, the temporal expression was determined in embryonic mice. Interestingly, TRPV2 expression was detected in spinal motor neurons in addition to the dorsal root ganglia from embryonic day 10.5 and was localized in axon shafts and growth cones, suggesting that the channel is important for axon outgrowth regulation. We revealed that endogenous TRPV2 was activated in a membrane stretch-dependent manner in developing neurons by knocking down the TRPV2 function with dominant-negative TRPV2 and TRPV2-specific shRNA and significantly promoted axon outgrowth. Thus, for the first time we revealed that TRPV2 is an important regulator for axon outgrowth through its activation by membrane stretch during development.

GIRK Channels Modulate Opioid-Induced Motor Activity in a Cell Type- and Subunit-Dependent Manner

PubMed Central

Kotecki, Lydia; Hearing, Matthew; McCall, Nora M.; Marron Fernandez de Velasco, Ezequiel; Pravetoni, Marco; Arora, Devinder; Victoria, Nicole C.; Munoz, Michaelanne B.; Xia, Zhilian; Slesinger, Paul A.; Weaver, C. David

2015-01-01

G-protein-gated inwardly rectifying K+ (GIRK/Kir3) channel activation underlies key physiological effects of opioids, including analgesia and dependence. GIRK channel activation has also been implicated in the opioid-induced inhibition of midbrain GABA neurons and consequent disinhibition of dopamine (DA) neurons in the ventral tegmental area (VTA). Drug-induced disinhibition of VTA DA neurons has been linked to reward-related behaviors and underlies opioid-induced motor activation. Here, we demonstrate that mouse VTA GABA neurons express a GIRK channel formed by GIRK1 and GIRK2 subunits. Nevertheless, neither constitutive genetic ablation of Girk1 or Girk2, nor the selective ablation of GIRK channels in GABA neurons, diminished morphine-induced motor activity in mice. Moreover, direct activation of GIRK channels in midbrain GABA neurons did not enhance motor activity. In contrast, genetic manipulations that selectively enhanced or suppressed GIRK channel function in midbrain DA neurons correlated with decreased and increased sensitivity, respectively, to the motor-stimulatory effect of systemic morphine. Collectively, these data support the contention that the unique GIRK channel subtype in VTA DA neurons, the GIRK2/GIRK3 heteromer, regulates the sensitivity of the mouse mesolimbic DA system to drugs with addictive potential. PMID:25948263

[The effect of modulators of SK channels on simple spike firing frequency in the discharge of the cerebellar Purkinje cells in laboratory mice].

PubMed

Egorova, P A; Karelina, T V; Vlasova, O L; Antonov, S M; Besprozvanny, I B

2014-01-01

The effect of CyPPA, a positive modulator of small conductance calcium-activated potassium channels of type 3 and 2 (SK3/SK2), and of NS309, an activator of intermediate and small conductance calcium-activated potassium channels (IK/SK), on the activity of cerebellar Purkinje cells was studied in 2-month-old male mice. The use of 1 mM of CyPPA has led to a decrease of simple spike firing frequency in the discharge of Purkinje cells by 25%, on average, during 1 h after application. At the same time, application of 100 μM of NS309 has promoted a decrease in simple spike firing frequency by 47 %, on average, during 1 h after the beginning of the action. The obtained results confirm the hypothesis that SK channels participate in regulation of simple spike firing frequency in the discharge of Purkinje cells and are responsible for restriction of signal frequency. The effect of NS309 on simple spike firing frequency was more pronounced; therefore, the IK/SK channels may be suggested to play the cardinal role in regulation of spike activity of Purkinje cells. Since increasing simple spike frequency in the discharge of Purkinje cells is observed at many disturbances of motor activity, in particular, at spinocerebellar ataxia, it can be suggested that the studied compounds or substances of similar action are of interest as potential medicinal agents.

Structural determinants of PIP(2) regulation of inward rectifier K(ATP) channels.

PubMed

Shyng, S L; Cukras, C A; Harwood, J; Nichols, C G

2000-11-01

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates K(ATP) and other inward rectifier (Kir) channels. To determine residues important for PIP(2) regulation, we have systematically mutated each positive charge in the COOH terminus of Kir6.2 to alanine. The effects of these mutations on channel function were examined using (86)Rb efflux assays on intact cells and inside-out patch-clamp methods. Both methods identify essentially the same basic residues in two narrow regions (176-222 and 301-314) in the COOH terminus that are important for the maintenance of channel function and interaction with PIP(2). Only one residue (R201A) simultaneously affected ATP and PIP(2) sensitivity, which is consistent with the notion that these ligands, while functionally competitive, are unlikely to bind to identical sites. Strikingly, none of 13 basic residues in the terminal portion (residues 315-390) of the COOH terminus affected channel function when neutralized. The data help to define the structural requirements for PIP(2) sensitivity of K(ATP) channels. Moreover, the regions and residues defined in this study parallel those uncovered in recent studies of PIP(2) sensitivity in other inward rectifier channels, indicating a common structural basis for PIP(2) regulation.

Testosterone decreases urinary bladder smooth muscle excitability via novel signaling mechanism involving direct activation of the BK channels

PubMed Central

Hristov, Kiril L.; Parajuli, Shankar P.; Provence, Aaron

2016-01-01

In addition to improving sexual function, testosterone has been reported to have beneficial effects in ameliorating lower urinary tract symptoms by increasing bladder capacity and compliance, while decreasing bladder pressure. However, the cellular mechanisms by which testosterone regulates detrusor smooth muscle (DSM) excitability have not been elucidated. Here, we used amphotericin-B perforated whole cell patch-clamp and single channel recordings on inside-out excised membrane patches to investigate the regulatory role of testosterone in guinea pig DSM excitability. Testosterone (100 nM) significantly increased the depolarization-induced whole cell outward currents in DSM cells. The selective pharmacological inhibition of the large-conductance voltage- and Ca2+-activated K+ (BK) channels with paxilline (1 μM) completely abolished this stimulatory effect of testosterone, suggesting a mechanism involving BK channels. At a holding potential of −20 mV, DSM cells exhibited transient BK currents (TBKCs). Testosterone (100 nM) significantly increased TBKC activity in DSM cells. In current-clamp mode, testosterone (100 nM) significantly hyperpolarized the DSM cell resting membrane potential and increased spontaneous transient hyperpolarizations. Testosterone (100 nM) rapidly increased the single BK channel open probability in inside-out excised membrane patches from DSM cells, clearly suggesting a direct BK channel activation via a nongenomic mechanism. Live-cell Ca2+ imaging showed that testosterone (100 nM) caused a decrease in global intracellular Ca2+ concentration, consistent with testosterone-induced membrane hyperpolarization. In conclusion, the data provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in DSM cells, independent from genomic testosterone receptors, and thus regulates DSM excitability. PMID:27605581

Cholesterol up-regulates neuronal G protein-gated inwardly rectifying potassium (GIRK) channel activity in the hippocampus.

PubMed

Bukiya, Anna N; Durdagi, Serdar; Noskov, Sergei; Rosenhouse-Dantsker, Avia

2017-04-14

Hypercholesterolemia is a well known risk factor for the development of neurodegenerative disease. However, the underlying mechanisms are mostly unknown. In recent years, it has become increasingly evident that cholesterol-driven effects on physiology and pathophysiology derive from its ability to alter the function of a variety of membrane proteins including ion channels. Yet, the effect of cholesterol on G protein-gated inwardly rectifying potassium (GIRK) channels expressed in the brain is unknown. GIRK channels mediate the actions of inhibitory brain neurotransmitters. As a result, loss of GIRK function can enhance neuron excitability, whereas gain of GIRK function can reduce neuronal activity. Here we show that in rats on a high-cholesterol diet, cholesterol levels in hippocampal neurons are increased. We also demonstrate that cholesterol plays a critical role in modulating neuronal GIRK currents. Specifically, cholesterol enrichment of rat hippocampal neurons resulted in enhanced channel activity. In accordance, elevated currents upon cholesterol enrichment were also observed in Xenopus oocytes expressing GIRK2 channels, the primary GIRK subunit expressed in the brain. Furthermore, using planar lipid bilayers, we show that although cholesterol did not affect the unitary conductance of GIRK2, it significantly enhanced the frequency of channel openings. Last, combining computational and functional approaches, we identified two putative cholesterol-binding sites in the transmembrane domain of GIRK2. These findings establish that cholesterol plays a critical role in modulating GIRK activity in the brain. Because up-regulation of GIRK function can reduce neuronal activity, our findings may lead to novel approaches for prevention and therapy of cholesterol-driven neurodegenerative disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

TRPV4 channels: physiological and pathological role in cardiovascular system.

PubMed

Randhawa, Puneet Kaur; Jaggi, Amteshwar Singh

2015-11-01

TRPV4 channels are non-selective cation channels permeable to Ca(2+), Na(+), and Mg(2+) ions. Recently, TRPV4 channels have received considerable attention as these channels are widely expressed in the cardiovascular system including endothelial cells, cardiac fibroblasts, vascular smooth muscles, and peri-vascular nerves. Therefore, these channels possibly play a pivotal role in the maintenance of cardiovascular homeostasis. TRPV4 channels critically regulate flow-induced arteriogenesis, TGF-β1-induced differentiation of cardiac fibroblasts into myofibroblasts, and heart failure-induced pulmonary edema. These channels also mediate hypoxia-induced increase in proliferation and migration of pulmonary artery smooth muscle cells and progression of pulmonary hypertension. These channels also maintain flow-induced vasodilation and preserve vascular function by directly activating Ca(2+)-dependent KCa channels. Furthermore, these may also induce vasodilation and maintain blood pressure indirectly by evoking the release of NO, CGRP, and substance P. The present review discusses the evidences and the potential mechanisms implicated in diverse responses including arteriogenesis, cardiac remodeling, congestive heart failure-induced pulmonary edema, pulmonary hypertension, flow-induced dilation, regulation of blood pressure, and hypoxic preconditioning.

Magnolol and honokiol regulate the calcium-activated potassium channels signaling pathway in Enterotoxigenic Escherichia coli-induced diarrhea mice.

PubMed

Deng, Yanli; Han, Xuefeng; Tang, Shaoxun; Xiao, Wenjun; Tan, Zhiliang; Zhou, Chuanshe; Wang, Min; Kang, Jinghe

2015-05-15

To explore the regulatory mechanisms of magnolol and honokiol on calcium-activated potassium channels signaling pathway in Enterotoxigenic Escherichia coli (ETEC)-induced diarrhea mice, the concentrations of serum chloride ion (Cl(-)), sodium ion (Na(+)), potassium ion (K(+)) and calcium ion (Ca(2+)) were measured. Additionally, the mRNA expressions of calmodulin 1 (CaM), calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) and beta subunit (CaMKIIβ), ryanodine receptor 1, inositol 1,4,5-trisphosphate receptors (IP3 receptors), protein kinases C (PKC), potassium intermediate/small conductance calcium-activated channels (SK) and potassium large conductance calcium-activated channels(BK)were determined. A diarrhea mouse model was established using ETEC suspensions (3.29×10(9)CFU/ml) at a dosage of 0.02ml/g live body weight (BW). Magnolol or honokiol was intragastrically administered at dosages of 100 (M100 or H100), 300 (M300 or H300) and 500 (M500 or H500) mg/kg BW according to a 3×3 factorial arrangement. Magnolol and honokiol increased the Cl(-) and K(+) concentrations, further, upregulated the CaM, BKα1 and BKβ3 mRNA levels but downregulated the IP3 receptors 1, PKC, SK1, SK2, SK3, SK4 and BKβ4 mRNA expressions. Magnolol and honokiol did not alter the CaMKIIα, CaMKIIβ, ryanodine receptor 1, IP3 receptor 2, IP3 receptor 3, BKβ1 and BKβ2 mRNA expressions. These results clarify that magnolol and honokiol, acting through Ca(2+) channel blockade, inhibit the activation of IP3 receptor 1 to regulate the IP3-Ca(2+) store release, activate CaM to inhibit SK channels, and effectively suppress PKC kinases to promote BKα1 and BKβ3 channels opening and BKβ4 channel closing, which modulates the intestinal ion secretion. Copyright © 2015 Elsevier B.V. All rights reserved.

Angiotensin II upregulates K(Ca)3.1 channels and stimulates cell proliferation in rat cardiac fibroblasts.

PubMed

Wang, Li-Ping; Wang, Yan; Zhao, Li-Mei; Li, Gui-Rong; Deng, Xiu-Ling

2013-05-15

The proliferation of cardiac fibroblasts is implicated in the pathogenesis of myocardial remodeling and fibrosis. Intermediate-conductance calcium-activated K⁺ channels (K(Ca)3.1 channels) have important roles in cell proliferation. However, it is unknown whether angiotensin II (Ang II), a potent profibrotic molecule, would regulate K(Ca)3.1 channels in cardiac fibroblasts and participate in cell proliferation. In the present study, we investigated whether K(Ca)3.1 channels were regulated by Ang II, and how the channel activity mediated cell proliferation in cultured adult rat cardiac fibroblasts using electrophysiology and biochemical approaches. It was found that mRNA, protein, and current density of K(Ca)3.1 channels were greatly enhanced in cultured cardiac fibroblasts treated with 1 μM Ang II, and the effects were countered by the angiotensin type 1 receptor (AT₁R) blocker losartan, the p38-MAPK inhibitor SB203580, the ERK1/2 inhibitor PD98059, and the PI3K/Akt inhibitor LY294002. Ang II stimulated cell proliferation and the effect was antagonized by the K(Ca)3.1 blocker TRAM-34 and siRNA targeting K(Ca)3.1. In addition, Ang II-induced increase of K(Ca)3.1 expression was attenuated by transfection of activator protein-1 (AP-1) decoy oligodeoxynucleotides. These results demonstrate for the first time that Ang II stimulates cell proliferation mediated by upregulating K(Ca)3.1 channels via interacting with the AT₁R and activating AP-1 complex through ERK1/2, p38-MAPK and PI3K/Akt signaling pathways in cultured adult rat cardiac fibroblasts. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

GABA/sub B/ receptor activation inhibits Ca/sup 2 +/-activated potassium channels in synaptosomes: involvement of G-proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ticku, M.K.; Delgado, A.

1989-01-01

/sup 86/Rb-efflux assay from preloaded synaptosomes of rat cerebral cortex was developed to study the effect of GABA/sub B/ receptor agonist baclofen on Ca/sup 2 +/-activated K/sup +/-channels. Depolarization of /sup 86/Rb-loaded synaptosomes in physiological buffer increased Ca/sup 2 +/-activated /sup 86/Rb-efflux by 400%. The /sup 86/Rb-efflux was blocked by quinine sulfate, tetraethylammonium, and La/sup 3 +/ indicating the involvement of Ca/sup 2 +/-activated K/sup +/-channels. (-)Baclofen inhibited Ca/sup 2 +/-activated /sup 86/Rb-efflux in a stereospecific manner. The inhibitory effect of (-)baclofen was mediated by GABA/sub B/ receptor activation, since it was blocked by GABA/sub B/ antagonist phaclofen, but notmore » by bicuculline. Further, pertussis toxin also blocked the ability of baclofen or depolarizing action to affect Ca/sup 2 +/-activated K/sup +/-channels. These results suggest that baclofen inhibits Ca/sup 2 +/-activated K/sup +/-channels in synaptosomes and these channels are regulated by G-proteins. This assay may provide an ideal in vitro model to study GABA/sub B/ receptor pharmacology.« less

Ginseng gintonin activates the human cardiac delayed rectifier K+ channel: involvement of Ca2+/calmodulin binding sites.

PubMed

Choi, Sun-Hye; Lee, Byung-Hwan; Kim, Hyeon-Joong; Jung, Seok-Won; Kim, Hyun-Sook; Shin, Ho-Chul; Lee, Jun-Hee; Kim, Hyoung-Chun; Rhim, Hyewhon; Hwang, Sung-Hee; Ha, Tal Soo; Kim, Hyun-Ji; Cho, Hana; Nah, Seung-Yeol

2014-09-01

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca(2+)]i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K(+) (I(Ks)) channel is a cardiac K(+) channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating I(Ks) channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human I(Ks) channel activity by expressing human I(Ks) channels in Xenopus oocytes. We found that gintonin enhances IKs channel currents in concentration- and voltage-dependent manners. The EC50 for the I(Ks) channel was 0.05 ± 0.01 μg/ml. Gintonin-mediated activation of the I(Ks) channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP3 receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the I(Ks) channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca(2+)]i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on I(Ks) channel. However, gintonin had no effect on hERG K(+) channel activity. These results show that gintonin-mediated enhancement of I(Ks) channel currents is achieved through binding of the [Ca(2+)]i/CaM complex to the C terminus of KCNQ1 subunit.

Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling.

PubMed

Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

2015-09-15

Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system.

Structure of the full-length TRPV2 channel by cryo-EM

NASA Astrophysics Data System (ADS)

Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

2016-03-01

Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a `minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ~5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

Structure of the full-length TRPV2 channel by cryo-EM

PubMed Central

Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

2016-01-01

Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a ‘minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2–6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels. PMID:27021073

Structure of the full-length TRPV2 channel by cryo-EM.

PubMed

Huynh, Kevin W; Cohen, Matthew R; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T; Zhou, Z Hong; Moiseenkova-Bell, Vera Y

2016-03-29

Transient receptor potential (TRP) proteins form a superfamily Ca(2+)-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a 'minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

Aluminium and hydrogen ions inhibit a mechanosensory calcium-selective cation channel

NASA Technical Reports Server (NTRS)

Ding, J. P.; Pickard, B. G.

1993-01-01

The tension-dependent activity of mechanosensory calcium-selective cation channels in excised plasmalemmal patches from onion bulb scale epidermis is modulated by pH in the physiologically meaningful range between 4.5 and 7.2. It is rapidly lowered by lowering pH and rapidly raised by raising pH. Channel activity is effectively inhibited by low levels of aluminium ions and activity can be partially restored by washing for a few minutes. We suggest that under normal conditions the sensitivity of the mechanosensory channels to pH of the wall free space plays important roles in regulation of plant activities such as growth. We further suggest that, when levels of acid and aluminium ions in the soil solution are high, they might inhibit similar sensory channels in cells of the root tip, thus contributing critically to the acid soil syndrome.

Contemplating the plasmalemmal control center model

NASA Technical Reports Server (NTRS)

Pickard, B. G.

1994-01-01

An abundant epidermal mechanosensory calcium-selective ion channel appears able not only to detect mechanical stimuli such as those that initiate gravitropism but also to detect thermal, electrical, and various chemical stimuli. Because it responds to multimodal input with a second messenger output, this channel system seems likely to be an integrator that can engage in feedbacks with many other systems of the cell--and feedback is the hallmark of regulation. In general, the mechanical tension required for channel activation is likely transmitted from the relatively rigid cell wall to the plasma membrane system via linkage or adhesion sites that display antigenicities recognized by antibodies to animal beta-1 integrin, vitronectin, and fibronectin and which have mechanical connections to the cytoskeleton. Thus, functionally, leverage exerted against any given adhesion site will tend to control channels within a surrounding domain. Reactions initiated by passage of calcium ions through the channels could presumably be more effectively regulated if channels within the domains were somewhat clustered and if appropriate receptors, kinases, porters, pumps, and some key cytoskeletal anchoring sites were in turn clustered about them. Accumulating evidence suggests not only that activity of clusters of channels may contribute to control of cytoskeletal architecture and of regulatory protein function within their domain, but also that both a variety of regulatory proteins and components of the cortical cytoskeleton may contribute to control of channel activity. The emerging capabilities of electronic optical microscopy are well suited for resolving the spatial distributions of many of these cytoskeletal and regulatory molecules in living cells, and for following some of their behaviors as channels are stimulated to open and cytosolic calcium builds in their vicinity. Such microscopy, coupled with biochemical and physiological probing, should help to establish the nature of the feedback loops putatively controlled by the linkage sites and their channel domains.

Regulation of Cardiac ATP-sensitive Potassium Channel Surface Expression by Calcium/Calmodulin-dependent Protein Kinase II*

PubMed Central

Sierra, Ana; Zhu, Zhiyong; Sapay, Nicolas; Sharotri, Vikas; Kline, Crystal F.; Luczak, Elizabeth D.; Subbotina, Ekaterina; Sivaprasadarao, Asipu; Snyder, Peter M.; Mohler, Peter J.; Anderson, Mark E.; Vivaudou, Michel; Zingman, Leonid V.; Hodgson-Zingman, Denice M.

2013-01-01

Cardiac ATP-sensitive potassium (KATP) channels are key sensors and effectors of the metabolic status of cardiomyocytes. Alteration in their expression impacts their effectiveness in maintaining cellular energy homeostasis and resistance to injury. We sought to determine how activation of calcium/calmodulin-dependent protein kinase II (CaMKII), a central regulator of calcium signaling, translates into reduced membrane expression and current capacity of cardiac KATP channels. We used real-time monitoring of KATP channel current density, immunohistochemistry, and biotinylation studies in isolated hearts and cardiomyocytes from wild-type and transgenic mice as well as HEK cells expressing wild-type and mutant KATP channel subunits to track the dynamics of KATP channel surface expression. Results showed that activation of CaMKII triggered dynamin-dependent internalization of KATP channels. This process required phosphorylation of threonine at 180 and 224 and an intact 330YSKF333 endocytosis motif of the KATP channel Kir6.2 pore-forming subunit. A molecular model of the μ2 subunit of the endocytosis adaptor protein, AP2, complexed with Kir6.2 predicted that μ2 docks by interaction with 330YSKF333 and Thr-180 on one and Thr-224 on the adjacent Kir6.2 subunit. Phosphorylation of Thr-180 and Thr-224 would favor interactions with the corresponding arginine- and lysine-rich loops on μ2. We concluded that calcium-dependent activation of CaMKII results in phosphorylation of Kir6.2, which promotes endocytosis of cardiac KATP channel subunits. This mechanism couples the surface expression of cardiac KATP channels with calcium signaling and reveals new targets to improve cardiac energy efficiency and stress resistance. PMID:23223335

Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function

PubMed Central

Concepcion, Axel R.; Vaeth, Martin; Wagner, Larry E.; Eckstein, Miriam; Hecht, Lee; Yang, Jun; Crottes, David; Seidl, Maximilian; Shin, Hyosup P.; Weidinger, Carl; Cameron, Scott; Turvey, Stuart E.; Issekutz, Thomas; Meyts, Isabelle; Lacruz, Rodrigo S.; Cuk, Mario; Yule, David I.

2016-01-01

Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release–activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel–deficient patients and mice with ectodermal tissue–specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice. PMID:27721237

Protease-Activated Receptor 2 Activation Inhibits N-Type Ca2+ Currents in Rat Peripheral Sympathetic Neurons

PubMed Central

Kim, Young-Hwan; Ahn, Duck-Sun; Kim, Myeong Ok; Joeng, Ji-Hyun; Chung, Seungsoo

2014-01-01

The protease-activated receptor (PAR)-2 is highly expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although several mechanisms have been suggested to explain PAR-2-induced hypotension, the precise mechanism remains to be elucidated. To investigate this possibility, we investigated the effects of PAR-2 activation on N-type Ca2+ currents (ICa-N) in isolated neurons of the celiac ganglion (CG), which is involved in the sympathetic regulation of mesenteric artery vascular tone. PAR-2 agonists irreversibly diminished voltage-gated Ca2+ currents (ICa), measured using the patch-clamp method, in rat CG neurons, whereas thrombin had little effect on ICa. This PAR-2-induced inhibition was almost completely prevented by ω-CgTx, a potent N-type Ca2+ channel blocker, suggesting the involvement of N-type Ca2+ channels in PAR-2-induced inhibition. In addition, PAR-2 agonists inhibited ICa–N in a voltage-independent manner in rat CG neurons. Moreover, PAR-2 agonists reduced action potential (AP) firing frequency as measured using the current-clamp method in rat CG neurons. This inhibition of AP firing induced by PAR-2 agonists was almost completely prevented by ω-CgTx, indicating that PAR-2 activation may regulate the membrane excitability of peripheral sympathetic neurons through modulation of N-type Ca2+ channels. In conclusion, the present findings demonstrate that the activation of PAR-2 suppresses peripheral sympathetic outflow by modulating N-type Ca2+ channel activity, which appears to be involved in PAR-2-induced hypotension, in peripheral sympathetic nerve terminals. PMID:25410909

A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus.

PubMed

Kajma, Anna; Szewczyk, Adam

2012-10-01

Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288±4pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150mM KCl). At positive voltages, this channel displayed a conductance of 67.84pS and a strong voltage dependence at holding potentials from -80mV to +80mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). Copyright © 2012 Elsevier B.V. All rights reserved.

Retinoschisin, a New Binding Partner for L-type Voltage-gated Calcium Channels in the Retina*

PubMed Central

Shi, Liheng; Jian, Kuihuan; Ko, Michael L.; Trump, Dorothy; Ko, Gladys Y.-P.

2009-01-01

The L-type voltage-gated calcium channels (L-VGCCs) are activated under high depolarization voltages. They are vital for diverse biological events, including cell excitability, differentiation, and synaptic transmission. In retinal photoreceptors, L-VGCCs are responsible for neurotransmitter release and are under circadian influences. However, the mechanism of L-VGCC regulation in photoreceptors is not fully understood. Here, we show that retinoschisin, a highly conserved extracellular protein, interacts with the L-VGCCα1D subunit and regulates its activities in a circadian manner. Mutations in the gene encoding retinoschisin (RS1) cause retinal disorganization that leads to early onset of macular degeneration. Since ion channel activities can be modulated through interactions with extracellular proteins, disruption of these interactions can alter physiology and be the root cause of disease states. Co-immunoprecipitation and mammalian two-hybrid assays showed that retinoschisin and the N-terminal fragment of the L-VGCCα1 subunit physically interacted with one another. The expression and secretion of retinoschisin are under circadian regulation with a peak at night and nadir during the day. Inhibition of L-type VGCCs decreased membrane-bound retinoschisin at night. Overexpression of a missense RS1 mutant gene, R141G, into chicken cone photoreceptors caused a decrease of L-type VGCC currents at night. Our findings demonstrate a novel bidirectional relationship between an ion channel and an extracellular protein; L-type VGCCs regulate the circadian rhythm of retinoschisin secretion, whereas secreted retinoschisin feeds back to regulate L-type VGCCs. Therefore, physical interactions between L-VGCCα1 subunits and retinoschisin play an important role in the membrane retention of L-VGCCα1 subunits and photoreceptor-bipolar synaptic transmission. PMID:19074145

GTP-Binding Proteins Inhibit cAMP Activation of Chloride Channels in Cystic Fibrosis Airway Epithelial Cells

NASA Astrophysics Data System (ADS)

Schwiebert, Erik M.; Kizer, Neil; Gruenert, Dieter C.; Stanton, Bruce A.

1992-11-01

Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl^- secretion by airway epithelial cells. In CF, cAMP does not activate Cl^- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[β-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl^- currents and restore cAMP-activated Cl^- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[γ-thio]triphosphate and AlF^-_4 reduce Cl^- currents and inhibit cAMP from activating Cl^- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[β-thio]diphosphate and in normal cells, cAMP activates a Cl^- conductance that has properties similar to CF transmembrane-conductance regulator Cl^- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl^- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl^- secretion in CF.

cGMP accumulation causes photoreceptor degeneration in CNG channel deficiency: evidence of cGMP cytotoxicity independently of enhanced CNG channel function.

PubMed

Xu, Jianhua; Morris, Lynsie; Thapa, Arjun; Ma, Hongwei; Michalakis, Stylianos; Biel, Martin; Baehr, Wolfgang; Peshenko, Igor V; Dizhoor, Alexander M; Ding, Xi-Qin

2013-09-11

Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca(2+) influx in rod and cone photoreceptors. cGMP, the native ligand of the photoreceptor CNG channels, has been associated with cytotoxicity when its levels rise above normal due to defects in photoreceptor phosphodiesterase (PDE6) or regulation of retinal guanylyl cyclase (retGC). We found a massive accumulation of cGMP in CNGA3-deficient retina and investigated whether cGMP accumulation plays a role in cone degeneration in CNG channel deficiency. The time course study showed that the retinal cGMP level in Cnga3(-/-);Nrl(-/-) mice with CNGA3 deficiency on a cone-dominant background was sharply increased at postnatal day 8 (P8), peaked around P10-P15, remained high through P30-P60, and returned to near control level at P90. This elevation pattern correlated with photoreceptor apoptotic death, which peaked around P15-P20. In Cnga3(-/-);Gucy2e(-/-) mice lacking retGC1, cone density and expression levels of cone-specific proteins were significantly increased compared with Cnga3(-/-), consistent with a role of cGMP accumulation as the major contributor to cone death caused by CNG channel deficiency. The activity and expression levels of cGMP-dependent protein kinase G (PKG) were significantly increased in Cnga3(-/-);Nrl(-/-) retina compared with Nrl(-/-), suggesting an involvement of PKG regulation in cell death. Our results indicate that cGMP accumulation in photoreceptors can itself exert cytotoxic effect in cones, independently of CNG channel activity and Ca(2+) influx.

TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion

PubMed Central

Togashi, Kazuya; Hara, Yuji; Tominaga, Tomoko; Higashi, Tomohiro; Konishi, Yasunobu; Mori, Yasuo; Tominaga, Makoto

2006-01-01

There are eight thermosensitive TRP (transient receptor potential) channels in mammals, and there might be other TRP channels sensitive to temperature stimuli. Here, we demonstrate that TRPM2 can be activated by exposure to warm temperatures (>35°C) apparently via direct heat-evoked channel gating. β-NAD+- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. We also show that, even though cyclic ADP-ribose (cADPR) does not activate TRPM2 at 25°C, co-application of heat and intracellular cADPR dramatically potentiates TRPM2 activity. Heat and cADPR evoke similar responses in rat insulinoma RIN-5F cells, which express TRPM2 endogenously. In pancreatic islets, TRPM2 is coexpressed with insulin, and mild heating of these cells evokes increases in both cytosolic Ca2+ and insulin release, which is KATP channel-independent and protein kinase A-mediated. Heat-evoked responses in both RIN-5F cells and pancreatic islets are significantly diminished by treatment with TRPM2-specific siRNA. These results identify TRPM2 as a potential molecular target for cADPR, and suggest that TRPM2 regulates Ca2+ entry into pancreatic β-cells at body temperature depending on the production of cADPR-related molecules, thereby regulating insulin secretion. PMID:16601673

Redox-Dependent Calcium-Mediated Signaling Networks that Control the Senescence-Associated Secretory Phenotype

NASA Astrophysics Data System (ADS)

Chandrasekaran, Akshaya

Cellular senescence has evolved as a protective mechanism to arrest growth of cells with oncogenic potential. While senescent cells have lost the ability to divide, they remain metabolically active and adapt a deleterious senescence associated secretory phenotype (SASP) central to the progression of several age-associated disease pathologies. The SASP is mechanistically regulated by the pro-inflammatory cytokine interleukin-1 alpha (IL-1alpha) whose expression and activity is responsive to the senescence associated (SA) oxidant production and the accompanying disruption of calcium (Ca2+) homeostasis. Using primary IMR-90 human fetal lung fibroblasts as a model of replicative senescence, we explored the molecular underpinnings driving Ca2+ dysregulation in senescent cells. We establish that the redox-responsive Transient Receptor Potential TRPC6 channel is compromised due to desensitization owing to SA increases in steady state hydrogen peroxide (H2O2) production. SA dysregulation of Ca2+ is also accompanied by loss of response to H2O2-induced Ca2+ influx that can be rescued with catalase pre-treatments. Senescent cells are also insensitive to Ca2+ entry induced by hyperforin, a specific activator of TRPC6, that can be restored by catalase pre-treatments, further suggesting redox regulation of TRPC6 in senescence. Inhibition of TRPC6 channel activity restores the ability of senescent cells to respond to peroxide-induced Ca2+ in addition to suppressing SASP gene expression. Furthermore, mammalian target of rapamycin (mTOR) signaling regulates SASP by means of modulating TRPC6 channel expression. Together, our findings provide compelling evidence that redox and mTOR-mediated regulation of TRPC6 channel modulate SASP gene expression. Further, the gain-of-function mutation of TRPC6 has pathological implications in several chronic pathologies and renders it a viable target in age-associated diseases.

Kv7 (KCNQ) channel openers induce hypothermia in the mouse.

PubMed

Kristensen, Line V; Sandager-Nielsen, Karin; Hansen, Henrik H

2011-01-20

Kv7 channels, encoded by corresponding kcnq genes, are expressed both centrally and peripherally where they serve to dampen neuronal activity. While Kv7 channel openers have shown efficacy in neurological and neuropsychiatric disease models, the impact of Kv7 channel activation on physiological endpoint markers have not been addressed in detail. In this study we assessed the effect of a range of Kv7 channel openers with different affinity for neuronal Kv7.2-5 channel subunits on body temperature regulation in mice. Female NMRI mice were acutely exposed to vehicle (10% Tween-80, i.p.), retigabine (3-30 mg/kg, i.p., pan-Kv7 channel opener), (S)BMS-204352 (60-240 mg/kg, i.p., Kv7.4/5 channel-preferring opener), ICA-27243 (1-10mg/kg, i.p., Kv7.2/3 channel-preferring opener), or S-(1) (10-60 mg/kg, i.p., Kv7.2/3 channel-preferring opener), and rectal body temperature was measured 15-120 min post-injection. Retigabine (>10mg/kg), ICA-27243 (≥ 10 mg/kg), and S-(1) (≥ 30 mg/kg) dose-dependently lowered rectal body temperature with maximal doses of each Kv7 channel opener inducing a marked drop (>4°C) in rectal temperature. The Kv7 channel openers showed differential temporal pharmacodynamics, which likely reflects their different pharmacokinetic profiles. Pretreatment with the pan-Kv7 channel blocker XE-991 (1.0mg/kg, i.p.) completely reversed the hypothermic effect of the pan-Kv7 opener, retigabine (15 mg/kg), whereas ICA-27243-induced hypothermia (10mg/kg) could only be partially prevented by XE-991. Because ICA-27743 and S-(1) are Kv7.2/3 channel subunit-preferring compounds, this suggests that the Kv7.2/3 channel isoform is the predominant substrate for Kv7 channel opener-evoked hypothermia. These data indicate the physiological relevance of Kv7 channel function on body temperature regulation which may potentially reside from central inhibitory Kv7 channel activity. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Dendritic Kv3.3 potassium channels in cerebellar purkinje cells regulate generation and spatial dynamics of dendritic Ca2+ spikes.

PubMed

Zagha, Edward; Manita, Satoshi; Ross, William N; Rudy, Bernardo

2010-06-01

Purkinje cell dendrites are excitable structures with intrinsic and synaptic conductances contributing to the generation and propagation of electrical activity. Voltage-gated potassium channel subunit Kv3.3 is expressed in the distal dendrites of Purkinje cells. However, the functional relevance of this dendritic distribution is not understood. Moreover, mutations in Kv3.3 cause movement disorders in mice and cerebellar atrophy and ataxia in humans, emphasizing the importance of understanding the role of these channels. In this study, we explore functional implications of this dendritic channel expression and compare Purkinje cell dendritic excitability in wild-type and Kv3.3 knockout mice. We demonstrate enhanced excitability of Purkinje cell dendrites in Kv3.3 knockout mice, despite normal resting membrane properties. Combined data from local application pharmacology, voltage clamp analysis of ionic currents, and assessment of dendritic Ca(2+) spike threshold in Purkinje cells suggest a role for Kv3.3 channels in opposing Ca(2+) spike initiation. To study the physiological relevance of altered dendritic excitability, we measured [Ca(2+)](i) changes throughout the dendritic tree in response to climbing fiber activation. Ca(2+) signals were specifically enhanced in distal dendrites of Kv3.3 knockout Purkinje cells, suggesting a role for dendritic Kv3.3 channels in regulating propagation of electrical activity and Ca(2+) influx in distal dendrites. These findings characterize unique roles of Kv3.3 channels in dendrites, with implications for synaptic integration, plasticity, and human disease.

Identification of a Mg2+-sensitive ORF in the 5′-leader of TRPM7 magnesium channel mRNA

PubMed Central

Nikonorova, Inna A.; Kornakov, Nikolay V.; Dmitriev, Sergey E.; Vassilenko, Konstantin S.; Ryazanov, Alexey G.

2014-01-01

TRPM7 is an essential and ubiquitous channel-kinase regulating cellular influx of Mg2+. Although TRPM7 mRNA is highly abundant, very small amount of the protein is detected in cells, suggesting post-transcriptional regulation of trpm7 gene expression. We found that TRPM7 mRNA 5′-leader contains two evolutionarily conserved upstream open reading frames that act together to drastically inhibit translation of the TRPM7 reading frame at high magnesium levels and ensure its optimal translation at low magnesium levels, when the activity of the channel-kinase is most required. The study provides the first example of magnesium channel synthesis being controlled by Mg2+ in higher eukaryotes. PMID:25326319

Properties and function of KCNQ1 K+ channels isolated from the rectal gland of Squalus acanthias.

PubMed

Kerst, G; Beschorner, U; Unsöld, B; von Hahn, T; Schreiber, R; Greger, R; Gerlach, U; Lang, H J; Kunzelmann, K; Bleich, M

2001-10-01

KCNQ1 (KVLQT1) K+ channels play an important role during electrolyte secretion in airways and colon. KCNQ1 was cloned recently from NaCl-secreting shark rectal glands. Here we study the properties and regulation of the cloned sKVLQT1 expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells and compare the results with those obtained from in vitro perfused rectal gland tubules (RGT). The expression of sKCNQ1 induced voltage-dependent, delayed activated K+ currents, which were augmented by an increase in intracellular cAMP and Ca2+. The chromanol derivatives 293B and 526B potently inhibited sKCNQ1 expressed in oocytes and CHO cells, but had little effect on RGT electrolyte transport. Short-circuit currents in RGT were activated by alkalinization and were decreased by acidification. In CHO cells an alkaline pH activated and an acidic pH inhibited 293B-sensitive KCNQ1 currents. Noise analysis of the cell-attached basolateral membrane of RGT indicated the presence of low-conductance (<3 pS) K+ channels, in parallel with other K+ channels. sKCNQ1 generated similar small-conductance K+ channels upon expression in CHO cells and Xenopus oocytes. The results suggest the presence of low-conductance KCNQ1 K+ channels in RGT, which are probably regulated by changes in intracellular cAMP, Ca2+ and pH.

DiBAC4(3) hits a “sweet spot” for the activation of arterial large-conductance Ca2+-activated potassium channels independently of the β1-subunit

PubMed Central

Scornik, Fabiana S.; Bucciero, Ronald S.; Wu, Yuesheng; Selga, Elisabet; Bosch Calero, Cristina; Brugada, Ramon

2013-01-01

The voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)] has been reported as a novel large-conductance Ca2+-activated K+ (BK) channel activator with selectivity for its β1- or β4-subunits. In arterial smooth muscle, BK channels are formed by a pore-forming α-subunit and a smooth muscle-abundant regulatory β1-subunit. This tissue specificity has driven extensive pharmacological research aimed at regulating arterial tone. Using animals with a disruption of the gene for the β1-subunit, we explored the effects of DiBAC4(3) in native channels from arterial smooth muscle. We tested the hypothesis that, in native BK channels, activation by DiBAC4(3) relies mostly on its α-subunit. We studied BK channels from wild-type and transgenic β1-knockout mice in excised patches. BK channels from brain arteries, with or without the β1-subunit, were similarly activated by DiBAC4(3). In addition, we found that saturating concentrations of DiBAC4(3) (∼30 μM) promote an unprecedented persistent activation of the channel that negatively shifts its voltage dependence by as much as −300 mV. This “sweet spot” for persistent activation is independent of Ca2+ and/or the β1–4-subunits and is fully achieved when DiBAC4(3) is applied to the intracellular side of the channel. Arterial BK channel response to DiBAC4(3) varies across species and/or vascular beds. DiBAC4(3) unique effects can reveal details of BK channel gating mechanisms and help in the rational design of BK channel activators. PMID:23542916

Mechanically Activated Ion Channels

PubMed Central

Ranade, Sanjeev S.; Syeda, Ruhma; Patapoutian, Ardem

2015-01-01

Mechanotransduction, the conversion of physical forces into biochemical signals, is an essential component of numerous physiological processes including not only conscious senses of touch and hearing, but also unconscious senses such as blood pressure regulation. Mechanically activated (MA) ion channels have been proposed as sensors of physical force, but the identity of these channels and an understanding of how mechanical force is transduced has remained elusive. A number of recent studies on previously known ion channels along with the identification of novel MA ion channels have greatly transformed our understanding of touch and hearing in both vertebrates and invertebrates. Here, we present an updated review of eukaryotic ion channel families that have been implicated in mechanotransduction processes and evaluate the qualifications of the candidate genes according to specified criteria. We then discuss the proposed gating models for MA ion channels and highlight recent structural studies of mechanosensitive potassium channels. PMID:26402601

Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels

PubMed Central

Ufret-Vincenty, Carmen A.; Klein, Rebecca M.; Collins, Marcus D.; Rosasco, Mario G.; Martinez, Gilbert Q.

2015-01-01

Although PI(4,5)P2 is believed to play an essential role in regulating the activity of numerous ion channels and transporters, the mechanisms by which it does so are unknown. Here, we used the ability of the TRPV1 ion channel to discriminate between PI(4,5)P2 and PI(4)P to localize the region of TRPV1 sequence that interacts directly with the phosphoinositide. We identified a point mutation in the proximal C-terminal region after the TRP box, R721A, that inverted the selectivity of TRPV1. Although the R721A mutation produced only a 30% increase in the EC50 for activation by PI(4,5)P2, it decreased the EC50 for activation by PI(4)P by more than two orders of magnitude. We used chemically induced and voltage-activated phosphatases to determine that PI(4)P continued to support TRPV1 activity even after depletion of PI(4,5)P2 from the plasma membrane. Our data cannot be explained by a purely electrostatic mechanism for interaction between the phosphoinositide and the protein, similar to that of the MARCKS (myristoylated alanine-rich C kinase substrate) effector domain or the EGF receptor. Rather, conversion of a PI(4,5)P2-selective channel to a PI(4)P-selective channel indicates that a structured phosphoinositide-binding site mediates the regulation of TRPV1 activity and that the amino acid at position 721 likely interacts directly with the moiety at the 5′ position of the phosphoinositide. PMID:25918361

Identification of Extracellular Domain Residues Required for Epithelial Na+ Channel Activation by Acidic pH

PubMed Central

Collier, Daniel M.; Peterson, Zerubbabel J.; Blokhin, Ilya O.; Benson, Christopher J.; Snyder, Peter M.

2012-01-01

A growing body of evidence suggests that the extracellular domain of the epithelial Na+ channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na+ self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γE455 is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with βE446A generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na+ self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH. PMID:23060445

Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death.

PubMed

Marks, A R

2001-04-01

Calcium (Ca2+) ions are second messengers in signaling pathways in all types of cells. They regulate muscle contraction, electrical signals which determine the cardiac rhythm and cell growth pathways in the heart. In the past decade cDNA cloning has provided clues as to the molecular structure of the intracellular Ca2+ release channels (ryanodine receptors, RyR, and inositol 1,4,5-trisphosphate receptors, IP3R) on the sarcoplasmic and endoplasmic reticulum (SR/ER) and an understanding of how these molecules regulate Ca2+ homeostasis in the heart is beginning to emerge. The intracellular Ca2+ release channels form a distinct class of ion channels distinguished by their structure, size, and function. Both RyRs and IP3Rs have gigantic cytoplasmic domains that serve as scaffolds for modulatory proteins that regulate the channel pore located in the carboxy terminal 10% of the channel sequence. The channels are tetramers comprised of four RyR or IP3R subunits. RyR2 is required for excitation-contraction (EC) coupling in the heart. Using co-sedimentation and co-immunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein mAKAP. We have shown that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (P(o)). In failing human hearts RyR2 is PKA hyperphosphorylated resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.

Extracellular protons enable activation of the calcium‐dependent chloride channel TMEM16A

PubMed Central

Cruz‐Rangel, Silvia; De Jesús‐Pérez, José J.; Aréchiga‐Figueroa, Iván A.; Rodríguez‐Menchaca, Aldo A.; Pérez‐Cornejo, Patricia; Hartzell, H. Criss

2017-01-01

Key points The calcium‐activated chloride channel TMEM16A provides a pathway for chloride ion movements that are key in preventing polyspermy, allowing fluid secretion, controlling blood pressure, and enabling gastrointestinal activity.TMEM16A is opened by voltage‐dependent calcium binding and regulated by permeant anions and intracellular protons.Here we show that a low proton concentration reduces TMEM16A activity while maximum activation is obtained when the external proton concentration is high.In addition, protonation conditions determine the open probability of TMEM16A without changing its calcium sensitivity. External glutamic acid 623 (E623) is key for TMEM16A's ability to respond to external protons.At physiological pH, E623 is un‐protonated and TMEM16A is activated when intracellular calcium increases; however, under acidic conditions E623 is partially protonated and works synergistically with intracellular calcium to activate the channel. These findings are critical for understanding physiological and pathological processes that involve changes in pH and chloride flux via TMEM16A. Abstract Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore‐forming subunit of a Ca2+‐dependent Cl− channel (CaCC), is activated by direct, voltage‐dependent, binding of intracellular Ca2+. Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H+]o) on mouse TMEM16A expressed in HEK‐293 cells using whole‐cell and inside‐out patch‐clamp recordings. We found that increasing the [H+]o from 10−10 to 10−5.5 m caused a progressive increase in the chloride current (I Cl) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage‐independent manner, regardless of channel state (open or closed), and without altering its apparent Ca2+ sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca2+]i. To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the I Cl was nearly insensitive to proton concentrations between 10−5.5 and 10−9.0 m. Additionally, I Cl of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10−5.5 m, but 10−9.0 m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non‐saturating [Ca2+]i. PMID:27859335

Extracellular protons enable activation of the calcium-dependent chloride channel TMEM16A.

PubMed

Cruz-Rangel, Silvia; De Jesús-Pérez, José J; Aréchiga-Figueroa, Iván A; Rodríguez-Menchaca, Aldo A; Pérez-Cornejo, Patricia; Hartzell, H Criss; Arreola, Jorge

2017-03-01

The calcium-activated chloride channel TMEM16A provides a pathway for chloride ion movements that are key in preventing polyspermy, allowing fluid secretion, controlling blood pressure, and enabling gastrointestinal activity. TMEM16A is opened by voltage-dependent calcium binding and regulated by permeant anions and intracellular protons. Here we show that a low proton concentration reduces TMEM16A activity while maximum activation is obtained when the external proton concentration is high. In addition, protonation conditions determine the open probability of TMEM16A without changing its calcium sensitivity. External glutamic acid 623 (E623) is key for TMEM16A's ability to respond to external protons. At physiological pH, E623 is un-protonated and TMEM16A is activated when intracellular calcium increases; however, under acidic conditions E623 is partially protonated and works synergistically with intracellular calcium to activate the channel. These findings are critical for understanding physiological and pathological processes that involve changes in pH and chloride flux via TMEM16A. Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore-forming subunit of a Ca 2+ -dependent Cl - channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca 2+ . Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H + ] o ) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch-clamp recordings. We found that increasing the [H + ] o from 10 -10 to 10 -5.5  m caused a progressive increase in the chloride current (I Cl ) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage-independent manner, regardless of channel state (open or closed), and without altering its apparent Ca 2+ sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca 2+ ] i . To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the I Cl was nearly insensitive to proton concentrations between 10 -5.5 and 10 -9.0  m. Additionally, I Cl of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10 -5.5  m, but 10 -9.0  m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca 2+ ] i . © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Regulator of G-protein signalling and GoLoco proteins suppress TRPC4 channel function via acting at Gαi/o.

PubMed

Jeon, Jae-Pyo; Thakur, Dhananjay P; Tian, Jin-Bin; So, Insuk; Zhu, Michael X

2016-05-15

Transient receptor potential canonical 4 (TRPC4) forms non-selective cation channels implicated in the regulation of diverse physiological functions. Previously, TRPC4 was shown to be activated by the Gi/o subgroup of heterotrimeric G-proteins involving Gαi/o, rather than Gβγ, subunits. Because the lifetime and availability of Gα-GTP are regulated by regulators of G-protein signalling (RGS) and Gαi/o-Loco (GoLoco) domain-containing proteins via their GTPase-activating protein (GAP) and guanine-nucleotide-dissociation inhibitor (GDI) functions respectively, we tested how RGS and GoLoco domain proteins affect TRPC4 currents activated via Gi/o-coupled receptors. Using whole-cell patch-clamp recordings, we show that both RGS and GoLoco proteins [RGS4, RGS6, RGS12, RGS14, LGN or activator of G-protein signalling 3 (AGS3)] suppress receptor-mediated TRPC4 activation without causing detectable basal current or altering surface expression of the channel protein. The inhibitory effects are dependent on the GAP and GoLoco domains and facilitated by enhancing membrane targeting of the GoLoco protein AGS3. In addition, RGS, but not GoLoco, proteins accelerate desensitization of receptor-activation evoked TRPC4 currents. The inhibitory effects of RGS and GoLoco domains are additive and are most prominent with RGS12 and RGS14, which contain both RGS and GoLoco domains. Our data support the notion that the Gα, but not Gβγ, arm of the Gi/o signalling is involved in TRPC4 activation and unveil new roles for RGS and GoLoco domain proteins in fine-tuning TRPC4 activities. The versatile and diverse functions of RGS and GoLoco proteins in regulating G-protein signalling may underlie the complexity of receptor-operated TRPC4 activation in various cell types under different conditions. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Hyperforin activates gene transcription involving transient receptor potential C6 channels.

PubMed

Thiel, Gerald; Rössler, Oliver G

2017-04-01

Hypericum perforatum is one of the most prominent medical plants. Hyperforin, a main ingredient of H. perforatum, has been shown to activate transient receptor potential canonical C6 (TRPC6) channels. Alternatively, it has been proposed that hyperforin functions as a protonophore in a TRPC6-independent manner. Here, we show that hyperforin stimulation activates the transcription factor AP-1 in HEK293 cells expressing TRPC6 (T6.11 cells), but did not substantially change the AP-1 activity in HEK293 cells lacking TRPC6. We identified the AP-1 binding site as a hyperforin-responsive element. AP-1 is composed of the transcription factors c-Jun and c-Fos, or other members of the c-Jun and c-Fos families of proteins. Hyperforin stimulation increased c-Jun and c-Fos promoter activities in T6.11 cells and induced an upregulation of c-Jun and c-Fos biosynthesis. The analysis of the c-Fos promoter revealed that the cAMP-response element also functions as a hyperforin-responsive element. Hyperforin-induced upregulation of AP-1 in T6.11 cells was attenuated by preincubation of the cells with either pregnenolone or progesterone, indicating that gene regulation via TRPC6 is under control of hormones or hormonal precursors. The signal transduction of hyperforin-induced AP-1 gene transcription required an influx of Ca 2+ ions into the cells, the activation of MAP kinases, and the activation of the transcription factors c-Jun and ternary complex factor. We conclude that hyperforin regulates gene transcription via activation of TRPC6 channels, involving stimulus-regulated protein kinases and stimulus-responsive transcription factors. The fact that hyperforin regulates gene transcription may explain many of the intracellular alterations induced by this compound. Copyright © 2017 Elsevier Inc. All rights reserved.

BAD-Dependent Regulation of Fuel Metabolism and KATP Channel Activity Confers Resistance to Epileptic Seizures

PubMed Central

Giménez-Cassina, Alfredo; Martínez-François, Juan Ramón; Fisher, Jill K.; Szlyk, Benjamin; Polak, Klaudia; Wiwczar, Jessica; Tanner, Geoffrey R.; Lutas, Andrew; Yellen, Gary; Danial, Nika N.

2012-01-01

Summary Neuronal excitation can be substantially modulated by alterations in metabolism, as evident from the anticonvulsant effect of diets that reduce glucose utilization and promote ketone body metabolism. We provide genetic evidence that BAD, a protein with dual functions in apoptosis and glucose metabolism, imparts reciprocal effects on metabolism of glucose and ketone bodies in brain cells. These effects involve phospho-regulation of BAD and are independent of its apoptotic function. BAD modifications that reduce glucose metabolism produce a marked increase in the activity of metabolically sensitive KATP channels in neurons, as well as resistance to behavioral and electrographic seizures in vivo. Seizure resistance is reversed by genetic ablation of the KATP channel, implicating the BAD-KATP axis in metabolic control of neuronal excitation and seizure responses. PMID:22632729

A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity

PubMed Central

Tien, Jason; Peters, Christian J; Wong, Xiu Ming; Cheng, Tong; Jan, Yuh Nung; Jan, Lily Yeh; Yang, Huanghe

2014-01-01

TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. DOI: http://dx.doi.org/10.7554/eLife.02772.001 PMID:24980701

OSR1 regulates a subset of inward rectifier potassium channels via a binding motif variant.

PubMed

Taylor, Clinton A; An, Sung-Wan; Kankanamalage, Sachith Gallolu; Stippec, Steve; Earnest, Svetlana; Trivedi, Ashesh T; Yang, Jonathan Zijiang; Mirzaei, Hamid; Huang, Chou-Long; Cobb, Melanie H

2018-04-10

The with-no-lysine (K) (WNK) signaling pathway to STE20/SPS1-related proline- and alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinase is an important mediator of cell volume and ion transport. SPAK and OSR1 associate with upstream kinases WNK 1-4, substrates, and other proteins through their C-terminal domains which interact with linear R-F-x-V/I sequence motifs. In this study we find that SPAK and OSR1 also interact with similar affinity with a motif variant, R-x-F-x-V/I. Eight of 16 human inward rectifier K + channels have an R-x-F-x-V motif. We demonstrate that two of these channels, Kir2.1 and Kir2.3, are activated by OSR1, while Kir4.1, which does not contain the motif, is not sensitive to changes in OSR1 or WNK activity. Mutation of the motif prevents activation of Kir2.3 by OSR1. Both siRNA knockdown of OSR1 and chemical inhibition of WNK activity disrupt NaCl-induced plasma membrane localization of Kir2.3. Our results suggest a mechanism by which WNK-OSR1 enhance Kir2.1 and Kir2.3 channel activity by increasing their plasma membrane localization. Regulation of members of the inward rectifier K + channel family adds functional and mechanistic insight into the physiological impact of the WNK pathway.

Heteromeric Slick/Slack K+ channels show graded sensitivity to cell volume changes

PubMed Central

Hashem, Nadia; Calloe, Kirstine; Klaerke, Dan A.

2017-01-01

Slick and Slack high-conductance K+ channels are found in the CNS, kidneys, pancreas, among other organs, where they play an important role in cell excitability as well as in ion transport processes. They are both activated by Na+ and Cl- but show a differential regulation by cell volume changes. Slick has been shown to be regulated by cell volume changes, whereas Slack is insensitive. α-subunits of these channels form homomeric as well as heteromeric channels. It is the aim of this work to explore whether the subunit composition of the Slick/Slack heteromeric channel affects the response to osmotic challenges. In order to provide with the adequate water permeability to the cell membrane of Xenopus laevis oocytes, mRNA of aquaporin 1 was co-expressed with homomeric or heteromeric Slick and Slack α-subunits. Oocytes were superfused with hypotonic or hypertonic buffers and changes in currents were measured by two-electrode voltage clamp. This work presents the first heteromeric K+ channel with a characteristic graded sensitivity to small and fast changes in cell volume. Our results show that the cell volume sensitivity of Slick/Slack heteromeric channels is dependent on the number of volume sensitive Slick α-subunits in the tetrameric channels, giving rise to graded cell volume sensitivity. Regulation of the subunit composition of a channel may constitute a novel mechanism to determine volume sensitivity of cells. PMID:28222129

Heteromeric Slick/Slack K+ channels show graded sensitivity to cell volume changes.

PubMed

Tejada, Maria A; Hashem, Nadia; Calloe, Kirstine; Klaerke, Dan A

2017-01-01

Slick and Slack high-conductance K+ channels are found in the CNS, kidneys, pancreas, among other organs, where they play an important role in cell excitability as well as in ion transport processes. They are both activated by Na+ and Cl- but show a differential regulation by cell volume changes. Slick has been shown to be regulated by cell volume changes, whereas Slack is insensitive. α-subunits of these channels form homomeric as well as heteromeric channels. It is the aim of this work to explore whether the subunit composition of the Slick/Slack heteromeric channel affects the response to osmotic challenges. In order to provide with the adequate water permeability to the cell membrane of Xenopus laevis oocytes, mRNA of aquaporin 1 was co-expressed with homomeric or heteromeric Slick and Slack α-subunits. Oocytes were superfused with hypotonic or hypertonic buffers and changes in currents were measured by two-electrode voltage clamp. This work presents the first heteromeric K+ channel with a characteristic graded sensitivity to small and fast changes in cell volume. Our results show that the cell volume sensitivity of Slick/Slack heteromeric channels is dependent on the number of volume sensitive Slick α-subunits in the tetrameric channels, giving rise to graded cell volume sensitivity. Regulation of the subunit composition of a channel may constitute a novel mechanism to determine volume sensitivity of cells.

Mechanisms of Calmodulin Regulation of Different Isoforms of Kv7.4 K+ Channels.

PubMed

Sihn, Choong-Ryoul; Kim, Hyo Jeong; Woltz, Ryan L; Yarov-Yarovoy, Vladimir; Yang, Pei-Chi; Xu, Jun; Clancy, Colleen E; Zhang, Xiao-Dong; Chiamvimonvat, Nipavan; Yamoah, Ebenezer N

2016-01-29

Calmodulin (CaM), a Ca(2+)-sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca(2+)-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca(2+)-independent manner but that only the long isoform (hKv7.4a) is regulated by Ca(2+)/CaM. Ca(2+)/CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca(2+)/CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca(2+) on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca(2+)/CaM regulation of the Kv7.4 channel under physiological and pathological conditions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Voltage-dependent and -independent titration of specific residues accounts for complex gating of a ClC chloride channel by extracellular protons

PubMed Central

Niemeyer, María Isabel; Cid, L Pablo; Yusef, Yamil R; Briones, Rodolfo; Sepúlveda, Francisco V

2009-01-01

The ClC transport protein family comprises both Cl− ion channel and H+/Cl− and H+/NO3− exchanger members. Structural studies on a bacterial ClC transporter reveal a pore obstructed at its external opening by a glutamate side-chain which acts as a gate for Cl− passage and in addition serves as a staging post for H+ exchange. This same conserved glutamate acts as a gate to regulate Cl− flow in ClC channels. The activity of ClC-2, a genuine Cl− channel, has a biphasic response to extracellular pH with activation by moderate acidification followed by abrupt channel closure at pH values lower than ∼7. We have now investigated the molecular basis of this complex gating behaviour. First, we identify a sensor that couples extracellular acidification to complete closure of the channel. This is extracellularly-facing histidine 532 at the N-terminus of transmembrane helix Q whose neutralisation leads to channel closure in a cooperative manner. We go on to show that acidification-dependent activation of ClC-2 is voltage dependent and probably mediated by protonation of pore gate glutamate 207. Intracellular Cl− acts as a voltage-independent modulator, as though regulating the pKa of the protonatable residue. Our results suggest that voltage dependence of ClC-2 is given by hyperpolarisation-dependent penetration of protons from the extracellular side to neutralise the glutamate gate deep within the channel, which allows Cl− efflux. This is reminiscent of a partial exchanger cycle, suggesting that the ClC-2 channel evolved from its transporter counterparts. PMID:19153159

Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels.

PubMed

Tuluc, Petronel; Benedetti, Bruno; Coste de Bagneaux, Pierre; Grabner, Manfred; Flucher, Bernhard E

2016-06-01

Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3-S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3-S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3-S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3-S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3-S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively. © 2016 Tuluc et al.

Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels

PubMed Central

Tuluc, Petronel; Benedetti, Bruno; Coste de Bagneaux, Pierre; Grabner, Manfred

2016-01-01

Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively. PMID:27185857

Drosophila QVR/SSS modulates the activation and C-type inactivation kinetics of Shaker K+ channels

PubMed Central

Dean, Terry; Xu, Rong; Joiner, William; Sehgal, Amita; Hoshi, Toshinori

2011-01-01

The quiver/sleepless (qvr/sss) gene encodes a small, glycosylphosphatidylinositol-anchored protein that plays a critical role in the regulation of sleep in Drosophila. Loss-of-function mutations in qvr/sss severely suppress sleep and effect multiple changes in in situ Shaker K+ currents, including decreased magnitude, slower time-to-peak, and cumulative inactivation. Recently, we demonstrated that SLEEPLESS (SSS) protein modulates Shaker channel activity, possibly through a direct interaction at the plasma membrane. We show here that SSS accelerates the activation of heterologously expressed Shaker channels with no effect on deactivation or fast N-type inactivation. Furthermore, this SSS-induced acceleration is sensitive to the pharmacological disruption of lipid rafts and sufficiently accounts for the slower time-to-peak of in situ Shaker currents seen in qvr/sss mutants. We also find that SSS decreases the rate of C-type inactivation of heterologously expressed Shaker channels, providing a potential mechanism for the cumulative inactivation phenotype induced by qvr/sss loss of function mutations. Kinetic modeling based on the in vitro results suggests that the SSS-dependent regulation of channel kinetics accounts for nearly 40% of the decrease in Shaker current magnitude in flies lacking SSS. Sleep duration in qvr/sss null mutants is restored to normal by a qvr/sss transgene that fully rescues the Shaker kinetic phenotypes but only partially rescues the decrease in current magnitude. Together, these results suggest that the role of SSS in the regulation of sleep in Drosophila correlates more strongly with the effects of SSS on Shaker kinetics than current magnitude. PMID:21813698

Drosophila QVR/SSS modulates the activation and C-type inactivation kinetics of Shaker K(+) channels.

PubMed

Dean, Terry; Xu, Rong; Joiner, William; Sehgal, Amita; Hoshi, Toshinori

2011-08-03

The quiver/sleepless (qvr/sss) gene encodes a small, glycosylphosphatidylinositol-anchored protein that plays a critical role in the regulation of sleep in Drosophila. Loss-of-function mutations in qvr/sss severely suppress sleep and effect multiple changes in in situ Shaker K(+) currents, including decreased magnitude, slower time-to-peak, and cumulative inactivation. Recently, we demonstrated that SLEEPLESS (SSS) protein modulates Shaker channel activity, possibly through a direct interaction at the plasma membrane. We show here that SSS accelerates the activation of heterologously expressed Shaker channels with no effect on deactivation or fast N-type inactivation. Furthermore, this SSS-induced acceleration is sensitive to the pharmacological disruption of lipid rafts and sufficiently accounts for the slower time-to-peak of in situ Shaker currents seen in qvr/sss mutants. We also find that SSS decreases the rate of C-type inactivation of heterologously expressed Shaker channels, providing a potential mechanism for the cumulative inactivation phenotype induced by qvr/sss loss-of-function mutations. Kinetic modeling based on the in vitro results suggests that the SSS-dependent regulation of channel kinetics accounts for nearly 40% of the decrease in Shaker current magnitude in flies lacking SSS. Sleep duration in qvr/sss-null mutants is restored to normal by a qvr/sss transgene that fully rescues the Shaker kinetic phenotypes but only partially rescues the decrease in current magnitude. Together, these results suggest that the role of SSS in the regulation of sleep in Drosophila correlates more strongly with the effects of SSS on Shaker kinetics than current magnitude.

Differential regulation of ASICs and TRPV1 by zinc in rat bronchopulmonary sensory neurons.

PubMed

Vysotskaya, Zhanna V; Moss, Charles R; Gu, Qihai

2014-12-01

Zinc has been known to act as a signaling molecule that regulates a variety of neuronal functions. In this study, we aimed to study the effect of zinc on two populations of acid-sensitive ion channels, acid-sensing ion channels (ASICs), and transient receptor potential vanilloid receptor-1 (TRPV1), in vagal bronchopulmonary sensory neurons. Rat vagal sensory neurons innervating lungs and airways were retrogradely labeled with a fluorescent tracer. Whole-cell perforated patch-clamp recordings were carried out in primarily cultured bronchopulmonary sensory neurons. The acid-evoked ASIC and TRPV1 currents were measured and compared between before and after the zinc pretreatment. ASIC currents were induced by a pH drop from 7.4 to 6.8 or 6.5 in the presence of capsazepine (10 µM), a specific TRPV1 antagonist. Pretreatment with zinc (50 or 300 µM, 2 min) displayed different effects on the two distinct phenotypes of ASIC currents: a marked potentiation on ASIC channels with fast kinetics of activation and inactivation or no significant effect on ASIC currents with slow activation and inactivation. On the other hand, pretreatment with zinc significantly inhibited the acid (pH 5.5 or 5.3)-induced TRPV1 currents. The inhibition was abolished by intracellular chelation of zinc by TPEN (25 µM), indicating that intracellular accumulation of zinc was likely required for its inhibitory effect on TRPV1 channels. Our study showed that zinc differentially regulates the activities of ASICs and TRPV1 channels in rat vagal bronchopulmonary sensory neurons.

Nicotinamide-rich diet improves physical endurance by up-regulating SUR2A in the heart

PubMed Central

Sukhodub, Andriy; Sudhir, Rajni; Du, Qingyou; Jovanović, Sofija; Reyes, Santiago; Jovanović, Aleksandar

2011-01-01

Abstract SUR2A is an ATP-binding protein that serves as a regulatory subunit of cardioprotective ATP-sensitive K+ (KATP) channels. Based on signalling pathway regulating SUR2A expression and SUR2A role in regulating numbers of fully assembled KATP channels, we have suggested that nicotinamide-rich diet could improve physical endurance by stimulating SUR2A expression. We have found that mice on nicotinamide-rich diet significantly improved physical endurance, which was associated with significant increase in expression of SUR2A. Transgenic mice with solely overexpressed SUR2A on control diet had increased physical endurance in a similar manner as the wild-type mice on nicotinamide-rich diet. The experiments focused on action membrane potential and intracellular Ca2+ concentration have demonstrated that increased SUR2A expression was associated with the activation of sarcolemmal KATP channels and steady Ca2+ levels in cardiomyocytes in response to β-adrenergic stimulation. In contrast, the same challenge in the wild-type was characterized by a lack of the channel activation and rise in intracellular Ca2+. Nicotinamide-rich diet was ineffective to increase physical endurance in mice lacking KATP channels. This study has shown that nicotinamide-rich diet improves physical endurance by increasing expression of SUR2A and that this is a sole mechanism of the nicotinamide-rich diet effect. The obtained results suggest that oral nicotinamide is a regulator of SUR2A expression and has a potential as a drug that can improve physical endurance in conditions where this effect would be desirable. PMID:20731746

BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization.

PubMed

Semenov, Iurii; Wang, Bin; Herlihy, Jeremiah T; Brenner, Robert

2011-04-01

The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges.

BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization

PubMed Central

Semenov, Iurii; Wang, Bin; Herlihy, Jeremiah T; Brenner, Robert

2011-01-01

Abstract The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges. PMID:21300746

cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice*

PubMed Central

Ma, Hongwei; Butler, Michael R.; Thapa, Arjun; Belcher, Josh; Yang, Fan; Baehr, Wolfgang; Biel, Martin; Michalakis, Stylianos; Ding, Xi-Qin

2015-01-01

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca2+ channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP3R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3−/−/Nrl−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP3R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca2+ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency. PMID:26124274

Impaired Cell Volume Regulation in Intestinal Crypt Epithelia of Cystic Fibrosis Mice

NASA Astrophysics Data System (ADS)

Valverde, M. A.; O'Brien, J. A.; Sepulveda, F. V.; Ratcliff, R. A.; Evans, M. J.; Colledge, W. H.

1995-09-01

Cystic fibrosis is a disease characterized by abnormalities in the epithelia of the lungs, intestine, salivary and sweat glands, liver, and reproductive systems, often as a result of inadequate hydration of their secretions. The primary defect in cystic fibrosis is the altered activity of a cAMP-activated Cl^- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) channel. However, it is not clear how a defect in the CFTR Cl^- channel function leads to the observed pathological changes. Although much is known about the structural properties and regulation of the CFTR, little is known of its relationship to cellular functions other than the cAMP-dependent Cl^- secretion. Here we report that cell volume regulation after hypotonic challenge is also defective in intestinal crypt epithelial cells isolated from CFTR -/- mutant mice. Moreover, the impairment of the regulatory volume decrease in CFTR -/- crypts appears to be related to the inability of a K^+ conductance to provide a pathway for the exit of this cation during the volume adjustments. This provides evidence that the lack of CFTR protein may have additional consequences for the cellular function other than the abnormal cAMP-mediated Cl^- secretion.

Potassium Channels and Uterine Vascular Adaptation to Pregnancy and Chronic Hypoxia

PubMed Central

Zhu, Ronghui; Xiao, DaLiao; Zhang, Lubo

2014-01-01

During a normal course of pregnancy, uterine vascular tone is significantly decreased resulting in a striking increase in uterine blood flow, which is essential for fetal development and fetal growth. Chronic hypoxia during gestation may adversely affect the normal adaptation of uterine vascular tone and increase the risk of preeclampsia and fetal intrauterine growth restriction. In this review, we present evidence that the regulation of K+ channels is an important mechanism in the adaptation of uterine vascular tone to pregnancy and hypoxia. There are four types of K+ channels identified in arterial smooth muscle cells: 1) voltage-dependent K+ (Kv) channels, 2) Ca2+-activated K+ (KCa) channels, 3) inward rectifier K+ (KIR) channels, and 4) ATP-sensitive K+ (KATP) channels. Pregnancy differentially augments the expression and activity of K+ channels via downregulation of protein kinase C signaling in uterine and other vascular beds, leading to decreased uterine vascular tone and increased uterine blood flow. Sex steroid hormones play an important role in the pregnancy-mediated alteration of K+ channels in the uterine vasculature. In addition, chronic hypoxia alters uterine vascular K+ channels expression and activities via modulation of steroid hormones/receptors-mediated signaling, resulting in increased uterine vascular tone during pregnancy. PMID:24063385

A conserved threonine in the S1-S2 loop of KV7.2 and K V7.3 channels regulates voltage-dependent activation.

PubMed

Füll, Yvonne; Seebohm, Guiscard; Lerche, Holger; Maljevic, Snezana

2013-06-01

The voltage-gated potassium channels KV7.2 and KV7.3 (KCNQ2/3 genes) play an important role in regulating neuronal excitability. More than 50 KCNQ2/3 mutations have been identified to cause an inherited form of epilepsy in newborns. For two of those (E119G and S122L) found in the S1-S2 region of KV7.2, we previously showed a decreased channel availability mainly at action potential subthreshold voltages caused by a slight depolarizing shift of the activation curve. Interestingly, recent studies revealed that a threonine residue within the S1-S2 loop, highly conserved among different classes of KV channels, is crucial for both their function and surface expression. To investigate the functional role of the homologous threonine residues in KV7.2 (T114) and KV7.3 (T144) channels, we replaced them with alanine and examined the electrophysiological properties using heterologous expression in CHO cells and whole cell patch clamping. Channels comprising mutant subunits yielded decreased potassium currents with slowed activation and accelerated deactivation kinetics. However, the most striking effect was a depolarizing shift in the voltage dependence of activation reaching +30 mV upon co-expression of both mutant subunits. Potential interactions of T114 within the channel were analyzed by creating a 3D homology model of KV7.2 in an open state suggesting that this residue plays a central role in the formation of a stable interface between the S1-S2 and the S5 segment helices. This could be the explanation why substitution of the conserved threonine in KV7.2 and KV7.3 channels destabilizes the open and favors the closed state of these channels.

Selectivity of prandial glucose regulators: nateglinide, but not repaglinide, accelerates exocytosis in rat pancreatic A-cells.

PubMed

Bokvist, K; Hoy, M; Buschard, K; Holst, J J; Thomsen, M K; Gromada, J

1999-12-10

The effects of the two prandial glucose regulators, repaglinide and nateglinide, on ATP-sensitive K(+) (K(ATP)) channel activity, membrane potential and exocytosis in single rat pancreatic A-cells were investigated using the patch-clamp technique. K(ATP) channel activity was reversibly blocked by repaglinide (K(d)=22 nM) and nateglinide (K(d)=410 nM) and this was associated with membrane depolarisation and initiation of electrical activity. The effect of repaglinide and nateglinide on stimulation of glucagon secretion by direct interference with the exocytotic machinery was investigated by the use of capacitance measurements. Nateglinide, but not repaglinide, at concentrations similar to those required to block K(ATP) channels potentiated Ca(2+)-evoked exocytosis 3-fold. In alphaTC1-9 glucagonoma cells addition of nateglinide, but not repaglinide, was associated with stimulation of glucagon secretion. These results indicate that the fast-acting insulin secretagogue nateglinide is glucagonotropic primarily by stimulating Ca(2+)-dependent exocytosis.

Lipid microdomains and the regulation of ion channel function

PubMed Central

Dart, Caroline

2010-01-01

Many types of ion channel localize to cholesterol and sphingolipid-enriched regions of the plasma membrane known as lipid microdomains or ‘rafts’. The precise physiological role of these unique lipid microenvironments remains elusive due largely to difficulties associated with studying these potentially extremely small and dynamic domains. Nevertheless, increasing evidence suggests that membrane rafts regulate channel function in a number of different ways. Raft-enriched lipids such as cholesterol and sphingolipids exert effects on channel activity either through direct protein–lipid interactions or by influencing the physical properties of the bilayer. Rafts also appear to selectively recruit interacting signalling molecules to generate subcellular compartments that may be important for efficient and selective signal transduction. Direct interaction with raft-associated scaffold proteins such as caveolin can also influence channel function by altering gating kinetics or by affecting trafficking and surface expression. Selective association of ion channels with specific lipid microenvironments within the membrane is thus likely to be an important and fundamental regulatory aspect of channel physiology. This brief review highlights some of the existing evidence for raft modulation of channel function. PMID:20519314

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moczydlowski, Edward G.

Ion channel proteins regulate complex patterns of cellular electrical activity and ionic signaling. Certain K+ channels play an important role in immunological biodefense mechanisms of adaptive and innate immunity. Most ion channel proteins are oligomeric complexes with the conductive pore located at the central subunit interface. The long-term activity of many K+ channel proteins is dependent on the concentration of extracellular K+; however, the mechanism is unclear. Thus, this project focused on mechanisms underlying structural stability of tetrameric K+ channels. Using KcsA of Streptomyces lividans as a model K+ channel of known structure, the molecular basis of tetramer stability wasmore » investigated by: 1. Bioinformatic analysis of the tetramer interface. 2. Effect of two local anesthetics (lidocaine, tetracaine) on tetramer stability. 3. Molecular simulation of drug docking to the ion conduction pore. The results provide new insights regarding the structural stability of K+ channels and its possible role in cell physiology.« less

Phospholipase C δ4 regulates cold sensitivity in mice.

PubMed

Yudin, Yevgen; Lutz, Brianna; Tao, Yuan-Xiang; Rohacs, Tibor

2016-07-01

The cold- and menthol-activated transient receptor potential melastatin 8 (TRPM8) channels are thought to be regulated by phospholipase C (PLC), but neither the specific PLC isoform nor the in vivo relevance of this regulation has been established. Here we identify PLCδ4 as the key PLC isoform involved in regulation of TRPM8 channels in vivo. We show that in small PLCδ4(-/-) TRPM8-positive dorsal root ganglion neurons cold, menthol and WS-12, a selective TRPM8 agonist, evoked significantly larger currents than in wild-type neurons, and action potential frequencies induced by menthol or by current injections were also higher in PLCδ4(-/-) neurons. PLCδ4(-/-) mice showed increased behavioural responses to evaporative cooling, and this effect was inhibited by a TRPM8 antagonist; behavioural responses to heat and mechanical stimuli were not altered. We provide evidence for the involvement of a specific PLC isoform in the regulation of cold sensitivity in mice by regulating TRPM8 activity. The transient receptor potential melastatin 8 (TRPM8) ion channel is a major sensor of environmental low temperatures. Ca(2+) -induced activation of phospholipase C (PLC) has been implied in the regulation of TRPM8 channels during menthol- and cold-induced desensitization in vitro. Here we identify PLCδ4 as the key PLC isoform involved in regulation of TRPM8 in sensory dorsal root ganglion (DRG) neurons. We identified two TRPM8-positive neuronal subpopulations, based on their cell body size. Most TRPM8-positive small neurons also responded to capsaicin, and had significantly larger menthol-induced inward current densities than medium-large cells, most of which did not respond to capsaicin. Small, but not medium-large, PLCδ4(-/-) neurons showed significantly larger currents induced by cold, menthol or WS-12, a specific TRPM8 agonist, compared to wild-type (WT) neurons, but TRPM8 protein levels were not different between the two groups. In current-clamp experiments small neurons had more depolarized resting membrane potentials, and required smaller current injections to generate action potentials (APs) than medium-large cells. In small PLCδ4(-/-) neurons, menthol application induced larger depolarizations and generation of APs with frequencies significantly higher compared to WT neurons. In behavioural experiments PLCδ4(-/-) mice showed greater sensitivity to evaporative cooling by acetone than control animals. Pretreatment with the TRPM8 antagonist PBMC reduced cold-induced responses, and the effect was more pronounced in the PLCδ4(-/-) group. Heat and mechanical sensitivity of the PLCδ4(-/-) mice was not different from WT animals. Our data support the involvement of PLCδ4 in the regulation of TRPM8 channel activity in vivo. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Basally activated nonselective cation currents regulate the resting membrane potential in human and monkey colonic smooth muscle

PubMed Central

Dwyer, Laura; Rhee, Poong-Lyul; Lowe, Vanessa; Zheng, Haifeng; Peri, Lauren; Ro, Seungil; Sanders, Kenton M.

2011-01-01

Resting membrane potential (RMP) plays an important role in determining the basal excitability of gastrointestinal smooth muscle. The RMP in colonic muscles is significantly less negative than the equilibrium potential of K+, suggesting that it is regulated not only by K+ conductances but by inward conductances such as Na+ and/or Ca2+. We investigated the contribution of nonselective cation channels (NSCC) to the RMP in human and monkey colonic smooth muscle cells (SMC) using voltage- and current-clamp techniques. Qualitative reverse transcriptase-polymerase chain reaction was performed to examine potential molecular candidates for these channels among the transient receptor potential (TRP) channel superfamily. Spontaneous transient inward currents and holding currents were recorded in human and monkey SMC. Replacement of extracellular Na+ with equimolar tetraethylammonium or Ca2+ with Mn2+ inhibited basally activated nonselective cation currents. Trivalent cations inhibited these channels. Under current clamp, replacement of extracellular Na+ with N-methyl-d-glucamine or addition of trivalent cations caused hyperpolarization. Three unitary conductances of NSCC were observed in human and monkey colonic SMC. Molecular candidates for basally active NSCC were TRPC1, C3, C4, C7, M2, M4, M6, M7, V1, and V2 in human and monkey SMC. Comparison of the biophysical properties of these TRP channels with basally active NSCC (bINSCC) suggests that TRPM4 and specific TRPC heteromultimer combinations may underlie the three single-channel conductances of bINSCC. In conclusion, these findings suggest that basally activated NSCC contribute to the RMP in human and monkey colonic SMC and therefore may play an important role in determining basal excitability of colonic smooth muscle. PMID:21566016

Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells.

PubMed

Yuan, X J; Wang, J; Juhaszova, M; Golovina, V A; Rubin, L J

1998-04-01

K(+)-channel activity-mediated alteration of the membrane potential and cytoplasmic free Ca2+ concentration ([Ca2+]cyt) is a pivotal mechanism in controlling pulmonary vasomotor tone. By using combined approaches of patch clamp, imaging fluorescent microscopy, and molecular biology, we examined the electrophysiological properties of K+ channels and the role of different K+ currents in regulating [Ca2+]cyt and explored the molecular identification of voltage-gated K+ (KV)- and Ca(2+)-activated K+ (KCa)-channel genes expressed in pulmonary arterial smooth muscle cells (PASMC). Two kinetically distinct KV currents [IK(V)], a rapidly inactivating (A-type) and a noninactivating delayed rectifier, as well as a slowly activated KCa current [IK(Ca)] were identified. IK(V) was reversibly inhibited by 4-aminopyridine (5 mM), whereas IK(Ca) was significantly inhibited by charybdotoxin (10-20 nM). K+ channels are composed of pore-forming alpha-subunits and auxiliary beta-subunits. Five KV-channel alpha-subunit genes from the Shaker subfamily (KV1.1, KV1.2, KV1.4, KV1.5, and KV1.6), a KV-channel alpha-subunit gene from the Shab subfamily (KV2.1), a KV-channel modulatory alpha-subunit (KV9.3), and a KCa-channel alpha-subunit gene (rSlo), as well as three KV-channel beta-subunit genes (KV beta 1.1, KV beta 2, and KV beta 3) are expressed in PASMC. The data suggest that 1) native K+ channels in PASMC are encoded by multiple genes; 2) the delayed rectifier IK(V) may be generated by the KV1.1, KV1.2, KV1.5, KV1.6, KV2.1, and/or KV2.1/KV9.3 channels; 3) the A-type IK(V) may be generated by the KV1.4 channel and/or the delayed rectifier KV channels (KV1 subfamily) associated with beta-subunits; and 4) the IK(Ca) may be generated by the rSlo gene product. The function of the KV channels plays an important role in the regulation of membrane potential and [Ca2+]cyt in PASMC.

Allosteric mechanism of water channel gating by Ca2+–calmodulin

PubMed Central

Reichow, Steve L.; Clemens, Daniel M.; Freites, J. Alfredo; Németh-Cahalan, Karin L.; Heyden, Matthias; Tobias, Douglas J.; Hall, James E.; Gonen, Tamir

2013-01-01

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits. PMID:23893133

How do voltage-gated sodium channels enhance migration and invasiveness in cancer cells?

PubMed

Besson, Pierre; Driffort, Virginie; Bon, Émeline; Gradek, Frédéric; Chevalier, Stéphan; Roger, Sébastien

2015-10-01

Voltage-gated sodium channels are abnormally expressed in tumors, often as neonatal isoforms, while they are not expressed, or only at a low level, in the matching normal tissue. The level of their expression and their activity is related to the aggressiveness of the disease and to the formation of metastases. A vast knowledge on the regulation of their expression and functioning has been accumulated in normal excitable cells. This helped understand their regulation in cancer cells. However, how voltage-gated sodium channels impose a pro-metastatic behavior to cancer cells is much less documented. This aspect will be addressed in the review. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers. Copyright © 2015 Elsevier B.V. All rights reserved.

TPC Proteins Are Phosphoinositide-activated Sodium-selective Ion Channels in Endosomes and Lysosomes

PubMed Central

Wang, Xiang; Zhang, Xiaoli; Dong, Xian-ping; Samie, Mohammad; Li, Xinran; Cheng, Xiping; Goschka, Andrew; Shen, Dongbiao; Zhou, Yandong; Harlow, Janice; Zhu, Michael X.; Clapham, David E.; Ren, Dejian; Xu, Haoxing

2012-01-01

Summary Mammalian Two-Pore Channels (TPC1, 2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P2, and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na+, not K+, as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes, and may explain the specificity of PI(3,5)P2 in regulating the fusogenic potential of intracellular organelles. PMID:23063126

Glucokinase activity in the arcuate nucleus regulates glucose intake

PubMed Central

Hussain, Syed; Richardson, Errol; Ma, Yue; Holton, Christopher; De Backer, Ivan; Buckley, Niki; Dhillo, Waljit; Bewick, Gavin; Zhang, Shuai; Carling, David; Bloom, Steve; Gardiner, James

2014-01-01

The brain relies on a constant supply of glucose, its primary fuel, for optimal function. A taste-independent mechanism within the CNS that promotes glucose delivery to the brain has been postulated to maintain glucose homeostasis; however, evidence for such a mechanism is lacking. Here, we determined that glucokinase activity within the hypothalamic arcuate nucleus is involved in regulation of dietary glucose intake. In fasted rats, glucokinase activity was specifically increased in the arcuate nucleus but not other regions of the hypothalamus. Moreover, pharmacologic and genetic activation of glucokinase in the arcuate nucleus of rodent models increased glucose ingestion, while decreased arcuate nucleus glucokinase activity reduced glucose intake. Pharmacologic targeting of potential downstream glucokinase effectors revealed that ATP-sensitive potassium channel and P/Q calcium channel activity are required for glucokinase-mediated glucose intake. Additionally, altered glucokinase activity affected release of the orexigenic neurotransmitter neuropeptide Y in response to glucose. Together, our results suggest that glucokinase activity in the arcuate nucleus specifically regulates glucose intake and that appetite for glucose is an important driver of overall food intake. Arcuate nucleus glucokinase activation may represent a CNS mechanism that underlies the oft-described phenomena of the “sweet tooth” and carbohydrate craving. PMID:25485685

Regulation of surface expression of TRPV2 channels in the retinal pigment epithelium.

PubMed

Reichhart, Nadine; Keckeis, Susanne; Fried, Frederik; Fels, Gabriele; Strauss, Olaf

2015-06-01

The retinal pigment epithelium (RPE) interacts closely with the photoreceptors in fulfilling tasks of visual function. Since an understanding of the RPE function is essential for understanding the patho-mechanisms involved in vision loss, we explored the regulation of the vanilloid receptor subtype transient receptor potential TRPV2 channels that trigger insulin-like growth factor-1 (IGF-1)-induced vascular endothelial growth factor A (VEGF-A) secretion. Immunohistochemistry was used to assess TRPV2 expression in retinal cross-sections or ARPE-19 cells, and surface expression of TRPV2 was quantified using confocal microscopy. Membrane currents of ARPE-19 cells were recorded using a whole-cell configuration of the patch-clamp technique. TRPV2 expression was detected in the RPE of the mouse retina as well as in ARPE-19 cells. Increasing the temperature to 45 °C activated membrane conductance sensitive to SKF-96365 and ruthenium red in 60 % of cells. Preincubation with either cannabidiol (CBD) or IGF-1 led to a three- or fourfold increase in current density, respectively, in all cells, which was blocked by SKF-96365. In contrast to IGF-1, CBD stimulation of membrane conductance was further increased by heat. TRPV2 surface expression was increased by both IGF-1 and CBD, with the increase by CBD twice as large as that by IGF-1. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished the effects on membrane conductance and surface expression. Both CBD and IGF-1 enhance TRPV2 channel activity by specific proportions of both channel activation and PI 3-kinase-dependent surface expression: IGF-1 predominantly increases ion channel activity, whereas CBD is more active in increasing TRPV2 surface expression. Thus, differential regulation of TRPV2 surface expression is an important mechanism for modulating the responsiveness of the RPE to growth factors.

Mechanism of the modulation of Kv4:KChIP-1 channels by external K+.

PubMed

Kaulin, Yu A; De Santiago-Castillo, J A; Rocha, C A; Covarrubias, M

2008-02-15

In response to a prolonged membrane depolarization, inactivation autoregulates the activity of voltage-gated ion channels. Slow inactivation involving a localized constriction of the selectivity filter (P/C-type mechanism) is prevalent in many voltage-gated K(+) channels of the Kv1 subfamily. However, the generalization of this mechanism to other Kv channel subfamilies has remained uncertain and controversial. In agreement with a "foot-in-the-door" mechanism and the presence of ion-ion interactions in the pore, elevated external K(+) slows the development of P/C-type inactivation and accelerates its recovery. In sharp contrast and resembling the regulation of the hippocampal A-type K(+) current, we found that Kv4.x channels associated with KChIP-1 (an auxiliary subunit) exhibit accelerated inactivation and unaffected recovery from inactivation when exposed to elevated external K(+). This regulation depends on the ability of a permeant ion to enter the selectivity filter (K(+) = Rb(+) = NH4(+) > Cs(+) > Na(+)); and the apparent equilibrium dissociation constant of a single regulatory site is 8 mM for K(+). By applying a robust quantitative global kinetic modeling approach to all macroscopic properties over a 210-mV range of membrane potentials, we determined that elevated external K(+) inhibits unstable closed states outside the main activation pathway and thereby promotes preferential closed-state inactivation. These results suggest the presence of a vestigial and unstable P/C-type mechanism of inactivation in Kv4 channels and strengthen the concept of novel mechanisms of closed-state inactivation. Regulation of Kv4 channel inactivation by hyperkalemia may help to explain the pathophysiology of electrolyte imbalances in excitable tissues.

Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase.

PubMed

Shyng, S L; Barbieri, A; Gumusboga, A; Cukras, C; Pike, L; Davis, J N; Stahl, P D; Nichols, C G

2000-01-18

ATP-sensitive potassium channels (K(ATP) channels) regulate cell excitability in response to metabolic changes. K(ATP) channels are formed as a complex of a sulfonylurea receptor (SURx), a member of the ATP-binding cassette protein family, and an inward rectifier K(+) channel subunit (Kir6.x). Membrane phospholipids, in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), activate K(ATP) channels and antagonize ATP inhibition of K(ATP) channels when applied to inside-out membrane patches. To examine the physiological relevance of this regulatory mechanism, we manipulated membrane PIP(2) levels by expressing either the wild-type or an inactive form of PI-4-phosphate 5-kinase (PIP5K) in COSm6 cells and examined the ATP sensitivity of coexpressed K(ATP) channels. Channels from cells expressing the wild-type PIP5K have a 6-fold lower ATP sensitivity (K(1/2), the half maximal inhibitory concentration, approximately 60 microM) than the sensitivities from control cells (K(1/2) approximately 10 microM). An inactive form of the PIP5K had little effect on the K(1/2) of wild-type channels but increased the ATP-sensitivity of a mutant K(ATP) channel that has an intrinsically lower ATP sensitivity (from K(1/2) approximately 450 microM to K(1/2) approximately 100 microM), suggesting a decrease in membrane PIP(2) levels as a consequence of a dominant-negative effect of the inactive PIP5K. These results show that PIP5K activity, which regulates PIP(2) and PI-3,4,5-P(3) levels, is a significant determinant of the physiological nucleotide sensitivity of K(ATP) channels.

Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase

PubMed Central

Shyng, S.-L.; Barbieri, A.; Gumusboga, A.; Cukras, C.; Pike, L.; Davis, J. N.; Stahl, P. D.; Nichols, C. G.

2000-01-01

ATP-sensitive potassium channels (KATP channels) regulate cell excitability in response to metabolic changes. KATP channels are formed as a complex of a sulfonylurea receptor (SURx), a member of the ATP-binding cassette protein family, and an inward rectifier K+ channel subunit (Kir6.x). Membrane phospholipids, in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP2), activate KATP channels and antagonize ATP inhibition of KATP channels when applied to inside-out membrane patches. To examine the physiological relevance of this regulatory mechanism, we manipulated membrane PIP2 levels by expressing either the wild-type or an inactive form of PI-4-phosphate 5-kinase (PIP5K) in COSm6 cells and examined the ATP sensitivity of coexpressed KATP channels. Channels from cells expressing the wild-type PIP5K have a 6-fold lower ATP sensitivity (K1/2, the half maximal inhibitory concentration, ≈ 60 μM) than the sensitivities from control cells (K1/2 ≈ 10 μM). An inactive form of the PIP5K had little effect on the K1/2 of wild-type channels but increased the ATP-sensitivity of a mutant KATP channel that has an intrinsically lower ATP sensitivity (from K1/2 ≈ 450 μM to K1/2 ≈ 100 μM), suggesting a decrease in membrane PIP2 levels as a consequence of a dominant-negative effect of the inactive PIP5K. These results show that PIP5K activity, which regulates PIP2 and PI-3,4,5-P3 levels, is a significant determinant of the physiological nucleotide sensitivity of KATP channels. PMID:10639183

Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling

PubMed Central

Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

2015-01-01

Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system. PMID:26370138

Serotonin 2C receptor activates a distinct population of arcuate pro-opiomelanocortin neurons via TRPC channels

USDA-ARS?s Scientific Manuscript database

Serotonin 2C receptors (5-HT2CRs) expressed by pro-opiomelanocortin (POMC) neurons of hypothalamic arcuate nucleus regulate food intake, energy homeostasis ,and glucose metabolism. However, the cellular mechanisms underlying the effects of 5-HT to regulate POMC neuronal activity via 5-HT2CRs have no...

Angiotensin II Inhibits the ROMK-like Small Conductance K Channel in Renal Cortical Collecting Duct during Dietary Potassium Restriction*

PubMed Central

Wei, Yuan; Zavilowitz, Beth; Satlin, Lisa M.; Wang, Wen-Hui

2010-01-01

Base-line urinary potassium secretion in the distal nephron is mediated by small conductance rat outer medullary K (ROMK)-like channels. We used the patch clamp technique applied to split-open cortical collecting ducts (CCDs) isolated from rats fed a normal potassium (NK) or low potassium (LK) diet to test the hypothesis that AngII directly inhibits ROMK channel activity. We found that AngII inhibited ROMK channel activity in LK but not NK rats in a dose-dependent manner. The AngII-induced reduction in channel activity was mediated by AT1 receptor (AT1R) binding, because pretreatment of CCDs with losartan but not PD123319 AT1 and AT2 receptor antagonists, respectively, blocked the response. Pretreatment of CCDs with U73122 and calphostin C, inhibitors of phospholipase C (PLC) and protein kinase C (PKC), respectively, abolished the AngII-induced decrease in ROMK channel activity, confirming a role of the PLC-PKC pathway in this response. Studies by others suggest that AngII stimulates an Src family protein-tyrosine kinase (PTK) via PKC-NADPH oxidase. PTK has been shown to regulate the ROMK channel. Inhibition of NADPH oxidase with diphenyliodonium abolished the inhibitory effect of AngII or the PKC activator phorbol 12-myristate 13-acetate on ROMK channels. Suppression of PTK by herbimycin A significantly attenuated the inhibitory effect of AngII on ROMK channel activity. We conclude that AngII inhibits ROMK channel activity through PKC-, NADPH oxidase-, and PTK-dependent pathways under conditions of dietary potassium restriction. PMID:17194699

Angiotensin II inhibits the ROMK-like small conductance K channel in renal cortical collecting duct during dietary potassium restriction.

PubMed

Wei, Yuan; Zavilowitz, Beth; Satlin, Lisa M; Wang, Wen-Hui

2007-03-02

Base-line urinary potassium secretion in the distal nephron is mediated by small conductance rat outer medullary K (ROMK)-like channels. We used the patch clamp technique applied to split-open cortical collecting ducts (CCDs) isolated from rats fed a normal potassium (NK) or low potassium (LK) diet to test the hypothesis that AngII directly inhibits ROMK channel activity. We found that AngII inhibited ROMK channel activity in LK but not NK rats in a dose-dependent manner. The AngII-induced reduction in channel activity was mediated by AT1 receptor (AT1R) binding, because pretreatment of CCDs with losartan but not PD123319 AT1 and AT2 receptor antagonists, respectively, blocked the response. Pretreatment of CCDs with U73122 and calphostin C, inhibitors of phospholipase C (PLC) and protein kinase C (PKC), respectively, abolished the AngII-induced decrease in ROMK channel activity, confirming a role of the PLC-PKC pathway in this response. Studies by others suggest that AngII stimulates an Src family protein-tyrosine kinase (PTK) via PKC-NADPH oxidase. PTK has been shown to regulate the ROMK channel. Inhibition of NADPH oxidase with diphenyliodonium abolished the inhibitory effect of AngII or the PKC activator phorbol 12-myristate 13-acetate on ROMK channels. Suppression of PTK by herbimycin A significantly attenuated the inhibitory effect of AngII on ROMK channel activity. We conclude that AngII inhibits ROMK channel activity through PKC-, NADPH oxidase-, and PTK-dependent pathways under conditions of dietary potassium restriction.

Disruption of CFTR chloride channel alters mechanical properties and cAMP-dependent Cl− transport of mouse aortic smooth muscle cells

PubMed Central

Robert, Renaud; Norez, Caroline; Becq, Frédéric

2005-01-01

Chloride (Cl−) channels expressed in vascular smooth muscle cells (VSMC) are important to control membrane potential equilibrium, intracellular pH, cell volume maintenance, contraction, relaxation and proliferation. The present study was designed to compare the expression, regulation and function of CFTR Cl− channels in aortic VSMC from Cftr+/+ and Cftr−/− mice. Using an iodide efflux assay we demonstrated stimulation of CFTR by VIP, isoproterenol, cAMP agonists and other pharmacological activators in cultured VSMC from Cftr+/+. On the contrary, in cultured VSMC from Cftr−/− mice these agonists have no effect, showing that CFTR is the dominant Cl− channel involved in the response to cAMP mediators. Angiotensin II and the calcium ionophore A23187 stimulated Ca2+-dependent Cl− channels in VSMCs from both genotypes. CFTR was activated in myocytes maintained in medium containing either high potassium or 5-hydroxytryptamine (5-HT) and was inhibited by CFTRinh-172, glibenclamide and diphenylamine-2,2′-dicarboxylic acid (DPC). We also examined the mechanical properties of aortas. Arteries with or without endothelium from Cftr−/− mice became significantly more constricted (∼2-fold) than that of Cftr+/+ mice in response to vasoactive agents. Moreover, in precontracted arteries of Cftr+/+ mice, VIP and CFTR activators induced vasorelaxation that was altered in Cftr−/− mice. Our findings suggest a novel mechanism for regulation of the vascular tone by cAMP-dependent CFTR chloride channels in VSMC. To our knowledge this study is the first to report the phenotypic consequences of the loss of a Cl− channel on vascular reactivity. PMID:16081479

Regulation of neuronal excitability by interaction of fragile X mental retardation protein with slack potassium channels.

PubMed

Zhang, Yalan; Brown, Maile R; Hyland, Callen; Chen, Yi; Kronengold, Jack; Fleming, Matthew R; Kohn, Andrea B; Moroz, Leonid L; Kaczmarek, Leonard K

2012-10-31

Loss of the RNA-binding protein fragile X mental retardation protein (FMRP) represents the most common form of inherited intellectual disability. Studies with heterologous expression systems indicate that FMRP interacts directly with Slack Na(+)-activated K(+) channels (K(Na)), producing an enhancement of channel activity. We have now used Aplysia bag cell (BC) neurons, which regulate reproductive behaviors, to examine the effects of Slack and FMRP on excitability. FMRP and Slack immunoreactivity were colocalized at the periphery of isolated BC neurons, and the two proteins could be reciprocally coimmunoprecipitated. Intracellular injection of FMRP lacking its mRNA binding domain rapidly induced a biphasic outward current, with an early transient tetrodotoxin-sensitive component followed by a slowly activating sustained component. The properties of this current matched that of the native Slack potassium current, which was identified using an siRNA approach. Addition of FMRP to inside-out patches containing native Aplysia Slack channels increased channel opening and, in current-clamp recordings, produced narrowing of action potentials. Suppression of Slack expression did not alter the ability of BC neurons to undergo a characteristic prolonged discharge in response to synaptic stimulation, but prevented recovery from a prolonged inhibitory period that normally follows the discharge. Recovery from the inhibited period was also inhibited by the protein synthesis inhibitor anisomycin. Our studies indicate that, in BC neurons, Slack channels are required for prolonged changes in neuronal excitability that require new protein synthesis, and raise the possibility that channel-FMRP interactions may link changes in neuronal firing to changes in protein translation.

Rapid activity-dependent modulation of the intrinsic excitability through up-regulation of KCNQ/Kv7 channel function in neonatal spinal motoneurons.

PubMed

Lombardo, Joseph; Sun, Jianli; Harrington, Melissa A

2018-01-01

Activity-dependent changes in the properties of the motor system underlie the necessary adjustments in its responsiveness on the basis of the environmental and developmental demands of the organism. Although plastic changes in the properties of the spinal cord have historically been neglected because of the archaic belief that the spinal cord is constituted by a hardwired network that simply relays information to muscles, plenty of evidence has been accumulated showing that synapses impinging on spinal motoneurons undergo short- and long-term plasticity. In the brain, brief changes in the activity level of the network have been shown to be paralleled by changes in the intrinsic excitability of the neurons and are suggested to either reinforce or stabilize the changes at the synaptic level. However, rapid activity-dependent changes in the intrinsic properties of spinal motoneurons have never been reported. In this study, we show that in neonatal mice the intrinsic excitability of spinal motoneurons is depressed after relatively brief but sustained changes in the spinal cord network activity. Using electrophysiological techniques together with specific pharmacological blockers of KCNQ/Kv7 channels, we demonstrate their involvement in the reduction of the intrinsic excitability of spinal motoneurons. This action results from an increased M-current, the product of the activation of KCNQ/Kv7 channels, which leads to a hyperpolarization of the resting membrane potential and a decrease in the input resistance of spinal motoneurons. Computer simulations showed that specific up-regulations in KCNQ/Kv7 channels functions lead to a modulation of the intrinsic excitability of spinal motoneurons as observed experimentally. These results indicate that KCNQ/Kv7 channels play a fundamental role in the activity-dependent modulation of the excitability of spinal motoneurons.

Identification of the functional binding pocket for compounds targeting small-conductance Ca2+-activated potassium channels

PubMed Central

Zhang, Miao; Pascal, John M.; Schumann, Marcel; Armen, Roger S.; Zhang, Ji-fang

2012-01-01

Small- and intermediate-conductance Ca2+-activated potassium channels, activated by Ca2+-bound calmodulin, play an important role in regulating membrane excitability. These channels are also linked to clinical abnormalities. A tremendous amount of effort has been devoted to developing small molecule compounds targeting these channels. However, these compounds often suffer from low potency and lack of selectivity, hindering their potentials for clinical use. A key contributing factor is the lack of knowledge of the binding site(s) for these compounds. Here we demonstrate by X-ray crystallography that the binding pocket for the compounds of the 1-EBIO class is located at the calmodulin-channel interface. We show that, based on structure data and molecular docking, mutations of the channel can effectively change the potency of these compounds. Our results provide insight into the molecular nature of the binding pocket and its contribution to the potency and selectivity of the compounds of the 1-EBIO class. PMID:22929778

Identification of the functional binding pocket for compounds targeting small-conductance Ca²⁺-activated potassium channels.

PubMed

Zhang, Miao; Pascal, John M; Schumann, Marcel; Armen, Roger S; Zhang, Ji-Fang

2012-01-01

Small- and intermediate-conductance Ca(2+)-activated potassium channels, activated by Ca(2+)-bound calmodulin, have an important role in regulating membrane excitability. These channels are also linked to clinical abnormalities. A tremendous amount of effort has been devoted to developing small molecule compounds targeting these channels. However, these compounds often suffer from low potency and lack of selectivity, hindering their potential for clinical use. A key contributing factor is the lack of knowledge of the binding site(s) for these compounds. Here we demonstrate by X-ray crystallography that the binding pocket for the compounds of the 1-ethyl-2-benzimidazolinone (1-EBIO) class is located at the calmodulin-channel interface. We show that, based on structure data and molecular docking, mutations of the channel can effectively change the potency of these compounds. Our results provide insight into the molecular nature of the binding pocket and its contribution to the potency and selectivity of the compounds of the 1-EBIO class.

Calcium-activated potassium channels in basolateral membranes of colon epithelial cells; reconstitution and functional properties.

PubMed

Wiener, H; Turnheim, K

1990-10-26

Using differential sedimentation, isopycnic and Ficoll-400 barrier centrifugation, basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon were enriched 34- and 9-fold, respectively. 86Rb(+)-uptake into these vesicles, driven by an electrical potential difference, was stimulated by submicromolar Ca2+ activities and inhibited by Ba2+. These findings indicate the presence of Ca2(+)-activated K+ channels. The K+ channels in surface and crypt cell membranes differed with respect to inhibition by the bee venom apamin, the scorpion venom charybdotoxin and tetraethylammonium and exhibited a different pH dependence. Fusion of basolateral membrane vesicles with planar phospholipid bilayers revealed the presence of high-conductance Ba2(+)-sensitive K+ channels which were activated by micromolar Ca2+ and inhibited by crude scorpion venom and trifluoperazine. These K+ channels may be involved in the coupling of apical and basolateral membrane conductances during Na+ absorption and Cl- secretion, but they may also play a role in cell volume regulation.

Guanylate cyclase-activating protein 2 contributes to phototransduction and light adaptation in mouse cone photoreceptors.

PubMed

Vinberg, Frans; Peshenko, Igor V; Chen, Jeannie; Dizhoor, Alexander M; Kefalov, Vladimir J

2018-05-11

Light adaptation of photoreceptor cells is mediated by Ca 2+ -dependent mechanisms. In darkness, Ca 2+ influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca 2+ extrusion via Na + /Ca 2+ , K + exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca 2+ levels in photoreceptor outer segment because of continuing Ca 2+ extrusion by NCKXs. Guanylate cyclase-activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca 2+ This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca 2+ -feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca 2+ Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca 2+ when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphatidic acid modulation of Kv channel voltage sensor function.

PubMed

Hite, Richard K; Butterwick, Joel A; MacKinnon, Roderick

2014-10-06

Membrane phospholipids can function as potent regulators of ion channel function. This study uncovers and investigates the effect of phosphatidic acid on Kv channel gating. Using the method of reconstitution into planar lipid bilayers, in which protein and lipid components are defined and controlled, we characterize two effects of phosphatidic acid. The first is a non-specific electrostatic influence on activation mediated by electric charge density on the extracellular and intracellular membrane surfaces. The second is specific to the presence of a primary phosphate group, acts only through the intracellular membrane leaflet and depends on the presence of a particular arginine residue in the voltage sensor. Intracellular phosphatidic acid accounts for a nearly 50 mV shift in the midpoint of the activation curve in a direction consistent with stabilization of the voltage sensor's closed conformation. These findings support a novel mechanism of voltage sensor regulation by the signaling lipid phosphatidic acid.

Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

PubMed Central

Parthier, Daniel; Frings, Stephan; Möhrlen, Frank

2015-01-01

Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum. PMID:26558388

S1-S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation.

PubMed

Groome, James R; Winston, Vern

2013-05-01

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1-S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1-S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I-III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.

Store-depletion and hyperforin activate distinct types of Ca(2+)-conducting channels in cortical neurons.

PubMed

Gibon, Julien; Tu, Peng; Bouron, Alexandre

2010-06-01

Cortical neurons embryos (E13) from murine brain have a wide diversity of plasma membrane Ca(2+)-conducting channels. For instance, they express several types of transient receptor potential channels of C-type (TRPC) and hyperforin, a potent TRPC6-channel activator, controls the activity of TRPC6-like channels. In addition, E13 cortical neurons possess plasma membrane channels activated in response to the depletion of internal Ca(2+) pools. Since some TRPC channels seem to be involved in the activity of store-depletion-activated channels, we investigated whether hyperforin and the depletion of the Ca(2+) stores control similar or distinct Ca(2+) routes. Calcium imaging experiments performed with the fluorescent Ca(2+) indicator Fluo-4 showed that the TRPC3 channel blocker Pyr3 potently inhibits with an IC(50) of 0.5microM the entry of Ca(2+) triggered in response to the thapsigargin-dependent depletion of the Ca(2+) stores. On the other hand, Pyr3 does not block the hyperforin-sensitive Ca(2+) entry. In contrast to the hyperforin responses, the Ca(2+) entry through the store-depletion-activated channels is down-regulated by the competitive tyrosine kinase inhibitors genistein and PP2. In addition, the immunosuppressant FK506, known to modulate several classes of Ca(2+)-conducting channels, strongly attenuates the entry of Ca(2+) through the store-depletion-activated channels, leaving the hyperforin-sensitive responses unaffected. Hence, the Zn(2+) chelator TPEN markedly attenuated the hyperforin-sensitive responses without modifying the thapsigargin-dependent Ca(2+) signals. Pyr3-insensitive channels are key components of the hyperforin-sensitive channels, whereas the thapsigargin-dependent depletion of the Ca(2+) stores of the endoplasmic reticulum activates Pyr3-sensitive channels. Altogether, these data support the notion that hyperforin and the depletion of the Ca(2+) pools control distinct plasma membrane Ca(2+)-conducting channels. This report further illustrates that, at the beginning of the corticogenesis, immature cortical neurons express diverse functional Ca(2+) channels. 2010 Elsevier Ltd. All rights reserved.

The role of an ancestral hyperpolarization-activated cyclic nucleotide-gated K+ channel in branchial acid-base regulation in the green crab, Carcinus maenas.

PubMed

Fehsenfeld, Sandra; Weihrauch, Dirk

2016-03-01

Numerous electrophysiological studies on branchial K(+) transport in brachyuran crabs have established an important role for potassium channels in osmoregulatory ion uptake and ammonia excretion in the gill epithelium of decapod crustaceans. However, hardly anything is known of the actual nature of these channels in crustaceans. In the present study, the identification of a hyperpolarization-activated cyclic nucleotide-gated potassium channel (HCN) in the transcriptome of the green crab Carcinus maenas and subsequent performance of quantitative real-time PCR revealed the ubiquitous expression of this channel in this species. Even though mRNA expression levels in the cerebral ganglion were found to be approximately 10 times higher compared with all other tissues, posterior gills still expressed significant levels of HCN, indicating an important role for this transporter in branchial ion regulation. The relatively unspecific K(+)-channel inhibitor Ba(2+), as well as the HCN-specific blocker ZD7288, as applied in gill perfusion experiments and electrophysiological studies employing the split gill lamellae revealed the presence of at least two different K(+)/NH4(+)-transporting structures in the branchial epithelium of C. maenas. Furthermore, HCN mRNA levels in posterior gill 7 decreased significantly in response to the respiratory or metabolic acidosis that was induced by acclimation of green crabs to high environmental PCO2 and ammonia, respectively. Consequently, the present study provides first evidence that HCN-promoted NH4(+) epithelial transport is involved in both branchial acid-base and ammonia regulation in an invertebrate. © 2016. Published by The Company of Biologists Ltd.

Nerve Growth Factor Regulates Transient Receptor Potential Vanilloid 2 via Extracellular Signal-Regulated Kinase Signaling To Enhance Neurite Outgrowth in Developing Neurons

PubMed Central

Cohen, Matthew R.; Johnson, William M.; Pilat, Jennifer M.; Kiselar, Janna; DeFrancesco-Lisowitz, Alicia; Zigmond, Richard E.

2015-01-01

Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca2+-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca2+-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca2+ signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca2+ signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function. PMID:26416880

Zn2+-dependent redox switch in the intracellular T1-T1 interface of a Kv channel.

PubMed

Wang, Guangyu; Strang, Candace; Pfaffinger, Paul J; Covarrubias, Manuel

2007-05-04

The thiol-based redox regulation of proteins plays a central role in cellular signaling. Here, we investigated the redox regulation at the Zn(2+) binding site (HX(5)CX(20)CC) in the intracellular T1-T1 inter-subunit interface of a Kv4 channel. This site undergoes conformational changes coupled to voltage-dependent gating, which may be sensitive to oxidative stress. The main results show that internally applied nitric oxide (NO) inhibits channel activity profoundly. This inhibition is reversed by reduced glutathione and suppressed by intracellular Zn(2+), and at least two Zn(2+) site cysteines are required to observe the NO-induced inhibition (Cys-110 from one subunit and Cys-132 from the neighboring subunit). Biochemical evidence suggests strongly that NO induces a disulfide bridge between Cys-110 and Cys-132 in intact cells. Finally, further mutational studies suggest that intra-subunit Zn(2+) coordination involving His-104, Cys-131, and Cys-132 protects against the formation of the inhibitory disulfide bond. We propose that the interfacial T1 Zn(2+) site of Kv4 channels acts as a Zn(2+)-dependent redox switch that may regulate the activity of neuronal and cardiac A-type K(+) currents under physiological and pathological conditions.

Ion channel-transporter interactions

PubMed Central

Neverisky, Daniel L.; Abbott, Geoffrey W.

2016-01-01

All living cells require membrane proteins that act as conduits for the regulated transport of ions, solutes and other small molecules across the cell membrane. Ion channels provide a pore that permits often rapid, highly selective, and tightly regulated movement of ions down their electrochemical gradient. In contrast, active transporters can move moieties up their electrochemical gradient. The secondary active transporters (such as SLC superfamily solute transporters) achieve this by coupling uphill movement of the substrate to downhill movement of another ion, such as sodium. The primary active transporters (including H+/K+-ATPases and Na+/K+-ATPases) utilize ATP hydrolysis as an energy source to power uphill transport. It is well known that proteins in each of these classes work in concert with members of the other classes to ensure, for example, ion homeostasis, ion secretion, and restoration of ion balance following action potentials. More recently, evidence is emerging of direct physical interaction between true ion channels, and some primary or secondary active transporters. Here, we review the first known members of this new class of macromolecular complexes that we term “chansporters”, explore their biological roles, and discuss the pathophysiological consequences of their disruption. We compare functional and/or physical interactions between the ubiquitous KCNQ1 potassium channel and various active transporters, and examine other newly discovered chansporter complexes that suggest we may be seeing the tip of the iceberg in a newly emerging signaling modality. PMID:27098917

Modeling the Impacts of Hydromodification (Conference paper)

EPA Science Inventory

Hydromodification is caused by anthropogenic activities driven by human population growth and resource consumption that alter watershed hydrologic responses. These activities include urbanization, channel modification, flow regulation by water impoundments, water withdrawal, and...

AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na+ transport across human airway epithelial cells.

PubMed

Tan, C D; Smolenski, R T; Harhun, M I; Patel, H K; Ahmed, S G; Wanisch, K; Yáñez-Muñoz, R J; Baines, D L

2012-09-01

Pulmonary transepithelial Na(+) transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na(+) channels and basolateral Na(+) K(+) ATPase activity. H441 human airway epithelial cells were used to examine the effects of hypoxia on Na(+) transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS. AMPK was activated by exposure to 3% or 0.2% O(2) for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm(-2) ) was added to the apical surface of cells grown at the air-liquid interface. Only 0.2% O(2) activated AMPK in cells grown at the air-liquid interface. AMPK activation was associated with elevation of cellular AMP:ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive I(sc) (I(ouabain) ) and apical amiloride-sensitive Na(+) conductance (G(Na+) ). Modification of AMPK activity prevented the effect of hypoxia on I(ouabain) (Na(+) K(+) ATPase) but not apical G(Na+) . Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical G(Na+) (epithelial Na(+) channels). Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na(+) channels and basolateral Na(+) K(+) ATPase activity to decrease transepithelial Na(+) transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

AMP-activated protein kinase (AMPK)–dependent and –independent pathways regulate hypoxic inhibition of transepithelial Na+ transport across human airway epithelial cells

PubMed Central

Tan, CD; Smolenski, RT; Harhun, MI; Patel, HK; Ahmed, SG; Wanisch, K; Yáñez-Muñoz, RJ; Baines, DL

2012-01-01

BACKGROUND AND PURPOSE Pulmonary transepithelial Na+ transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na+ channels and basolateral Na+K+ ATPase activity. EXPERIMENTAL APPROACH H441 human airway epithelial cells were used to examine the effects of hypoxia on Na+ transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS. KEY RESULTS AMPK was activated by exposure to 3% or 0.2% O2 for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm−2) was added to the apical surface of cells grown at the air–liquid interface. Only 0.2% O2 activated AMPK in cells grown at the air–liquid interface. AMPK activation was associated with elevation of cellular AMP : ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive Isc (Iouabain) and apical amiloride-sensitive Na+ conductance (GNa+). Modification of AMPK activity prevented the effect of hypoxia on Iouabain (Na+K+ ATPase) but not apical GNa+. Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical GNa+ (epithelial Na+ channels). CONCLUSIONS AND IMPLICATIONS Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na+ channels and basolateral Na+K+ ATPase activity to decrease transepithelial Na+ transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions. PMID:22509822

Chloride and potassium channels in cystic fibrosis airway epithelia

NASA Astrophysics Data System (ADS)

Welsh, Michael J.; Liedtke, Carole M.

1986-07-01

Cystic fibrosis, the most common lethal genetic disease in Caucasians, is characterized by a decreased permeability in sweat gland duct and airway epithelia. In sweat duct epithelium, a decreased Cl- permeability accounts for the abnormally increased salt content of sweat1. In airway epithelia a decreased Cl- permeability, and possibly increased sodium absorption, may account for the abnormal respiratory tract fluid2,3. The Cl- impermeability has been localized to the apical membrane of cystic fibrosis airway epithelial cells4. The finding that hormonally regulated Cl- channels make the apical membrane Cl- permeable in normal airway epithelial cells5 suggested abnormal Cl- channel function in cystic fibrosis. Here we report that excised, cell-free patches of membrane from cystic fibrosis epithelial cells contain Cl- channels that have the same conductive properties as Cl- channels from normal cells. However, Cl- channels from cystic fibrosis cells did not open when they were attached to the cell. These findings suggest defective regulation of Cl- channels in cystic fibrosis epithelia; to begin to address this issue, we performed two studies. First, we found that isoprenaline, which stimulates Cl- secretion, increases cellular levels of cyclic AMP in a similar manner in cystic fibrosis and non-cystic fibrosis epithelial cells. Second, we show that adrenergic agonists open calcium-activated potassium channels, indirectly suggesting that calcium-dependent stimulus-response coupling is intact in cystic fibrosis. These data suggest defective regulation of Cl- channels at a site distal to cAMP accumulation.

Use-dependent activation of neuronal Kv1.2 channel complexes.

PubMed

Baronas, Victoria A; McGuinness, Brandon R; Brigidi, G Stefano; Gomm Kolisko, Rachel N; Vilin, Yury Y; Kim, Robin Y; Lynn, Francis C; Bamji, Shernaz X; Yang, Runying; Kurata, Harley T

2015-02-25

In excitable cells, ion channels are frequently challenged by repetitive stimuli, and their responses shape cellular behavior by regulating the duration and termination of bursts of action potentials. We have investigated the behavior of Shaker family voltage-gated potassium (Kv) channels subjected to repetitive stimuli, with a particular focus on Kv1.2. Genetic deletion of this subunit results in complete mortality within 2 weeks of birth in mice, highlighting a critical physiological role for Kv1.2. Kv1.2 channels exhibit a unique property described previously as "prepulse potentiation," in which activation by a depolarizing step facilitates activation in a subsequent pulse. In this study, we demonstrate that this property enables Kv1.2 channels to exhibit use-dependent activation during trains of very brief depolarizations. Also, Kv subunits usually assemble into heteromeric channels in the central nervous system, generating diversity of function and sensitivity to signaling mechanisms. We demonstrate that other Kv1 channel types do not exhibit use-dependent activation, but this property is conferred in heteromeric channel complexes containing even a single Kv1.2 subunit. This regulatory mechanism is observed in mammalian cell lines as well as primary cultures of hippocampal neurons. Our findings illustrate that use-dependent activation is a unique property of Kv1.2 that persists in heteromeric channel complexes and may influence function of hippocampal neurons. Copyright © 2015 the authors 0270-6474/15/353515-10$15.00/0.

Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits

PubMed Central

Torres, Yolima P.; Granados, Sara T.; Latorre, Ramón

2014-01-01

Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca2+-activated K+ channel (BK) is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels) and a large C terminus composed of two regulators of K+ conductance domains (RCK domains), where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca2+ sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above. PMID:25346693

Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits.

PubMed

Torres, Yolima P; Granados, Sara T; Latorre, Ramón

2014-01-01

Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca(2+) concentration, the large conductance voltage- and Ca(2+)-activated K(+) channel (BK) is unique among the superfamily of K(+) channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K(+) channels) and a large C terminus composed of two regulators of K(+) conductance domains (RCK domains), where the Ca(2+)-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca(2+) sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above.

Functional significance of M-type potassium channels in nociceptive cutaneous sensory endings

PubMed Central

Passmore, Gayle M.; Reilly, Joanne M.; Thakur, Matthew; Keasberry, Vanessa N.; Marsh, Stephen J.; Dickenson, Anthony H.; Brown, David A.

2012-01-01

M-channels carry slowly activating potassium currents that regulate excitability in a variety of central and peripheral neurons. Functional M-channels and their Kv7 channel correlates are expressed throughout the somatosensory nervous system where they may play an important role in controlling sensory nerve activity. Here we show that Kv7.2 immunoreactivity is expressed in the peripheral terminals of nociceptive primary afferents. Electrophysiological recordings from single afferents in vitro showed that block of M-channels by 3 μM XE991 sensitized Aδ- but not C-fibers to noxious heat stimulation and induced spontaneous, ongoing activity at 32°C in many Aδ-fibers. These observations were extended in vivo: intraplantar injection of XE991 selectively enhanced the response of deep dorsal horn (DH) neurons to peripheral mid-range mechanical and higher range thermal stimuli, consistent with a selective effect on Aδ-fiber peripheral terminals. These results demonstrate an important physiological role of M-channels in controlling nociceptive Aδ-fiber responses and provide a rationale for the nocifensive behaviors that arise following intraplantar injection of the M-channel blocker XE991. PMID:22593734

Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P2 affinity, phosphoinositide selectivity, and PI(4,5)P2 pool accessibility

PubMed Central

Rjasanow, Alexandra; Leitner, Michael G.; Thallmair, Veronika; Halaszovich, Christian R.; Oliver, Dominik

2015-01-01

The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P2 to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P2. Because cellular PI(4,5)P2 is resynthesized rapidly, steady state PI(4,5)P2 changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P2 with Ci-VSP allows for the quantification of relative PI(4,5)P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K+ channels to Ci-VSP allowed for comparison of PI(4,5)P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P2 and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P2 that differ in their accessibility to PLC and VSPs. PMID:26150791

Ion channel remodeling in vascular smooth muscle during hypertension: Implications for novel therapeutic approaches

PubMed Central

Joseph, Biny K.; Thakali, Keshari M.; Moore, Christopher L.; Rhee, Sung W.

2013-01-01

Ion channels are multimeric, transmembrane proteins that selectively mediate ion flux across the plasma membrane in a variety of cells including vascular smooth muscle cells (VSMCs). The dynamic interplay of Ca2+ and K+ channels on the plasma membrane of VSMCs plays a pivotal role in modulating the vascular tone of small arteries and arterioles. The abnormally-elevated arterial tone observed in hypertension thus points to an aberrant expression and function of Ca2+ and K+ channels in the VSMCs. In this short review, we focus on the three well-studied ion channels in VSMCs, namely the L-type Ca2+ (CaV1.2) channels, the voltage-gated K+ (KV) channels, and the large-conductance Ca2+-activated K+ (BK) channels. First, we provide a brief overview on the physiological role of vascular CaV1.2, KV and BK channels in regulating arterial tone. Second, we discuss the current understanding of the expression changes and regulation of CaV1.2, KV and BK channels in the vasculature during hypertension. Third, based on available proof-of-concept studies, we describe the potential therapeutic approaches targeting these vascular ion channels in order to restore blood pressure to normotensive levels. PMID:23376354

Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+ channels.

PubMed

Hallaq, Haifa; Wang, Dao W; Kunic, Jennifer D; George, Alfred L; Wells, K Sam; Murray, Katherine T

2012-02-01

Na(+) current derived from expression of the cardiac isoform SCN5A is reduced by receptor-mediated or direct activation of protein kinase C (PKC). Previous work has suggested a possible role for loss of Na(+) channels at the plasma membrane in this effect, but the results are controversial. In this study, we tested the hypothesis that PKC activation acutely modulates the intracellular distribution of SCN5A channels and that this effect can be visualized in living cells. In human embryonic kidney cells that stably expressed SCN5A with green fluorescent protein (GFP) fused to the channel COOH-terminus (SCN5A-GFP), Na(+) currents were suppressed by an exposure to PKC activation. Using confocal microscopy, colocalization of SCN5A-GFP channels with the plasma membrane under control and stimulated conditions was quantified. A separate population of SCN5A channels containing an extracellular epitope was immunolabeled to permit temporally stable labeling of the plasma membrane. Our results demonstrated that Na(+) channels were preferentially trafficked away from the plasma membrane by PKC activation, with a major contribution by Ca(2+)-sensitive or conventional PKC isoforms, whereas stimulation of protein kinase A (PKA) had the opposite effect. Removal of the conserved PKC site Ser(1503) or exposure to the NADPH oxidase inhibitor apocynin eliminated the PKC-mediated effect to alter channel trafficking, indicating that both channel phosphorylation and ROS were required. Experiments using fluorescence recovery after photobleaching demonstrated that both PKC and PKA also modified channel mobility in a manner consistent with the dynamics of channel distribution. These results demonstrate that the activation of protein kinases can acutely regulate the intracellular distribution and molecular mobility of cardiac Na(+) channels in living cells.

CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions

PubMed Central

Dai, Gucan

2013-01-01

Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositides (PIPn) such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIPn regulation. We determined the functional consequences of this mutation using electrophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIPn inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIPn sensitivity depended on an intact amino (NH2) terminal PIPn regulation module. These observations led us to hypothesize that L633P enhances PIPn inhibition by altering the coupling between NH2- and COOH-terminal regions of CNGA3. A recombinant COOH-terminal fragment partially restored normal PIPn sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the L633P mutation. PMID:23552282

CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions.

PubMed

Dai, Gucan; Varnum, Michael D

2013-07-15

Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositides (PIPn) such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIPn regulation. We determined the functional consequences of this mutation using electrophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIPn inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIPn sensitivity depended on an intact amino (NH2) terminal PIPn regulation module. These observations led us to hypothesize that L633P enhances PIPn inhibition by altering the coupling between NH2- and COOH-terminal regions of CNGA3. A recombinant COOH-terminal fragment partially restored normal PIPn sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the L633P mutation.

H2O2 Regulates Lung Epithelial Sodium Channel (ENaC) via Ubiquitin-like Protein Nedd8

PubMed Central

Downs, Charles A.; Kumar, Amrita; Kreiner, Lisa H.; Johnson, Nicholle M.; Helms, My N.

2013-01-01

Redundancies in both the ubiquitin and epithelial sodium transport pathways allude to their importance of proteolytic degradation and ion transport in maintaining normal cell function. The classical pathway implicated in ubiquitination of the epithelial sodium channel (ENaC) involves Nedd4-2 regulation of sodium channel subunit expression and has been studied extensively studied. However, less attention has been given to the role of the ubiquitin-like protein Nedd8. Here we show that Nedd8 plays an important role in the ubiquitination of ENaC in alveolar epithelial cells. We report that the Nedd8 pathway is redox-sensitive and that under oxidizing conditions Nedd8 conjugation to Cullin-1 is attenuated, resulting in greater surface expression of α-ENaC. This observation was confirmed in our electrophysiology studies in which we inhibited Nedd8-activating enzyme using MLN4924 (a specific Nedd8-activating enzyme inhibitor) and observed a marked increase in ENaC activity (measured as the product of the number of channels (N) and the open probability (Po) of a channel). These results suggest that ubiquitination of lung ENaC is redox-sensitive and may have significant implications for our understanding of the role of ENaC in pulmonary conditions where oxidative stress occurs, such as pulmonary edema and acute lung injury. PMID:23362276

Functional link between muscarinic receptors and large-conductance Ca2+-activated K+ channels in freshly-isolated human detrusor smooth muscle cells

PubMed Central

Parajuli, Shankar P.; Hristov, Kiril L.; Cheng, Qiuping; Malysz, John; Rovner, Eric S.; Petkov, Georgi V.

2014-01-01

Activation of muscarinic acetylcholine receptors (mAChRs) constitutes the primary mechanism for enhancing excitability and contractility of human detrusor smooth muscle (DSM). Since the large conductance Ca2+-activated K+ (KCa1.1) channels are key regulators of human DSM function, we investigated whether mAChR activation increases human DSM excitability by inhibiting KCa1.1 channels. We used the mAChR agonist, carbachol, to determine the changes in KCa1.1 channel activity upon mAChR activation in freshly-isolated human DSM cells obtained from open bladder surgeries using the perforated whole cell and single KCa1.1 channel patch-clamp recordings. Human DSM cells were collected from 29 patients (23 males and 6 females, average age of 65.9±1.5 years). Carbachol inhibited the amplitude and frequency of KCa1.1 channel-mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, which are triggered by the release of Ca2+ from ryanodine receptors. Carbachol also caused membrane potential depolarization, which was not observed in the presence of iberiotoxin, a KCa1.1 channel inhibitor, indicating the critical role of the KCa1.1 channels. The potential direct carbachol effects on KCa1.1channels were examined under conditions of removing the major cellular Ca2+ sources for KCa1.1 channel activation with pharmacological inhibitors (thapsigargin, ryanodine, and nifedipine). In the presence of these inhibitors, carbachol did not affect the single KCa1.1 channel open probability and mean KCa1.1 channel conductance (cell-attached configuration) or depolarization-induced whole cell steady-state KCa1.1 currents. The data support the concept that mAChR activation triggers indirect functional KCa1.1 channel inhibition mediated by intracellular Ca2+, thus increasing the excitability in human DSM cells. PMID:24867682

A self-limiting regulation of vasoconstrictor-activated TRPC3/C6/C7 channels coupled to PI(4,5)P2-diacylglycerol signalling

PubMed Central

Imai, Yuko; Itsuki, Kyohei; Okamura, Yasushi; Inoue, Ryuji; Mori, Masayuki X

2012-01-01

Activation of transient receptor potential (TRP) canonical TRPC3/C6/C7 channels by diacylglycerol (DAG) upon stimulation of phospholipase C (PLC)-coupled receptors results in the breakdown of phosphoinositides (PIPs). The critical importance of PIPs to various ion-transporting molecules is well documented, but their function in relation to TRPC3/C6/C7 channels remains controversial. By using an ectopic voltage-sensing PIP phosphatase (DrVSP), we found that dephosphorylation of PIPs robustly inhibits currents induced by carbachol (CCh), 1-oleolyl-2-acetyl-sn-glycerol (OAG) or RHC80267 in TRPC3, TRPC6 and TRPC7 channels, though the strength of the DrVSP-mediated inhibition (VMI) varied among the channels with a rank order of C7 > C6 > C3. Pharmacological and molecular interventions suggest that depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is most likely the critical event for VMI in all three channels. When the PLC catalytic signal was vigorously activated through overexpression of the muscarinic type-I receptor (M1R), the inactivation of macroscopic TRPC currents was greatly accelerated in the same rank order as the VMI, and VMI of these currents was attenuated or lost. VMI was also rarely detected in vasopressin-induced TRPC6-like currents in A7r5 vascular smooth muscle cells, indicating that the inactivation by PI(4,5)P2 depletion underlies the physiological condition. Simultaneous fluorescence resonance energy transfer (FRET)-based measurement of PI(4,5)P2 levels and TRPC6 currents confirmed that VMI magnitude reflects the degree of PI(4,5)P2 depletion. These results demonstrate that TRPC3/C6/C7 channels are differentially regulated by depletion of PI(4,5)P2, and that the bimodal signal produced by PLC activation controls these channels in a self-limiting manner. PMID:22183723

Activation of TRPM7 channels by small molecules under physiological conditions.

PubMed

Hofmann, T; Schäfer, S; Linseisen, M; Sytik, L; Gudermann, T; Chubanov, V

2014-12-01

Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a cation channel covalently linked to a protein kinase domain. TRPM7 is ubiquitously expressed and regulates key cellular processes such as Mg(2+) homeostasis, motility, and proliferation. TRPM7 is involved in anoxic neuronal death, cardiac fibrosis, and tumor growth. The goal of this work was to identify small molecule activators of the TRPM7 channel and investigate their mechanism of action. We used an aequorin bioluminescence-based assay to screen for activators of the TRPM7 channel. Valid candidates were further characterized using patch clamp electrophysiology. We identified 20 drug-like compounds with various structural backbones that can activate the TRPM7 channel. Among them, the δ opioid antagonist naltriben was studied in greater detail. Naltriben's action was selective among the TRP channels tested. Naltriben activates TRPM7 currents without prior depletion of intracellular Mg(2+) even under conditions of low PIP2. Moreover, naltriben interfered with the effect of the TRPM7 inhibitor NS8593. Finally, our experiments with TRPM7 variants carrying mutations in the pore, TRP, and kinase domains indicate that the site of TRPM7 activation by this small-molecule ligand is most likely located in or near the TRP domain. In conclusion, we identified the first organic small-molecule activators of TRPM7 channels, thus providing new experimental tools to study TRPM7 function in native cellular environments.

Sediment and channel-geometry investigations for the Kansas River bank stabilization study, Kansas, Nebraska, and Colorado

USGS Publications Warehouse

Osterkamp, W.R.; Curtis, R.E.; Crowther, H.G.

1982-01-01

Analysis of hydrologic data from the Kansas River basin suggests that the channels of the lower Solomon, Saline, and Smoky Hill Rivers have narrowed and stabilized as a result of construction of upstream reservoirs. The Kansas River channel, however, remains relatively unstable and locally active. Streamflow regulation and sediment trapping by reservoirs are possible causes of changes occurring at various Kansas River sites. An inferred deficiency of the suspended-sediment load, however, is likely to cause continuing instability. Suspended sediment in the Kansas River apparently is too sparse to form and maintain stable alluvial banks. The deficiency probably results in an increase of bed material movement, general channel widening, and local braiding. Significant channel degradation is lacking at most sites, but may occur in response to long-term (decades-to-centuries) regulation. Recent degradation near Bonner Springs, Kans., may be the result of sand and gravel removal. Any imposed changes that shorten the channel or reduce the suspended-sediment discharge of the Kansas River are expected to cause additional channel instability. (USGS)

Evidence of K+ channel function in epithelial cell migration, proliferation, and repair

PubMed Central

Girault, Alban

2013-01-01

Efficient repair of epithelial tissue, which is frequently exposed to insults, is necessary to maintain its functional integrity. It is therefore necessary to better understand the biological and molecular determinants of tissue regeneration and to develop new strategies to promote epithelial repair. Interestingly, a growing body of evidence indicates that many members of the large and widely expressed family of K+ channels are involved in regulation of cell migration and proliferation, key processes of epithelial repair. First, we briefly summarize the complex mechanisms, including cell migration, proliferation, and differentiation, engaged after epithelial injury. We then present evidence implicating K+ channels in the regulation of these key repair processes. We also describe the mechanisms whereby K+ channels may control epithelial repair processes. In particular, changes in membrane potential, K+ concentration, cell volume, intracellular Ca2+, and signaling pathways following modulation of K+ channel activity, as well as physical interaction of K+ channels with the cytoskeleton or integrins are presented. Finally, we discuss the challenges to efficient, specific, and safe targeting of K+ channels for therapeutic applications to improve epithelial repair in vivo. PMID:24196531

Endolymphatic Sodium Homeostasis by Extramacular Epithelium of the Saccule

PubMed Central

Kim, Sung Huhn

2009-01-01

The saccule is a vestibular sensory organ that depends upon regulation of its luminal fluid, endolymph, for normal transduction of linear acceleration into afferent neural transmission. Previous studies suggested that endolymph in the saccule was merely derived from cochlear endolymph. We developed and used a preparation of isolated mouse saccule to measure transepithelial currents from the extramacular epithelium with a current density probe. The direction and pharmacology of transepithelial current was consistent with Na+ absorption by the epithelial Na+ channel (ENaC) and was blocked by the ENaC-specific inhibitors benzamil and amiloride. Involvement of Na+,K+-ATPase and K+ channels was demonstrated by reduction of the current by ouabain and the K+ channel blockers Ba2+, XE991, and 4-AP. Glucocorticoids upregulated the current via glucocorticoid receptors. Dexamethasone stimulated the current after 24 h and the stimulation was blocked by mifepristone but not spironolactone. No acute response was observed to elevated cAMP in the presence of amiloride nor to bumetanide, a blocker of Na+,K+,2Cl− cotransporter. The results are consistent with a canonical model of corticosteroid-regulated Na+ absorption that includes entry of luminal Na+ through apical membrane Na+ channels and active basolateral exit of Na+ via a Na+ pump, with recycling of K+ at the basolateral membrane via K+-permeable channels. These observations provide our first understanding of the active role played by saccular epithelium in the local regulation of the [Na+] of endolymph for maintenance of our sense of balance. PMID:20016101

Reconstitution of CO2 Regulation of SLAC1 Anion Channel and Function of CO2-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor

PubMed Central

Zeise, Brian; Xu, Danyun; Rappel, Wouter-Jan; Boron, Walter F.; Schroeder, Julian I.

2016-01-01

Dark respiration causes an increase in leaf CO2 concentration (Ci), and the continuing increases in atmospheric [CO2] further increases Ci. Elevated leaf CO2 concentration causes stomatal pores to close. Here, we demonstrate that high intracellular CO2/HCO3− enhances currents mediated by the Arabidopsis thaliana guard cell S-type anion channel SLAC1 upon coexpression of any one of the Arabidopsis protein kinases OST1, CPK6, or CPK23 in Xenopus laevis oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the βCA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation, and coimmunoprecipitation experiments. PIP2;1 exhibited CO2 permeability. Mutation of PIP2;1 in planta alone was insufficient to impair CO2- and abscisic acid-induced stomatal closing, likely due to redundancy. Interestingly, coexpression of βCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 in oocytes enabled extracellular CO2 enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified that abrogated water and CO2 permeability and extracellular CO2 regulation of SLAC1 activity. These findings identify the CO2-permeable PIP2;1 as key interactor of βCA4 and demonstrate functional reconstitution of extracellular CO2 signaling to ion channel regulation upon coexpression of PIP2;1, βCA4, SLAC1, and protein kinases. These data further implicate SLAC1 as a bicarbonate-responsive protein contributing to CO2 regulation of S-type anion channels. PMID:26764375

Oligomeric Bax Is a Component of the Putative Cytochrome c Release Channel MAC, Mitochondrial Apoptosis-induced Channel

PubMed Central

Dejean, Laurent M.; Martinez-Caballero, Sonia; Guo, Liang; Hughes, Cynthia; Teijido, Oscar; Ducret, Thomas; Ichas, François; Korsmeyer, Stanley J.; Antonsson, Bruno; Jonas, Elizabeth A.; Kinnally, Kathleen W.

2005-01-01

Bcl-2 family proteins regulate apoptosis, in part, by controlling formation of the mitochondrial apoptosis-induced channel (MAC), which is a putative cytochrome c release channel induced early in the intrinsic apoptotic pathway. This channel activity was never observed in Bcl-2–overexpressing cells. Furthermore, MAC appears when Bax translocates to mitochondria and cytochrome c is released in cells dying by intrinsic apoptosis. Bax is a component of MAC of staurosporine-treated HeLa cells because MAC activity is immunodepleted by Bax antibodies. MAC is preferentially associated with oligomeric, not monomeric, Bax. The single channel behavior of recombinant oligomeric Bax and MAC is similar. Both channel activities are modified by cytochrome c, consistent with entrance of this protein into the pore. The mean conductance of patches of mitochondria isolated after green fluorescent protein-Bax translocation is significantly higher than those from untreated cells, consistent with onset of MAC activity. In contrast, the mean conductance of patches of mitochondria indicates MAC activity is present in apoptotic cells deficient in Bax but absent in apoptotic cells deficient in both Bax and Bak. These findings indicate Bax is a component of MAC in staurosporine-treated HeLa cells and suggest Bax and Bak are functionally redundant as components of MAC. PMID:15772159

Chronic Hypoxia Suppresses Pregnancy-Induced Upregulation of Large-Conductance Ca2+-Activated K+ Channel Activity in Uterine Arteries

PubMed Central

Hu, Xiang-Qun; Xiao, Daliao; Zhu, Ronghui; Huang, Xiaohui; Yang, Shumei; Wilson, Sean M.; Zhang, Lubo

2013-01-01

Our previous study demonstrated that increased Ca2+-activated K+ (BKCa) channel activity played a key role in the normal adaptation of reduced myogenic tone of uterine arteries in pregnancy. The present study tested the hypothesis that chronic hypoxia during gestation inhibits pregnancy-induced upregulation of BKCa channel function in uterine arteries. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (≈300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Hypoxia during gestation significantly inhibited pregnancy-induced upregulation of BKCa channel activity and suppressed BKCa channel current density in pregnant uterine arteries. This was mediated by a selective downregulation of BKCa channel β1 subunit in the uterine arteries. In accordance, hypoxia abrogated the role of the BKCa channel in regulating pressure-induced myogenic tone of uterine arteries that was significantly elevated in pregnant animals acclimatized to chronic hypoxia. In addition, hypoxia abolished the steroid hormone-mediated increase in the β1 subunit and BKCa channel current density observed in nonpregnant uterine arteries. Although the activation of protein kinase C inhibited BKCa channel current density in pregnant uterine arteries of normoxic sheep, this effect was ablated in the hypoxic animals. The results demonstrate that selectively targeting BKCa channel β1 subunit plays a critical role in the maladaption of uteroplacental circulation caused by chronic hypoxia, which contributes to the increased incidence of preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia. PMID:22665123

Acid-sensing ion channels: trafficking and synaptic function.

PubMed

Zha, Xiang-ming

2013-01-02

Extracellular acidification occurs in the brain with elevated neural activity, increased metabolism, and neuronal injury. This reduction in pH can have profound effects on brain function because pH regulates essentially every single biochemical reaction. Therefore, it is not surprising to see that Nature evolves a family of proteins, the acid-sensing ion channels (ASICs), to sense extracellular pH reduction. ASICs are proton-gated cation channels that are mainly expressed in the nervous system. In recent years, a growing body of literature has shown that acidosis, through activating ASICs, contributes to multiple diseases, including ischemia, multiple sclerosis, and seizures. In addition, ASICs play a key role in fear and anxiety related psychiatric disorders. Several recent reviews have summarized the importance and therapeutic potential of ASICs in neurological diseases, as well as the structure-function relationship of ASICs. However, there is little focused coverage on either the basic biology of ASICs or their contribution to neural plasticity. This review will center on these topics, with an emphasis on the synaptic role of ASICs and molecular mechanisms regulating the spatial distribution and function of these ion channels.

Gating, Regulation, and Structure in K2P K+ Channels: In Varietate Concordia?

PubMed

Niemeyer, María Isabel; Cid, L Pablo; González, Wendy; Sepúlveda, Francisco V

2016-09-01

K2P K(+) channels with two pore domains in tandem associate as dimers to produce so-called background conductances that are regulated by a variety of stimuli. Whereas gating in K2P channels has been poorly understood, recent developments have provided important clues regarding the gating mechanism for this family of proteins. Two modes of gating present in other K(+) channels have been considered. The first is the so-called activation gating that occurs by bundle crossing and the splaying apart of pore-lining helices commanding ion passage. The second mode involves a change in conformation at the selectivity filter (SF), which impedes ion flow at this narrow portion of the conduction pathway and accounts for extracellular pH modulation of several K2P channels. Although some evidence supports the existence of an activation gate in K2P channels, recent results suggest that perhaps all stimuli, even those sensed at a distant location in the protein, are also mediated by SF gating. Recently resolved crystal structures of K2P channels in conductive and nonconductive conformations revealed that the nonconductive state is reached by blockade by a lipid acyl chain that gains access to the channel cavity through intramembrane fenestrations. Here we discuss whether this novel type of gating, proposed so far only for membrane tension gating, might mediate gating in response to other stimuli or whether SF gating is the only type of opening/closing mechanism present in K2P channels. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Novel inhibitors of the Gardos channel for the treatment of sickle cell disease.

PubMed

McNaughton-Smith, Grant A; Burns, J Ford; Stocker, Jonathan W; Rigdon, Gregory C; Creech, Christopher; Arrington, Susan; Shelton, Tara; de Franceschi, Lucia

2008-02-28

Sickle cell disease (SCD) is a hereditary condition characterized by deformation of red blood cells (RBCs). This phenomenon is due to the presence of abnormal hemoglobin that polymerizes upon deoxygenation. This effect is exacerbated when dehydrated RBCs experience a loss of both water and potassium salts. One critical pathway for the regulation of potassium efflux from RBCs is the Gardos channel, a calcium-activated potassium channel. This paper describes the synthesis and biological evaluation of a series of potent inhibitors of the Gardos channel. The goal was to identify compounds that were potent and selective inhibitors of the channel but had improved pharmacokinetic properties compared to 1, Clotrimazole. Several triarylamides such as 10 and 21 were potent inhibitors of the Gardos channel (IC50 of <10 nM) and active in a mouse model of SCD. Compound 21 (ICA-17043) was advanced into phase 3 clinical trials for SCD.

Distance constraints on activation of TRPV4 channels by AKAP150-bound PKCα in arterial myocytes

PubMed Central

Moreno, Claudia M.; O’Dwyer, Samantha; Woods, Sean

2017-01-01

TRPV4 (transient receptor potential vanilloid 4) channels are Ca2+-permeable channels that play a key role in regulating vascular tone. In arterial myocytes, opening of TRPV4 channels creates local increases in Ca2+ influx, detectable optically as “TRPV4 sparklets.” TRPV4 sparklet activity can be enhanced by the action of the vasoconstrictor angiotensin II (AngII). This modulation depends on the activation of subcellular signaling domains that comprise protein kinase C α (PKCα) bound to the anchoring protein AKAP150. Here, we used super-resolution nanoscopy, patch-clamp electrophysiology, Ca2+ imaging, and mathematical modeling approaches to test the hypothesis that AKAP150-dependent modulation of TRPV4 channels is critically dependent on the distance between these two proteins in the sarcolemma of arterial myocytes. Our data show that the distance between AKAP150 and TRPV4 channel clusters varies with sex and arterial bed. Consistent with our hypothesis, we further find that basal and AngII-induced TRPV4 channel activity decays exponentially as the distance between TRPV4 and AKAP150 increases. Our data suggest a maximum radius of action of ∼200 nm for local modulation of TRPV4 channels by AKAP150-associated PKCα. PMID:28507079

Activation of muscarinic M3 receptors inhibits large-conductance voltage- and Ca2+-activated K+ channels in rat urinary bladder smooth muscle cells

PubMed Central

Parajuli, Shankar P.

2013-01-01

Large conductance voltage- and Ca2+-activated K+ (BK) channels are key regulators of detrusor smooth muscle (DSM) contraction and relaxation during urine voiding and storage. Here, we explored whether BK channels are regulated by muscarinic receptors (M-Rs) in native freshly isolated rat DSM cells under physiological conditions using the perforated whole cell patch-clamp technique and pharmacological inhibitors. M-R activation with carbachol (1 μM) initially evoked large transient outward BK currents, followed by inhibition of the spontaneous transient outward BK currents (STBKCs) in DSM cells. Carbachol (1 μM) also inhibited the amplitude and frequency of spontaneous transient hyperpolarizations (STHs) and depolarized the DSM cell membrane potential. Selective inhibition of the muscarinic M3 receptors (M3-Rs) with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 0.1 μM), but not muscarinic M2 receptors with methoctramine (1 μM), blocked the carbachol inhibitory effects on STBKCs. Furthermore, blocking the inositol 1,4,5-triphosphate (IP3) receptors with xestospongin-C (1 μM) inhibited the carbachol-induced large transient outward BK currents without affecting carbachol inhibitory effects on STBKCs. Upon pharmacological inhibition of all known cellular sources of Ca2+ for BK channel activation, carbachol (1 μM) did not affect the voltage-step-induced steady-state BK currents, suggesting that the muscarinic effects in DSM cells are mediated by mobilization of intracellular Ca2+. In conclusion, our findings provide strong evidence that activation of M3-Rs leads to inhibition of the STBKCs, STHs, and depolarization of DSM cells. Collectively, the data suggest the existence of functional interactions between BK channels and M3-Rs at a cellular level in DSM. PMID:23703523

Different rate-limiting activities of intracellular pH regulators for HCO3- secretion stimulated by forskolin and carbachol in rat parotid intralobular ducts.

PubMed

Ueno, Kaori; Hirono, Chikara; Kitagawa, Michinori; Shiba, Yoshiki; Sugita, Makoto

2016-11-01

Intracellular pH (pH i ) regulation fundamentally participates in maintaining HCO 3 - release from HCO 3 - -secreting epithelia. We used parotid intralobular ducts loaded with BCECF to investigate the contributions of a carbonic anhydrase (CA), anion channels and a Na + -H + exchanger (NHE) to pH i regulation for HCO 3 - secretion by cAMP and Ca 2+ signals. Resting pH i was dispersed between 7.4 and 7.9. Forskolin consistently decreased pH i showing the dominance of pH i -lowering activities, but carbachol gathered pH i around 7.6. CA inhibition suppressed the forskolin-induced decrease in pH i , while it allowed carbachol to consistently increase pH i by revealing that carbachol prominently activated NHE via Ca 2+ -calmodulin. Under NHE inhibition, forskolin and carbachol induced the remarkable decreases in pH i , which were slowed predominantly by CA inhibition and by CA or anion channel inhibition, respectively. Our results suggest that forskolin and carbachol primarily activate the pH i -lowering CA and pH i -raising NHE, respectively, to regulate pH i for HCO 3 - secretion.

Functional identification of activity-regulated, high-affinity glutamine transport in hippocampal neurons inhibited by riluzole.

PubMed

Erickson, Jeffrey D

2017-07-01

Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC 50 1.3 ± 0.5 μM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K + -stimulated MeAIB transport displays an affinity (K m ) for MeAIB of 37 ± 1.2 μM, saturates at ~ 200 μM, is dependent on extracellular Ca 2+ , and is blocked by inhibition of voltage-gated Ca 2+ channels. Spontaneous MeAIB transport is also dependent on extracellullar Ca 2+ and voltage-gated calcium channels, but is also blocked by the Na + channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca 2+ , but on Na + ions, and is pH sensitive. Activity-regulated, riluzole-sensitive spontaneous and K + -stimulated transport is minimal at 7-8 days in vitro, coordinately induced during the next 2 weeks and is maximally expressed by days in vitro > 20; the known period for maturation of the Glu/Gln cycle and regulated pre-synaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity-stimulated pre-synaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity. Cover Image for this issue: doi: 10.1111/jnc.13805. © 2017 International Society for Neurochemistry.

Role of phospholipase A2 pathway in regulating activation of Bufo arenarum oocytes.

PubMed

Ajmat, M T; Bonilla, F; Hermosilla, P C; Zelarayán, L; Bühler, M I

2013-08-01

Transient increases in the concentration of cytosolic Ca(2+) are essential for triggering egg activation events. Increased Ca(2+) results from its rapid release from intracellular stores, mainly mediated by one or both intracellular calcium channels: the inositol trisphosphate receptor (IP3R) and the ryanodine receptor (RyR). Several regulatory pathways that tailor the response of these channels to the specific cell type have been proposed. Among its many modulatory actions, calcium can serve as an activator of a cytosolic phospholipase A(2) (cPLA2), which releases arachidonic acid from phospholipids of the endoplasmic reticulum as well as from the nuclear envelope. Previous studies have suggested that arachidonic acid and/or its metabolites were able to modulate the activity of several ion channels. Based on these findings, we have studied the participation of the phospholipase A(2) (PLA(2)) pathway in the process of Bufo arenarum oocyte activation and the interrelation between any of its metabolites and the ion channels involved in the calcium release from the intracellular reservoirs at fertilization. We found that addition of both melittin, a potent PLA(2) activator, and arachidonic acid, the main PLA(2) reaction metabolite, was able to induce activation events in a bell-shaped manner. Differential regulation of IP3Rs and RyRs by arachidonic acid and its products could explain melittin and arachidonic acid behaviour in Bufo arenarum egg activation. The concerted action of arachidonic acid and/or its metabolites could provide controlled mobilization of calcium from intracellular reservoirs and useful tools for understanding calcium homeostasis in eggs that express both types of receptors.

The Potassium Channel, Kir3.4 Participates in Angiotensin II-Stimulated Aldosterone Production by a Human Adrenocortical Cell Line

PubMed Central

Oki, Kenji; Plonczynski, Maria W.; Lam, Milay Luis; Gomez-Sanchez, Elise P.

2012-01-01

Angiotensin II (A-II) regulation of aldosterone secretion is initiated by inducing cell membrane depolarization, thereby increasing intracellular calcium and activating the calcium calmodulin/calmodulin kinase cascade. Mutations in the selectivity filter of the KCNJ5 gene coding for inward rectifying potassium channel (Kir)3.4 has been found in about one third of aldosterone-producing adenomas. These mutations result in loss of selectivity of the inward rectifying current for potassium, which causes membrane depolarization and opening of calcium channels and activation of the calcium calmodulin/calmodulin kinase cascade and results in an increase in aldosterone secretion. In this study we show that A-II and a calcium ionophore down-regulate the expression of KCNJ5 mRNA and protein. Activation of Kir3.4 by naringin inhibits A-II-stimulated membrane voltage and aldosterone secretion. Overexpression of KCNJ5 in the HAC15 cells using a lentivirus resulted in a decrease in membrane voltage, intracellular calcium, expression of steroidogenic acute regulatory protein, 3-β-hydroxysteroid dehydrogenase 3B2, cytochrome P450 11B1 and cytochrome P450 11B2 mRNA, and aldosterone synthesis. In conclusion, A-II appears to stimulate aldosterone secretion by depolarizing the membrane acting in part through the regulation of the expression and activity of Kir3.4. PMID:22798349

Human EAG channels are directly modulated by PIP2 as revealed by electrophysiological and optical interference investigations

PubMed Central

Han, Bo; He, Kunyan; Cai, Chunlin; Tang, Yin; Yang, Linli; Heinemann, Stefan H.; Hoshi, Toshinori; Hou, Shangwei

2016-01-01

Voltage-gated ether à go-go (EAG) K+ channels are expressed in various types of cancer cells and also in the central nervous system. Aberrant overactivation of human EAG1 (hEAG1) channels is associated with cancer and neuronal disorders such as Zimmermann-Laband and Temple-Baraitser syndromes. Although hEAG1 channels are recognized as potential therapeutic targets, regulation of their functional properties is only poorly understood. Here, we show that the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is a potent inhibitory gating modifier of hEAG1 channels. PIP2 inhibits the channel activity by directly binding to a short N-terminal segment of the channel important for Ca2+/calmodulin (CaM) binding as evidenced by bio-layer interferometry measurements. Conversely, depletion of endogenous PIP2 either by serotonin-induced phospholipase C (PLC) activation or by a rapamycin-induced translocation system enhances the channel activity at physiological membrane potentials, suggesting that PIP2 exerts a tonic inhibitory influence. Our study, combining electrophysiological and direct binding assays, demonstrates that hEAG1 channels are subject to potent inhibitory modulation by multiple phospholipids and suggests that manipulations of the PIP2 signaling pathway may represent a strategy to treat hEAG1 channel-associated diseases. PMID:27005320

Stimulation effect of wide type CFTR chloride channel by the naturally occurring flavonoid tangeretin.

PubMed

Jiang, Yu; Yu, Bo; Wang, Xue; Sui, Yujie; Zhang, Yaofang; Yang, Shuang; Yang, Hong; Ma, Tonghui

2014-12-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed in the apical membrane of serous epithelial cells. Both deficiency and overactivation of CFTR may cause fluid and salt secretion related diseases. In the present study, we identified tangeretin from Pericarpium Citri Reticulatae Viride as a CFTR activator using high-throughput screening based on FRT cell-based fluorescence assay. The activation effect of tangeretin on CFTR chloride channel and the possible underlying mechanisms were investigated. Fluorescence quenching tests showed that tangeretin dose- and time-dependently activated CFTR chloride channel, the activity had rapid and reversible characteristics and the activation effect could be completely reversed by the CFTR specific blocker CFTRinh-172. Primary mechanism studies indicated that the activation effect of tangeretin on CFTR chloride channel was FSK dependent as well as had additional effect with FSK and IBMX suggesting that tangeretin activates CFTR by direct interacting with the protein. Ex-vivo tests revealed that tangeretin could accelerate the speed of the submucosal gland fluid secretion. Short-circuit current measurement demonstrated that tangeretin activated rat colonic mucosa chloride current. Thus, CFTR Cl(-) channel is a molecular target of natural compound tangeretin. Tangeretin may have potential use for the treatment of CFTR-related diseases like cystic fibrosis, bronchiectasis and habitual constipation. Copyright © 2014 Elsevier B.V. All rights reserved.

K+ channel openers restore verapamil-inhibited lung fluid resolution and transepithelial ion transport

PubMed Central

2010-01-01

Background Lung epithelial Na+ channels (ENaC) are regulated by cell Ca2+ signal, which may contribute to calcium antagonist-induced noncardiogenic lung edema. Although K+ channel modulators regulate ENaC activity in normal lungs, the therapeutical relevance and the underlying mechanisms have not been completely explored. We hypothesized that K+ channel openers may restore calcium channel blocker-inhibited alveolar fluid clearance (AFC) by up-regulating both apical and basolateral ion transport. Methods Verapamil-induced depression of heterologously expressed human αβγ ENaC in Xenopus oocytes, apical and basolateral ion transport in monolayers of human lung epithelial cells (H441), and in vivo alveolar fluid clearance were measured, respectively, using the two-electrode voltage clamp, Ussing chamber, and BSA protein assays. Ca2+ signal in H441 cells was analyzed using Fluo 4AM. Results The rate of in vivo AFC was reduced significantly (40.6 ± 6.3% of control, P < 0.05, n = 12) in mice intratracheally administrated verapamil. KCa3.1 (1-EBIO) and KATP (minoxidil) channel openers significantly recovered AFC. In addition to short-circuit current (Isc) in intact H441 monolayers, both apical and basolateral Isc levels were reduced by verapamil in permeabilized monolayers. Moreover, verapamil significantly altered Ca2+ signal evoked by ionomycin in H441 cells. Depletion of cytosolic Ca2+ in αβγ ENaC-expressing oocytes completely abolished verapamil-induced inhibition. Intriguingly, KV (pyrithione-Na), K Ca3.1 (1-EBIO), and KATP (minoxidil) channel openers almost completely restored the verapamil-induced decrease in Isc levels by diversely up-regulating apical and basolateral Na+ and K+ transport pathways. Conclusions Our observations demonstrate that K+ channel openers are capable of rescuing reduced vectorial Na+ transport across lung epithelial cells with impaired Ca2+ signal. PMID:20507598

Identification and validation of midbrain Kcnq4 regulation of heavy alcohol consumption in rodents.

PubMed

McGuier, Natalie S; Rinker, Jennifer A; Cannady, Reginald; Fulmer, Diana B; Jones, Sara R; Hoffman, Michaela; Mulholland, Patrick J

2018-05-24

Currently available pharmacotherapies for treating alcohol use disorder (AUD) suffer from deleterious side effects and are not efficacious in diverse populations. Clinical and preclinical studies provide evidence that the Kcnq family of genes that encode K V 7 channels influence alcohol intake and dependence. K V 7 channels are a class of slowly activating voltage-dependent K + channels that regulate neuronal excitability. Studies indicate that the K V 7 channel positive modulator retigabine can decrease dopaminergic neuron firing, alter dopamine (DA) release, and reduce alcohol intake in heavy drinking rodents. Given the critical nature of ventral tegmental area (VTA) DA to the addiction process and predominant expression of Kcnq4 in DA neurons, we investigated the role of midbrain Kcnq genes and K V 7 channels in the VTA of genetically diverse mice and long-term heavy drinking rats, respectively. Integrative bioinformatics analysis identified negative correlations between midbrain Kcnq4 expression and alcohol intake and seeking behaviors. Kcnq4 expression levels were also correlated with dopaminergic-related phenotypes in BXD strains, and Kcnq4 was present in support intervals for alcohol sensitivity and alcohol withdrawal severity QTLs in rodents. Pharmacological validation studies revealed that VTA K V 7 channels regulate excessive alcohol intake in rats with a high-drinking phenotype. Administration of a novel and selective K V 7.2/4 channel positive modulator also reduced alcohol drinking in rats. Together, these findings indicate that midbrain Kcnq4 expression regulates alcohol-related behaviors in genetically diverse mice and provide evidence that K V 7.4 channels are a critical mediator of excessive alcohol drinking. Copyright © 2018 Elsevier Ltd. All rights reserved.

Tip-localized Ca2+ -permeable channels control pollen tube growth via kinase-dependent R- and S-type anion channel regulation.

PubMed

Gutermuth, Timo; Herbell, Sarah; Lassig, Roman; Brosché, Mikael; Romeis, Tina; Feijó, José Alberto; Hedrich, Rainer; Konrad, Kai Robert

2018-05-01

Pollen tubes (PTs) are characterized by having tip-focused cytosolic calcium ion (Ca 2+ ) concentration ([Ca 2+ ] cyt ) gradients, which are believed to control PT growth. However, the mechanisms by which the apical [Ca 2+ ] cyt orchestrates PT growth are not well understood. Here, we aimed to identify these mechanisms by combining reverse genetics, cell biology, electrophysiology, and live-cell Ca 2+ and anion imaging. We triggered Ca 2+ -channel activation by applying hyperpolarizing voltage pulses and observed that the evoked [Ca 2+ ] cyt increases were paralleled by high anion channel activity and a decrease in the cytosolic anion concentration at the PT tip. We confirmed a functional correlation between these patterns by showing that inhibition of Ca 2+ -permeable channels eliminated the [Ca 2+ ] cyt increase, resulting in the abrogation of anion channel activity via Ca 2+ -dependent protein kinases (CPKs). Functional characterization of CPK and anion-channel mutants revealed a CPK2/20/6-dependent activation of SLAH3 and ALMT12/13/14 anion channels. The impaired growth phenotypes of anion channel and CPK mutants support the physiological significance of a kinase- and Ca 2+ -dependent pathway to control PT growth via anion channel activation. Other than unveiling this functional link, our membrane hyperpolarization method allows for unprecedented manipulation of the [Ca 2+ ] cyt gradient or oscillations in the PT tips and opens an array of opportunities for channel screenings. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

The function and regulation of acid‐sensing ion channels (ASICs) and the epithelial Na+ channel (ENaC): IUPHAR Review 19

PubMed Central

Boscardin, Emilie; Alijevic, Omar; Hummler, Edith

2016-01-01

Acid‐sensing ion channels (ASICs) and the epithelial Na+ channel (ENaC) are both members of the ENaC/degenerin family of amiloride‐sensitive Na+ channels. ASICs act as proton sensors in the nervous system where they contribute, besides other roles, to fear behaviour, learning and pain sensation. ENaC mediates Na+ reabsorption across epithelia of the distal kidney and colon and of the airways. ENaC is a clinically used drug target in the context of hypertension and cystic fibrosis, while ASIC is an interesting potential target. Following a brief introduction, here we will review selected aspects of ASIC and ENaC function. We discuss the origin and nature of pH changes in the brain and the involvement of ASICs in synaptic signalling. We expose how in the peripheral nervous system, ASICs cover together with other ion channels a wide pH range as proton sensors. We introduce the mechanisms of aldosterone‐dependent ENaC regulation and the evidence for an aldosterone‐independent control of ENaC activity, such as regulation by dietary K+. We then provide an overview of the regulation of ENaC by proteases, a topic of increasing interest over the past few years. In spite of the profound differences in the physiological and pathological roles of ASICs and ENaC, these channels share many basic functional and structural properties. It is likely that further research will identify physiological contexts in which ASICs and ENaC have similar or overlapping roles. PMID:27278329

Serum and Glucocorticoid Regulated Kinase 1 in Sodium Homeostasis

PubMed Central

Lou, Yiyun; Zhang, Fan; Luo, Yuqin; Wang, Liya; Huang, Shisi; Jin, Fan

2016-01-01

The ubiquitously expressed serum and glucocorticoid regulated kinase 1 (SGK1) is tightly regulated by osmotic and hormonal signals, including glucocorticoids and mineralocorticoids. Recently, SGK1 has been implicated as a signal hub for the regulation of sodium transport. SGK1 modulates the activities of multiple ion channels and carriers, such as epithelial sodium channel (ENaC), voltage-gated sodium channel (Nav1.5), sodium hydrogen exchangers 1 and 3 (NHE1 and NHE3), sodium-chloride symporter (NCC), and sodium-potassium-chloride cotransporter 2 (NKCC2); as well as the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase) and type A natriuretic peptide receptor (NPR-A). Accordingly, SGK1 is implicated in the physiology and pathophysiology of Na+ homeostasis. Here, we focus particularly on recent findings of SGK1’s involvement in Na+ transport in renal sodium reabsorption, hormone-stimulated salt appetite and fluid balance and discuss the abnormal SGK1-mediated Na+ reabsorption in hypertension, heart disease, edema with diabetes, and embryo implantation failure. PMID:27517916

Voltage-Gated Na+ Channels are Modulated by Glucose and Involved in Regulating Cellular Insulin Content of INS-1 Cells.

PubMed

Chen, Chong; Wang, Songhua; Hu, Qingjuan; Zeng, Lvming; Peng, Hailong; Liu, Chao; Huang, Li-Ping; Song, Hao; Li, Yuping; Yao, Li-Hua; Meng, Wei

2018-01-01

Islet beta cells (β-cells) are unique cells that play a critical role in glucose homeostasis by secreting insulin in response to increased glucose levels. Voltage-gated ion channels in β-cells, such as K+ and Ca2+ channels, contribute to insulin secretion. The response of voltage-gated Na+ channels (VGSCs) in β-cells to the changes in glucose levels remains unknown. This work aims to determine the role of extracellular glucose on the regulation of VGSC. The effect of glucose on VGSC currents (INa) was investigated in insulin-secreting β-cell line (INS-1) cells of rats using whole-cell patch clamp techniques, and the effects of glucose on insulin content and cell viability were determined using Enzyme-Linked Immunosorbent Assay (ELISA) and Methylthiazolyldiphenyl-tetrazolium Bromide (MTT) assay methods respectively. Our results show that extracellular glucose application can inhibit the peak of INa in a concentration-dependent manner. Glucose concentration of 18 mM reduced the amplitude of INa, suppressed the INa of steady-state activation, shifted the steady-state inactivation curves of INa to negative potentials, and prolonged the time course of INa recovery from inactivation. Glucose also enhanced the activity-dependent attenuation of INa and reduced the fraction of activated channels. Furthermore, 18 mM glucose or low concentration of tetrodotoxin (TTX, a VGSC-specific blocker) partially inhibited the activity of VGSC and also improved insulin synthesis. These results revealed that extracellular glucose application enhances the insulin synthesis in INS-1 cells and the mechanism through the partial inhibition on INa channel is involved. Our results innovatively suggest that VGSC plays a vital role in modulating glucose homeostasis. © 2018 The Author(s). Published by S. Karger AG, Basel.

Conserved Allosteric Hot Spots in the Transmembrane Domains of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels and Multidrug Resistance Protein (MRP) Pumps*

PubMed Central

Wei, Shipeng; Roessler, Bryan C.; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L.; Kirk, Kevin L.

2014-01-01

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. PMID:24876383

Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

PubMed

Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

2014-07-18

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Inward-rectifying potassium (Kir) channels regulate pacemaker activity in spinal nociceptive circuits during early life

PubMed Central

Li, Jie; Blankenship, Meredith L.; Baccei, Mark L.

2013-01-01

Pacemaker neurons in neonatal spinal nociceptive circuits generate intrinsic burst-firing and are distinguished by a lower “leak” membrane conductance compared to adjacent, non-bursting neurons. However, little is known about which subtypes of leak channels regulate the level of pacemaker activity within the developing rat superficial dorsal horn (SDH). Here we demonstrate that a hallmark feature of lamina I pacemaker neurons is a reduced conductance through inward-rectifying potassium (Kir) channels at physiological membrane potentials. Differences in the strength of inward rectification between pacemakers and non-pacemakers indicate the presence of functionally distinct Kir currents in these two populations at room temperature. However, Kir currents in both groups showed high sensitivity to block by extracellular Ba2+ (IC50 ~ 10 µM), which suggests the presence of ‘classical’ Kir (Kir2.x) channels in the neonatal SDH. The reduced Kir conductance within pacemakers is unlikely to be explained by an absence of particular Kir2.x isoforms, as immunohistochemical analysis revealed the expression of Kir2.1, Kir2.2 and Kir2.3 within spontaneously bursting neurons. Importantly, Ba2+ application unmasked rhythmic burst-firing in ~42% of non-bursting lamina I neurons, suggesting that pacemaker activity is a latent property of a sizeable population of SDH cells during early life. In addition, the prevalence of spontaneous burst-firing within lamina I was enhanced in the presence of high internal concentrations of free Mg2+, consistent with its documented ability to block Kir channels from the intracellular side. Collectively, the results indicate that Kir channels are key modulators of pacemaker activity in newborn central pain networks. PMID:23426663

Structural Changes Fundamental to Gating of the Cystic Fibrosis Transmembrane Conductance Regulator Anion Channel Pore.

PubMed

Linsdell, Paul

2017-01-01

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial cell anion channel. Potentiator drugs used in the treatment of cystic fibrosis act on the channel to increase overall channel function, by increasing the stability of its open state and/or decreasing the stability of its closed state. The structure of the channel in either the open state or the closed state is not currently known. However, changes in the conformation of the protein as it transitions between these two states have been studied using functional investigation and molecular modeling techniques. This review summarizes our current understanding of the architecture of the transmembrane channel pore that controls the movement of chloride and other small anions, both in the open state and in the closed state. Evidence for different kinds of changes in the conformation of the pore as it transitions between open and closed states is described, as well as the mechanisms by which these conformational changes might be controlled to regulate normal channel gating. The ways that key conformational changes might be targeted by small compounds to influence overall CFTR activity are also discussed. Understanding the changes in pore structure that might be manipulated by such small compounds is key to the development of novel therapeutic strategies for the treatment of cystic fibrosis.

Select α-arrestins control cell-surface abundance of the mammalian Kir2.1 potassium channel in a yeast model.

PubMed

Hager, Natalie A; Krasowski, Collin J; Mackie, Timothy D; Kolb, Alexander R; Needham, Patrick G; Augustine, Andrew A; Dempsey, Alison; Szent-Gyorgyi, Christopher; Bruchez, Marcel P; Bain, Daniel J; Kwiatkowski, Adam V; O'Donnell, Allyson F; Brodsky, Jeffrey L

2018-05-21

Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K+ inwardly rectifying channel Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorescence-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin-mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Novel targets for Huntington’s disease in an mTOR-independent autophagy pathway

PubMed Central

Williams, Andrea; Sarkar, Sovan; Cuddon, Paul; Ttofi, Evangelia K.; Saiki, Shinji; Siddiqi, Farah H.; Jahreiss, Luca; Fleming, Angeleen; Pask, Dean; Goldsmith, Paul; O’Kane, Cahir J.; Floto, R. Andres; Rubinsztein, David C.

2009-01-01

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases like Huntington’s disease. Autophagy induction with the mTOR inhibitor, rapamycin, accelerates clearance of these toxic substrates. As rapamycin has non-trivial side effects, we screened FDA-approved drugs to identify novel autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the Gi signaling activator clonidine, induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, where cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating Gsα, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced and we provide proof-of-principle for therapeutic relevance in Huntington’s disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+, like excitotoxicity, will inhibit autophagy, thus retarding clearance of aggregate-prone proteins. PMID:18391949

Formation of a physiological complex between TRPV2 and RGA protein promotes cell surface expression of TRPV2.

PubMed

Stokes, Alexander J; Wakano, Clay; Del Carmen, Kimberly A; Koblan-Huberson, Murielle; Turner, Helen

2005-03-01

The transient receptor potential, sub-family Vanilloid (TRPV)(2) cation channel is activated in response to extreme temperature elevations in sensory neurons. However, TRPV2 is widely expressed in tissues with no sensory function, including cells of the immune system. Regulation of GRC, the murine homolog of TRPV2 has been studied in insulinoma cells and myocytes. GRC is activated in response to certain growth factors and neuropeptides, via a mechanism that involves regulated access of the channel to the plasma membrane. This is likely to be an important primary control mechanism for TRPV2 outside the CNS. Here, we report that a regulated trafficking step controls the access of TRPV2 to the cell surface in mast cells. In mast cells, elevations in cytosolic cAMP are sufficient to drive plasma membrane localization of TRPV2. We have previously proposed that the recombinase gene activator protein (RGA), a four-transmembrane domain, intracellular protein, associates with TRPV2 during the biosynthesis and early trafficking of the channel. We use a polyclonal antibody to RGA to confirm the formation of a physiological complex between RGA and TRPV2. Finally, we show that over-expression of the RGA protein potentiates the basal surface localization of TRPV2. We propose that trafficking and activation mechanisms intersect for TRPV2, and that cAMP mobilizing stimuli may regulate TRPV2 localization in non-sensory cells. RGA participates in the control of TRPV2 surface levels, and co-expression of RGA may be a key component of experimental systems that seek to study TRPV2 physiology.

TRPM4 channel: a new player in urinary bladder smooth muscle function in rats

PubMed Central

Smith, Amy C.; Parajuli, Shankar P.; Hristov, Kiril L.; Cheng, Qiuping; Soder, Rupal P.; Afeli, Serge A. Y.; Earley, Scott; Xin, Wenkuan; Malysz, John

2013-01-01

The TRPM4 channel is a Ca2+-activated, monovalent cation-selective channel of the melastatin transient receptor potential (TRPM) family. The TRPM4 channel is implicated in the regulation of many cellular processes including the immune response, insulin secretion, and pressure-induced vasoconstriction of cerebral arteries. However, the expression and function of the TRPM4 channels in detrusor smooth muscle (DSM) have not yet been explored. Here, we provide the first molecular, electrophysiological, and functional evidence for the presence of TRPM4 channels in rat DSM. We detected the expression of TRPM4 channels at mRNA and protein levels in freshly isolated DSM single cells and DSM tissue using RT-PCR, Western blotting, immunohistochemistry, and immunocytochemistry. 9-Hydroxyphenanthrene (9-phenanthrol), a novel selective inhibitor of TRPM4 channels, was used to examine their role in DSM function. In perforated patch-clamp recordings using freshly isolated rat DSM cells, 9-phenanthrol (30 μM) decreased the spontaneous inward current activity at −70 mV. Real-time DSM live-cell Ca2+ imaging showed that selective inhibition of TRPM4 channels with 9-phenanthrol (30 μM) significantly reduced the intracellular Ca2+ levels. Isometric DSM tension recordings revealed that 9-phenanthrol (0.1–30 μM) significantly inhibited the amplitude, muscle force integral, and frequency of the spontaneous phasic and pharmacologically induced contractions of rat DSM isolated strips. 9-Phenanthrol also decreased the amplitude and muscle force integral of electrical field stimulation-induced contractions. In conclusion, this is the first study to examine the expression and provide evidence for TRPM4 channels as critical regulators of rat DSM excitability and contractility. PMID:23283997

Vinpocetine regulates cation channel permeability of inner retinal neurons in the ischaemic retina.

PubMed

Nivison-Smith, Lisa; Acosta, Monica L; Misra, Stuti; O'Brien, Brendan J; Kalloniatis, Michael

2014-01-01

Vinpocetine is a natural drug which exerts neuroprotective effects in ischaemia of the brain through actions on cation channels, glutamate receptors and other pathways. This study investigated the effect of vinpocetine on cation channel permeability of inner retinal neurons after acute retinal metabolic insult. We focused on amacrine and ganglion cells immunoreactive for calretinin or parvalbumin due to their previously documented susceptibility to ischaemia. Using the probe, 1-amino-4-guanidobutane (AGB), we observed increased cation channel permeability across amacrine and ganglion cells under ischaemia and hypoglycaemia but not anoxia. Calretinin and parvalbumin immunoreactivity was also reduced during ischaemia and hypoglyacemia but not anoxia. Vinpocetine decreased AGB entry into ischaemic and hypoglycaemic ganglion cells indicating that the drug can modulate unregulated cation entry. In addition, vinpocetine prevented the loss of calretinin and parvalbumin immunoreactivity following ischaemia suggesting it may indirectly regulate intracellular calcium. Vinpocetine also reduced AGB permeability in selected amacrine and ganglion cell populations following N-methyl-D-aspartate (NMDA) but not kainate activation suggesting that vinpocetine's regulation of cation channel permeability may partly involve NMDA sensitive glutamate receptors. Copyright © 2014 Elsevier Ltd. All rights reserved.

Kinetic Studies of Calcium-Induced Calcium Release in Cardiac Sarcoplasmic Reticulum Vesicles

PubMed Central

Sánchez, Gina; Hidalgo, Cecilia; Donoso, Paulina

2003-01-01

Fast Ca2+ release kinetics were measured in cardiac sarcoplasmic reticulum vesicles actively loaded with Ca2+. Release was induced in solutions containing 1.2 mM free ATP and variable free [Ca2+] and [Mg2+]. Release rate constants (k) were 10-fold higher at pCa 6 than at pCa 5 whereas Ryanodine binding was highest at pCa ≤5. These results suggest that channels respond differently when exposed to sudden [Ca2+] changes than when exposed to Ca2+ for longer periods. Vesicles with severalfold different luminal calcium contents exhibited double exponential release kinetics at pCa 6, suggesting that channels undergo time-dependent activity changes. Addition of Mg2+ produced a marked inhibition of release kinetics at pCa 6 (K0.5 = 63 μM) but not at pCa 5. Coexistence of calcium activation and inhibition sites with equally fast binding kinetics is proposed to explain this behavior. Thimerosal activated release kinetics at pCa 5 at all [Mg2+] tested and increased at pCa 6 the K0.5 for Mg2+ inhibition, from 63 μM to 136 μM. We discuss the possible relevance of these results, which suggest release through RyR2 channels is subject to fast regulation by Ca2+ and Mg2+ followed by time-dependent regulation, to the physiological mechanisms of cardiac channel opening and closing. PMID:12668440

TRPC6 channel-mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK, PI3K, and CAMKIV signaling.

PubMed

Heiser, Jeanine H; Schuwald, Anita M; Sillani, Giacomo; Ye, Lian; Müller, Walter E; Leuner, Kristina

2013-11-01

The non-selective cationic transient receptor canonical 6 (TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin-induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient-mediated changes in synaptic plasticity, ranging from calmodulin-mediated Ras-induced signaling cascades comprising the mitogen-activated protein kinase, PI3K signal transduction pathways as well as Ca(2+) /calmodulin-dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6-mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/mitogen-activated protein kinase/extracellular signal-regulated kinases, phosphatidylinositide 3-kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP-response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract-mediated antidepressant activity. Alterations in synaptic plasticity are considered to play an important role in the pathogenesis of depression. Beside several other proteins, TRPC6 channels regulate synaptic plasticity. This study demonstrates that different pathways including Ras/MEK/ERK, PI3K/Akt, and CAMKIV are involved in the improvement of synaptic plasticity by the TRPC6 activator hyperforin, the antidepressant active constituent of St. John's wort extract. © 2013 International Society for Neurochemistry.

Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis

PubMed Central

Lewis, Michele D.; Park, Hyun Woo; Brand, Randall E.; Gelrud, Andres; Anderson, Michelle A.; Banks, Peter A.; Conwell, Darwin; Lawrence, Christopher; Romagnuolo, Joseph; Baillie, John; Alkaade, Samer; Cote, Gregory; Gardner, Timothy B.; Amann, Stephen T.; Slivka, Adam; Sandhu, Bimaljit; Aloe, Amy; Kienholz, Michelle L.; Yadav, Dhiraj; Barmada, M. Michael; Bahar, Ivet; Lee, Min Goo; Whitcomb, David C.

2014-01-01

CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system. PMID:25033378

Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.

PubMed

LaRusch, Jessica; Jung, Jinsei; General, Ignacio J; Lewis, Michele D; Park, Hyun Woo; Brand, Randall E; Gelrud, Andres; Anderson, Michelle A; Banks, Peter A; Conwell, Darwin; Lawrence, Christopher; Romagnuolo, Joseph; Baillie, John; Alkaade, Samer; Cote, Gregory; Gardner, Timothy B; Amann, Stephen T; Slivka, Adam; Sandhu, Bimaljit; Aloe, Amy; Kienholz, Michelle L; Yadav, Dhiraj; Barmada, M Michael; Bahar, Ivet; Lee, Min Goo; Whitcomb, David C

2014-07-01

CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.

A Ca(2)+ signaling pathway regulates a K(+) channel for low-K response in Arabidopsis.

PubMed

Li, Legong; Kim, Beom-Gi; Cheong, Yong Hwa; Pandey, Girdhar K; Luan, Sheng

2006-08-15

Nutrient sensing is critical for plant adaptation to the environment. Because of extensive farming and erosion, low content of mineral nutrients such as potassium (K(+)) in soils becomes a limiting factor for plant growth. In response to low-K conditions, plants enhance their capability of K(+) uptake through an unknown signaling mechanism. Here we report the identification of a Ca(2+)-dependent pathway for low-K response in Arabidopsis. We are not aware of any other example of a molecular pathway for a nutrient response in plants. Earlier genetic analyses revealed three genes encoding two Ca(2+) sensors (CBL1 and CBL9) and their target protein kinase (CIPK23) to be critical for plant growth on low-K media and for stomatal regulation, indicating that these calcium signaling components participate in the low-K response and turgor regulation. In this study, we show that the protein kinase CIPK23 interacted with, and phosphorylated, a voltage-gated inward K(+) channel (AKT1) required for K(+) acquisition in Arabidopsis. In the Xenopus oocyte system, our studies showed that interacting calcium sensors (CBL1 and CBL9) together with target kinase CIPK23, but not either component alone, activated the AKT1 channel in a Ca(2+)-dependent manner, connecting the Ca(2+) signal to enhanced K(+) uptake through activation of a K(+) channel. Disruption of both CBL1 and CBL9 or CIPK23 gene in Arabidopsis reduced the AKT1 activity in the mutant roots, confirming that the Ca(2+)-CBL-CIPK pathway functions to orchestrate transporting activities in planta according to external K(+) availability.

Bell-shaped calcium-response curves of lns(l,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum

NASA Astrophysics Data System (ADS)

Bezprozvanny, Llya; Watras, James; Ehrlich, Barbara E.

1991-06-01

RELEASE of calcium from intracellular stores occurs by two pathways, an inositol 1,4,5-trisphosphate (InsP3)-gated channel1-3 and a calcium-gated channel (ryanodine receptor)4-6. Using specific antibodies, both receptors were found in Purkinje cells of cerebellum7,8. We have now compared the functional properties of the channels corresponding to the two receptors by incorporating endoplasmic reticulum vesicles from canine cerebellum into planar bilayers. InsP3-gated channels were observed most frequently. Another channel type was activated by adenine nucleotides or caffeine, inhibited by ruthenium red, and modified by ryanodine, characteristics of the ryanodine receptor/channel6. The open probability of both channel types displayed a bell-shaped curve for dependence on calcium. For the InsP3-gated channel, the maximum probability of opening occurred at 0.2 µM free calcium, with sharp decreases on either side of the maximum. Maximum activity for the ryanodine receptor/channel was maintained between 1 and 100 µM calcium. Thus, within the physiological range of cytoplasmic calcium, the InsP3-gated channel itself allows positive feed-back and then negative feedback for calcium release, whereas the ryanodine receptor/channel behaves solely as a calcium-activated channel. The existence in the same cell of two channels with different responses to calcium and different ligand sensitivities provides a basis for complex patterns of intracellular calcium regulation.

Estrogens and human papilloma virus oncogenes regulate human ether-à-go-go-1 potassium channel expression.

PubMed

Díaz, Lorenza; Ceja-Ochoa, Irais; Restrepo-Angulo, Iván; Larrea, Fernando; Avila-Chávez, Euclides; García-Becerra, Rocío; Borja-Cacho, Elizabeth; Barrera, David; Ahumada, Elías; Gariglio, Patricio; Alvarez-Rios, Elizabeth; Ocadiz-Delgado, Rodolfo; Garcia-Villa, Enrique; Hernández-Gallegos, Elizabeth; Camacho-Arroyo, Ignacio; Morales, Angélica; Ordaz-Rosado, David; García-Latorre, Ethel; Escamilla, Juan; Sánchez-Peña, Luz Carmen; Saqui-Salces, Milena; Gamboa-Dominguez, Armando; Vera, Eunice; Uribe-Ramírez, Marisela; Murbartián, Janet; Ortiz, Cindy Sharon; Rivera-Guevara, Claudia; De Vizcaya-Ruiz, Andrea; Camacho, Javier

2009-04-15

Ether-à-go-go-1 (Eag1) potassium channels are potential tools for detection and therapy of numerous cancers. Here, we show human Eag1 (hEag1) regulation by cancer-associated factors. We studied hEag1 gene expression and its regulation by estradiol, antiestrogens, and human papillomavirus (HPV) oncogenes (E6/E7). Primary cultures from normal placentas and cervical cancer tissues; tumor cell lines from cervix, choriocarcinoma, keratinocytes, and lung; and normal cell lines from vascular endothelium, keratinocytes, and lung were used. Reverse transcription-PCR (RT-PCR) experiments and Southern blot analysis showed Eag1 expression in all of the cancer cell types, normal trophoblasts, and vascular endothelium, in contrast to normal keratinocytes and lung cells. Estradiol and antiestrogens regulated Eag1 in a cell type-dependent manner. Real-time RT-PCR experiments in HeLa cells showed that Eag1 estrogenic regulation was strongly associated with the expression of estrogen receptor-alpha. Eag1 protein was detected by monoclonal antibodies in normal placenta and placental blood vessels. Patch-clamp recordings in normal trophoblasts treated with estradiol exhibited potassium currents resembling Eag1 channel activity. Eag1 gene expression in keratinocytes depended either on cellular immortalization or the presence of HPV oncogenes. Eag1 protein was found in keratinocytes transfected with E6/E7 HPV oncogenes. Cell proliferation of E6/E7 keratinocytes was decreased by Eag1 antibodies inhibiting channel activity and by the nonspecific Eag1 inhibitors imipramine and astemizole; the latter also increased apoptosis. Our results propose novel oncogenic mechanisms of estrogen/antiestrogen use and HPV infection. We also suggest Eag1 as an early indicator of cell proliferation leading to malignancies and a therapeutic target at early stages of cellular hyperproliferation.

Fe(2+) substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization.

PubMed

Behera, Rabindra K; Torres, Rodrigo; Tosha, Takehiko; Bradley, Justin M; Goulding, Celia W; Theil, Elizabeth C

2015-09-01

Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3·H2O), by moving cytoplasmic Fe(2+) through intracage ion channels to cage-embedded enzyme (2Fe(2+)/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe(2+) movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one -CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650 nm (DFP λ max). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe(3+)-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: (1) narrower interior ion channel openings/pores; (2) increased numbers of ion channel protein-metal binding sites, and (3) a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells.

Fe2+ Substrate Transport through Ferritin Protein Cage Ion Channels Influences Enzyme Activity and Biomineralization

PubMed Central

Behera, Rabindra K.; Torres, Rodrigo; Tosha, Takehiko; Bradley, Justin M.; Goulding, Celia W.; Theil, Elizabeth C.

2015-01-01

Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3.H2O), by moving cytoplasmic Fe2+ through intracage ion channels to cage-embedded enzyme (2Fe2+/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe2+ movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one – CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650nm (DFP λmax). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe3+-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: 1. narrower interior ion channel openings/pores, 2. increased numbers of ion channel protein-metal binding sites, and 3. a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells. PMID:26202907

The Transmembrane Region of Guard Cell SLAC1 Channels Perceives CO2 Signals via an ABA-Independent Pathway in Arabidopsis.

PubMed

Yamamoto, Yoshiko; Negi, Juntaro; Wang, Cun; Isogai, Yasuhiro; Schroeder, Julian I; Iba, Koh

2016-02-01

The guard cell S-type anion channel, SLOW ANION CHANNEL1 (SLAC1), a key component in the control of stomatal movements, is activated in response to CO2 and abscisic acid (ABA). Several amino acids existing in the N-terminal region of SLAC1 are involved in regulating its activity via phosphorylation in the ABA response. However, little is known about sites involved in CO2 signal perception. To dissect sites that are necessary for the stomatal CO2 response, we performed slac1 complementation experiments using transgenic plants expressing truncated SLAC1 proteins. Measurements of gas exchange and stomatal apertures in the truncated transgenic lines in response to CO2 and ABA revealed that sites involved in the stomatal CO2 response exist in the transmembrane region and do not require the SLAC1 N and C termini. CO2 and ABA regulation of S-type anion channel activity in guard cells of the transgenic lines confirmed these results. In vivo site-directed mutagenesis experiments targeted to amino acids within the transmembrane region of SLAC1 raise the possibility that two tyrosine residues exposed on the membrane are involved in the stomatal CO2 response. © 2016 American Society of Plant Biologists. All rights reserved.

Leptin-mediated ion channel regulation: PI3K pathways, physiological role, and therapeutic potential.

PubMed

Gavello, Daniela; Carbone, Emilio; Carabelli, Valentina

2016-07-03

Leptin is produced by adipose tissue and identified as a "satiety signal," informing the brain when the body has consumed enough food. Specific areas of the hypothalamus express leptin receptors (LEPRs) and are the primary site of leptin action for body weight regulation. In response to leptin, appetite is suppressed and energy expenditure allowed. Beside this hypothalamic action, leptin targets other brain areas in addition to neuroendocrine cells. LEPRs are expressed also in the hippocampus, neocortex, cerebellum, substantia nigra, pancreatic β-cells, and chromaffin cells of the adrenal gland. It is intriguing how leptin is able to activate different ionic conductances, thus affecting excitability, synaptic plasticity and neurotransmitter release, depending on the target cell. Most of the intracellular pathways activated by leptin and directed to ion channels involve PI3K, which in turn phosphorylates different downstream substrates, although parallel pathways involve AMPK and MAPK. In this review we will describe the effects of leptin on BK, KATP, KV, CaV, TRPC, NMDAR and AMPAR channels and clarify the landscape of pathways involved. Given the ability of leptin to influence neuronal excitability and synaptic plasticity by modulating ion channels activity, we also provide a short overview of the growing potentiality of leptin as therapeutic agent for treating neurological disorders.

The Transmembrane Region of Guard Cell SLAC1 Channels Perceives CO2 Signals via an ABA-Independent Pathway in Arabidopsis

PubMed Central

Yamamoto, Yoshiko; Negi, Juntaro; Isogai, Yasuhiro; Schroeder, Julian I.; Iba, Koh

2016-01-01

The guard cell S-type anion channel, SLOW ANION CHANNEL1 (SLAC1), a key component in the control of stomatal movements, is activated in response to CO2 and abscisic acid (ABA). Several amino acids existing in the N-terminal region of SLAC1 are involved in regulating its activity via phosphorylation in the ABA response. However, little is known about sites involved in CO2 signal perception. To dissect sites that are necessary for the stomatal CO2 response, we performed slac1 complementation experiments using transgenic plants expressing truncated SLAC1 proteins. Measurements of gas exchange and stomatal apertures in the truncated transgenic lines in response to CO2 and ABA revealed that sites involved in the stomatal CO2 response exist in the transmembrane region and do not require the SLAC1 N and C termini. CO2 and ABA regulation of S-type anion channel activity in guard cells of the transgenic lines confirmed these results. In vivo site-directed mutagenesis experiments targeted to amino acids within the transmembrane region of SLAC1 raise the possibility that two tyrosine residues exposed on the membrane are involved in the stomatal CO2 response. PMID:26764376

Structural and Biochemical Consequences of Disease-Causing Mutations in the Ankyrin Repeat Domain of the Human TRPV4 Channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inada, Hitoshi; Procko, Erik; Sotomayor, Marcos

2012-10-23

The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Fingermore » 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.« less

Targeting G-Protein Signaling for the Therapeutics of Prostate Tumor Bone Metastases and the Associated Chronic Bone Pain

DTIC Science & Technology

2014-07-01

sensing receptor channels, such as TRPV1 , such that the channels are constitutively activated, leading to the sensation of chronic pain without any...Cancer Pain, Heterotrimeric G protein betagamma subunits, G protein coupled receptors (GPCRs), TRPV1 , Nociceptor Sensitization 3. Overall project...well as mediating GPCR-regulated TRPV1 channel function in cultured mouse sensory neurons (Aim 1). Major Goal/Objective 1: Determine the role of G

Role for the TRPV1 channel in insulin secretion from pancreatic beta cells.

PubMed

Diaz-Garcia, Carlos Manlio; Morales-Lázaro, Sara L; Sánchez-Soto, Carmen; Velasco, Myrian; Rosenbaum, Tamara; Hiriart, Marcia

2014-06-01

Transient receptor potential channels have been put forward as regulators of insulin secretion. A role for the TRPV1 ion channel in insulin secretion has been suggested in pancreatic beta cell lines. We explored whether TRPV1 is functionally expressed in RINm5F and primary beta cells from neonate and adult rats. We examined if capsaicin could activate cationic non-selective currents. Our results show that TRPV1 channels are not functional in insulin-secreting cells, since capsaicin did not produce current activation, not even under culture conditions known to induce the expression of other ion channels in these cells. Although TRPV1 channels seem to be irrelevant for the physiology of isolated beta cells, they may play a role in glucose homeostasis acting through the nerve fibers that regulate islet function. At the physiological level, we observed that Trpv1 (-/-) mice presented lower fasting insulin levels than their wild-type littermates, however, we did not find differences between these experimental groups nor in the glucose tolerance test or in the insulin secretion. However, we did find that the Trpv1 (-/-) mice exhibited a higher insulin sensitivity compared to their wild-type counterparts. Our results demonstrate that TRPV1 does not contribute to glucose-induced insulin secretion in beta cells as was previously thought, but it is possible that it may control insulin sensitivity.

24 CFR 4100.3 - Field activities.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 5 2010-04-01 2010-04-01 false Field activities. 4100.3 Section 4100.3 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) NEIGHBORHOOD REINVESTMENT CORPORATION ORGANIZATION AND CHANNELING OF FUNCTIONS § 4100.3 Field activities. The...

24 CFR 4100.3 - Field activities.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 5 2011-04-01 2011-04-01 false Field activities. 4100.3 Section 4100.3 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) NEIGHBORHOOD REINVESTMENT CORPORATION ORGANIZATION AND CHANNELING OF FUNCTIONS § 4100.3 Field activities. The...

The role of ATP-sensitive potassium channels in cellular function and protection in the cardiovascular system.

PubMed

Tinker, Andrew; Aziz, Qadeer; Thomas, Alison

2014-01-01

ATP-sensitive potassium channels (K(ATP)) are widely distributed and present in a number of tissues including muscle, pancreatic beta cells and the brain. Their activity is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels. Thus, they link cellular metabolism with membrane excitability. Recent studies using genetically modified mice and genomic studies in patients have implicated K(ATP) channels in a number of physiological and pathological processes. In this review, we focus on their role in cellular function and protection particularly in the cardiovascular system. © 2013 The British Pharmacological Society.

VOLTAGE-GATED POTASSIUM CHANNELS AT THE CROSSROADS OF NEURONAL FUNCTION, ISCHEMIC TOLERANCE, AND NEURODEGENERATION

PubMed Central

Shah, Niyathi Hegde; Aizenman, Elias

2013-01-01

Voltage-gated potassium (Kv) channels are widely expressed in the central and peripheral nervous system, and are crucial mediators of neuronal excitability. Importantly, these channels also actively participate in cellular and molecular signaling pathways that regulate the life and death of neurons. Injury-mediated increased K+ efflux through Kv2.1 channels promotes neuronal apoptosis, contributing to widespread neuronal loss in neurodegenerative disorders such as Alzheimer’s disease and stroke. In contrast, some forms of neuronal activity can dramatically alter Kv2.1 channel phosphorylation levels and influence their localization. These changes are normally accompanied by modifications in channel voltage-dependence, which may be neuroprotective within the context of ischemic injury. Kv1 and Kv7 channel dysfunction leads to neuronal hyperexcitability that critically contributes to the pathophysiology of human clinical disorders such as episodic ataxia and epilepsy. This review summarizes the neurotoxic, neuroprotective, and neuroregulatory roles of Kv channels, and highlights the consequences of Kv channel dysfunction on neuronal physiology. The studies described in this review thus underscore the importance of normal Kv channel function in neurons, and emphasize the therapeutic potential of targeting Kv channels in the treatment of a wide range of neurological diseases. PMID:24323720

Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels

PubMed Central

Castillo, Karen; Contreras, Gustavo F.; Pupo, Amaury; Torres, Yolima P.; Neely, Alan; González, Carlos; Latorre, Ramon

2015-01-01

Being activated by depolarizing voltages and increases in cytoplasmic Ca2+, voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation. PMID:25825713

Questioning regulation of two-pore channels by NAADP

PubMed Central

Marchant, Jonathan S.; Patel, Sandip

2014-01-01

NAADP is a potent Ca2+ mobilizing messenger [1–3]. Since its discovery in 1995 [4] a considerable volume of literature has shown that NAADP couples cell stimulation to endolysosomal Ca2+ release and thereby the regulation of many cellular functions [5]. However definition of its molecular mechanism of action has proved far from easy. Since 2009, a consensus emerged as several independent groups coalesced upon the two-pore channel (TPC) family as NAADP-activated channels essential for Ca2+ release from endolysosomal Ca2+ stores [6–8]. However this view has been recently challenged by data clearly showing that TPCs function as Na+-selective channels apparently insensitive to NAADP [9;10]. Given the two fundamental characteristics defining an ion channel comprise the opening stimulus and the nature of the permeant ions, scrutiny of these seeming irreconcilable viewpoints is essential. The purpose of this commentary is to distil the remaining consensus while interrogating these divergent viewpoints. From this analysis, critical experimental needs are identified. PMID:24829847

Connexin 43 and ATP-sensitive potassium channels crosstalk: a missing link in hypoxia/ischemia stress.

PubMed

Ahmad Waza, Ajaz; Ahmad Bhat, Shabir; Ul Hussain, Mahboob; Ganai, Bashir A

2018-02-01

Connexin 43 (Cx43) is a gap junction protein expressed in various tissues and organs of vertebrates. Besides functioning as a gap junction, Cx43 also regulates diverse cellular processes like cell growth and differentiation, cell migration, cell survival, etc. Cx43 is critical for normal cardiac functioning and is therefore abundantly expressed in cardiomyocytes. On the other hand, ATP-sensitive potassium (K ATP ) channels are metabolic sensors converting metabolic changes into electrical activity. These channels are important in maintaining the neurotransmitter release, smooth muscle relaxation, cardiac action potential repolarization, normal physiology of cellular repolarization, insulin secretion and immune function. Cx43 and K ATP channels are part of the same signaling pathway, regulating cell survival during stress conditions and ischemia/hypoxia preconditioning. However, the underlying molecular mechanism for their combined role in ischemia/hypoxia preconditioning is largely unknown. The current review focuses on understanding the molecular mechanism responsible for the coordinated role of Cx43 and K ATP channel protein in protecting cardiomyocytes against ischemia/hypoxia stress.

Opioid, cannabinoid, and transient receptor potential (TRP) systems: effects on body temperature

PubMed Central

Rawls, Scott M.; Benamar, Khalid

2014-01-01

Cannabinoid and opioid drugs produce marked changes in body temperature. Recent findings have extended our knowledge about the thermoregulatory effects of cannabinoids and opioids, particularly as related to delta opioid receptors, endogenous systems, and transient receptor potential (TRP) channels. Although delta opioid receptors were originally thought to play only a minor role in thermoregulation compared to mu and kappa opioid receptors, their activation has been shown to produce hypothermia in multiple species. Endogenous opioids and cannabinoids also regulate body temperature. Mu and kappa opioid receptors are thought to be in tonic balance, with mu and kappa receptor activation producing hyperthermia and hypothermia, respectively. Endocannabinoids participate in the febrile response, but more studies are needed to determine if a cannabinoid CB1 receptor tone exerts control over basal body temperature. A particularly intense research focus is TRP channels, where TRPV1 channel activation produces hypothermia whereas TRPA1 and TRPM8 channel activation causes hyperthermia. The marked hyperthermia produced by TRPV1 channel antagonists suggests these warm channels tonically control body temperature. A better understanding of the roles of cannabinoid, opioid, and TRP systems in thermoregulation may have broad clinical implications and provide insights into interactions among neurotransmitter systems involved in thermoregulation. PMID:21622235

Loss of Ca2+-mediated ion transport during colitis correlates with reduced ion transport responses to a Ca2+-activated K+ channel opener

PubMed Central

Hirota, Christina L; McKay, Derek M

2009-01-01

Background and purpose: Epithelial surface hydration is critical for proper gut function. However, colonic tissues from individuals with inflammatory bowel disease or animals with colitis are hyporesponsive to Cl− secretagogues. The Cl− secretory responses to the muscarinic receptor agonist bethanechol are virtually absent in colons of mice with dextran sodium sulphate (DSS)-induced colitis. Our aim was to define the mechanism underlying this cholinergic hyporesponsiveness. Experimental approach: Colitis was induced by 4% DSS water, given orally. Epithelial ion transport was measured in Ussing chambers. Colonic crypts were isolated and processed for mRNA expression via RT-PCR and protein expression via immunoblotting and immunolocalization. Key results: Expression of muscarinic M3 receptors in colonic epithelium was not decreased during colitis. Short-circuit current (ISC) responses to other Ca2+-dependent secretagogues (histamine, thapsigargin, cyclopiazonic acid and calcium ionophore) were either absent or severely attenuated in colonic tissue from DSS-treated mice. mRNA levels of several ion transport molecules (a Ca2+-regulated Cl− channel, the intermediate-conductance Ca2+-activated K+ channel, the cystic fibrosis transmembrane conductance regulator, the Na+/K+-ATPase pump or the Na+/K+/2Cl− co-transporter) were not reduced in colonic crypts from DSS-treated mice. However, protein expression of Na+/K+-ATPase α1 subunits was decreased twofold during colitis. Activation of Ca2+-activated K+ channels increased ISC significantly less in DSS colons compared with control, as did the protein kinase C activator, phorbol 12-myristate 13-acetate. Conclusions and implications: Decreased Na+/K+-ATPase expression probably contributes to overall epithelial hyporesponsiveness during colitis, while dysfunctional K+ channels may account, at least partially, for lack of epithelial secretory responses to Ca2+-mediated secretagogues. PMID:19298254

Endothelins activate Ca(2+)-gated K(+) channels via endothelin B receptors in CD-1 mouse erythrocytes.

PubMed

Rivera, A; Rotter, M A; Brugnara, C

1999-10-01

Cell dehydration mediated by Ca(2+)-activated K(+) channels plays an important role in the pathogenesis of sickle cell disease. CD-1 mouse erythrocytes possess a Ca(2+)-activated K(+) channel (Gardos channel) with maximal velocity (V(max)) of 0.154 +/- 0.02 mmol. l cells(-1). min(-1) and an affinity constant (K(0.5)) for Ca(2+) of 286 +/- 83 nM in the presence of A-23187. Cells pretreated with 500 nM endothelin-1 (ET-1) increased their V(max) by 88 +/- 9% (n = 8) and decreased their K(0.5) for Ca(2+) to 139 +/- 63 nM (P < 0.05; n = 4). Activation of the Gardos channel resulted in an EC(50) of 75 +/- 20 nM for ET-1 and 374 +/- 97 nM for ET-3. Analysis of the affinity of unlabeled ET-1 for its receptor showed two classes of binding sites with apparent dissociation constants of 167 +/- 51 and 785 +/- 143 nM and with capacity of binding sites of 298 +/- 38 and 1,568 +/- 211 sites/cell, respectively. The Gardos channel was activated by the endothelin B (ET(B)) receptor agonist IRL 1620 and inhibited by BQ-788, demonstrating the involvement of ET(B) receptors. Calphostin C inhibited 73% of ET-1-induced Gardos activation and 84% of the ET-1-induced membrane protein kinase C activity. Thus endothelins regulate erythrocyte Gardos channels via ET(B) receptors and a calphostin-sensitive mechanism.

Control of Excitation/Inhibition Balance in a Hippocampal Circuit by Calcium Sensor Protein Regulation of Presynaptic Calcium Channels.

PubMed

Nanou, Evanthia; Lee, Amy; Catterall, William A

2018-05-02

Activity-dependent regulation controls the balance of synaptic excitation to inhibition in neural circuits, and disruption of this regulation impairs learning and memory and causes many neurological disorders. The molecular mechanisms underlying short-term synaptic plasticity are incompletely understood, and their role in inhibitory synapses remains uncertain. Here we show that regulation of voltage-gated calcium (Ca 2+ ) channel type 2.1 (Ca V 2.1) by neuronal Ca 2+ sensor (CaS) proteins controls synaptic plasticity and excitation/inhibition balance in a hippocampal circuit. Prevention of CaS protein regulation by introducing the IM-AA mutation in Ca V 2.1 channels in male and female mice impairs short-term synaptic facilitation at excitatory synapses of CA3 pyramidal neurons onto parvalbumin (PV)-expressing basket cells. In sharp contrast, the IM-AA mutation abolishes rapid synaptic depression in the inhibitory synapses of PV basket cells onto CA1 pyramidal neurons. These results show that CaS protein regulation of facilitation and inactivation of Ca V 2.1 channels controls the direction of short-term plasticity at these two synapses. Deletion of the CaS protein CaBP1/caldendrin also blocks rapid depression at PV-CA1 synapses, implicating its upregulation of inactivation of Ca V 2.1 channels in control of short-term synaptic plasticity at this inhibitory synapse. Studies of local-circuit function revealed reduced inhibition of CA1 pyramidal neurons by the disynaptic pathway from CA3 pyramidal cells via PV basket cells and greatly increased excitation/inhibition ratio of the direct excitatory input versus indirect inhibitory input from CA3 pyramidal neurons to CA1 pyramidal neurons. This striking defect in local-circuit function may contribute to the dramatic impairment of spatial learning and memory in IM-AA mice. SIGNIFICANCE STATEMENT Many forms of short-term synaptic plasticity in neuronal circuits rely on regulation of presynaptic voltage-gated Ca 2+ (Ca V ) channels. Regulation of Ca V 2.1 channels by neuronal calcium sensor (CaS) proteins controls short-term synaptic plasticity. Here we demonstrate a direct link between regulation of Ca V 2.1 channels and short-term synaptic plasticity in native hippocampal excitatory and inhibitory synapses. We also identify CaBP1/caldendrin as the calcium sensor interacting with Ca V 2.1 channels to mediate rapid synaptic depression in the inhibitory hippocampal synapses of parvalbumin-expressing basket cells to CA1 pyramidal cells. Disruption of this regulation causes altered short-term plasticity and impaired balance of hippocampal excitatory to inhibitory circuits. Copyright © 2018 the authors 0270-6474/18/384430-11$15.00/0.

Naringin directly activates inwardly rectifying potassium channels at an overlapping binding site to tertiapin-Q

PubMed Central

Yow, Tin T; Pera, Elena; Absalom, Nathan; Heblinski, Marika; Johnston, Graham AR; Hanrahan, Jane R; Chebib, Mary

2011-01-01

BACKGROUND G protein-coupled inwardly rectifying potassium (KIR3) channels are important proteins that regulate numerous physiological processes including excitatory responses in the CNS and the control of heart rate. Flavonoids have been shown to have significant health benefits and are a diverse source of compounds for identifying agents with novel mechanisms of action. EXPERIMENTAL APPROACH The flavonoid glycoside, naringin, was evaluated on recombinant human KIR3.1–3.4 and KIR3.1–3.2 expressed in Xenopus oocytes using two-electrode voltage clamp methods. In addition, we evaluated the activity of naringin alone and in the presence of the KIR3 channel blocker tertiapin-Q (0.5 nM, 1 nM and 3 nM) at recombinant KIR3.1–3.4 channels. Site-directed mutagenesis was used to identify amino acids within the M1–M2 loop of the KIR3.1F137S mutant channel important for naringin's activity. KEY RESULTS Naringin (100 µM) had minimal effect on uninjected oocytes but activated KIR3.1–3.4 and KIR3.1–3.2 channels. The activation by naringin of KIR3.1–3.4 channels was inhibited by tertiapin-Q in a competitive manner. An alanine-scan performed on the KIR3.1F137S mutant channel, replacing one by one aromatic amino acids within the M1–M2 loop, identified tyrosines 148 and 150 to be significantly contributing to the affinity of naringin as these mutations reduced the activity of naringin by 20- and 40-fold respectively. CONCLUSIONS AND IMPLICATIONS These results show that naringin is a direct activator of KIR3 channels and that tertiapin-Q shares an overlapping binding site on the KIR3.1–3.4. This is the first example of a ligand that activates KIR3 channels by binding to the extracellular M1–M2 linker of the channel. PMID:21391982

Convergent phosphomodulation of the major neuronal dendritic potassium channel Kv4.2 by pituitary adenylate cyclase-activating polypeptide.

PubMed

Gupte, Raeesa P; Kadunganattil, Suraj; Shepherd, Andrew J; Merrill, Ronald; Planer, William; Bruchas, Michael R; Strack, Stefan; Mohapatra, Durga P

2016-02-01

The endogenous neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is secreted by both neuronal and non-neuronal cells in the brain and spinal cord, in response to pathological conditions such as stroke, seizures, chronic inflammatory and neuropathic pain. PACAP has been shown to exert various neuromodulatory and neuroprotective effects. However, direct influence of PACAP on the function of intrinsically excitable ion channels that are critical to both hyperexcitation as well as cell death, remain largely unexplored. The major dendritic K(+) channel Kv4.2 is a critical regulator of neuronal excitability, back-propagating action potentials in the dendrites, and modulation of synaptic inputs. We identified, cloned and characterized the downstream signaling originating from the activation of three PACAP receptor (PAC1) isoforms that are expressed in rodent hippocampal neurons that also exhibit abundant expression of Kv4.2 protein. Activation of PAC1 by PACAP leads to phosphorylation of Kv4.2 and downregulation of channel currents, which can be attenuated by inhibition of either PKA or ERK1/2 activity. Mechanistically, this dynamic downregulation of Kv4.2 function is a consequence of reduction in the density of surface channels, without any influence on the voltage-dependence of channel activation. Interestingly, PKA-induced effects on Kv4.2 were mediated by ERK1/2 phosphorylation of the channel at two critical residues, but not by direct channel phosphorylation by PKA, suggesting a convergent phosphomodulatory signaling cascade. Altogether, our findings suggest a novel GPCR-channel signaling crosstalk between PACAP/PAC1 and Kv4.2 channel in a manner that could lead to neuronal hyperexcitability. Copyright © 2015 Elsevier Ltd. All rights reserved.

Diverse Roles for Auxiliary Subunits in Phosphorylation-Dependent Regulation of Mammalian Brain Voltage-Gated Potassium Channels

PubMed Central

Vacher, Helene; Trimmer, James S.

2012-01-01

Voltage-gated ion channels are a diverse family of signaling proteins that mediate rapid electrical signaling events. Among these, voltage-gated potassium or Kv channels are the most diverse, in part due to the large number of principal (or α) subunits and auxiliary subunits that can assemble in different combinations to generate Kv channel complexes with distinct structures and functions. The diversity of Kv channels underlies much of the variability in the active properties between different mammalian central neurons, and the dynamic changes that lead to experience-dependent plasticity in intrinsic excitability. Recent studies have revealed that Kv channel α subunits and auxiliary subunits are extensively phosphorylated, contributing to additional structural and functional diversity. Here we highlight recent studies that show that auxiliary subunits exert some of their profound effects on dendritic Kv4 and axonal Kv1 channels through phosphorylation-dependent mechanisms, either due to phosphorylation on the auxiliary subunit itself, or by influencing the extent and/or impact of α subunit phosphorylation. The complex effects of auxiliary subunits and phosphorylation provide a potent mechanism to generate additional diversity in the structure and function of Kv4 and Kv1 channels, as well as allowing for dynamic reversible regulation of these important ion channels. PMID:21822597

Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density

NASA Astrophysics Data System (ADS)

Ferron, Laurent; Nieto-Rostro, Manuela; Cassidy, John S.; Dolphin, Annette C.

2014-04-01

Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (CaV) channels. Here we show that the functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases CaV channel density in somata and in presynaptic terminals. We then show that FMRP controls CaV2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and CaV2.2 occurs between the carboxy-terminal domain of FMRP and domains of CaV2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via CaV2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.

75 FR 67214 - Special Local Regulations for Marine Events; Wrightsville Channel, Wrightsville Beach, NC

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-02

... side activities in the event area. The category of water activities includes but is not limited to sail boat regattas, boat parades, power boat racing, swimming events, crew racing, and sail board racing...

Channeling the Central Dogma

PubMed Central

Calabrese, Ronald L.

2014-01-01

How do neurons and networks achieve their characteristic electrical activity, regulate this activity homeostatically, and yet show population variability in expression? O'Leary et al. address some of these thorny questions in this theoretical analysis that starts with the Central Dogma. PMID:24853932

Cystic Fibrosis Gene Encodes a cAMP-Dependent Chloride Channel in Heart

NASA Astrophysics Data System (ADS)

Hart, Padraig; Warth, John D.; Levesque, Paul C.; Collier, Mei Lin; Geary, Yvonne; Horowitz, Burton; Hume, Joseph R.

1996-06-01

cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significnatly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.

DOR activation inhibits anoxic/ischemic Na+ influx through Na+ channels via PKC mechanisms in the cortex.

PubMed

Chao, Dongman; He, Xiaozhou; Yang, Yilin; Bazzy-Asaad, Alia; Lazarus, Lawrence H; Balboni, Gianfranco; Kim, Dong H; Xia, Ying

2012-08-01

Activation of delta-opioid receptors (DOR) is neuroprotective against hypoxic/ischemic injury in the cortex, which is at least partially related to its action against hypoxic/ischemic disruption of ionic homeostasis that triggers neuronal injury. Na(+) influx through TTX-sensitive voltage-gated Na(+) channels may be a main mechanism for hypoxia-induced disruption of K(+) homeostasis, with DOR activation attenuating the disruption of ionic homeostasis by targeting voltage-gated Na(+) channels. In the present study we examined the role of DOR in the regulation of Na(+) influx in anoxia and simulated ischemia (oxygen-glucose deprivation) as well as the effect of DOR activation on the Na(+) influx induced by a Na(+) channel opener without anoxic/ischemic stress and explored a potential PKC mechanism underlying the DOR action. We directly measured extracellular Na(+) activity in mouse cortical slices with Na(+) selective electrodes and found that (1) anoxia-induced Na(+) influx occurred mainly through TTX-sensitive Na(+) channels; (2) DOR activation inhibited the anoxia/ischemia-induced Na(+) influx; (3) veratridine, a Na(+) channel opener, enhanced the anoxia-induced Na(+) influx; this could be attenuated by DOR activation; (4) DOR activation did not reduce the anoxia-induced Na(+) influx in the presence of chelerythrine, a broad-spectrum PKC blocker; and (5) DOR effects were blocked by PKCβII peptide inhibitor, and PKCθ pseudosubstrate inhibitor, respectively. We conclude that DOR activation inhibits anoxia-induced Na(+) influx through Na(+) channels via PKC (especially PKCβII and PKCθ isoforms) dependent mechanisms in the cortex. Copyright © 2012 Elsevier Inc. All rights reserved.

Altered expression and function of small-conductance (SK) Ca2+-activated K+ channels in pilocarpine-treated epileptic rats

PubMed Central

Oliveira, Mauro S.; Skinner, Frank; Arshadmansab, Massoud F.; Garcia, Ileana; Mello, Carlos F.; Knaus, Hans-Günther; Ermolinsky, Boris S.; Pacheco Otalora, Luis F.; Garrido-Sanabria, Emilio R.

2010-01-01

Small conductance calcium (Ca2+) activated SK channels are critical regulators of neuronal excitability in hippocampus. Accordingly, these channels are thought to play a key role in controlling neuronal activity in acute models of epilepsy. In this study, we investigate the expression and function of SK channels in the pilocarpine model of mesial temporal lobe epilepsy. For this purpose, protein expression was assessed using western blotting assays and gene expression was analyzed using TaqMan-based probes and the quantitative real-time polymerase chain reaction (qPCR) comparative method delta-delta cycle threshold (ΔΔCT) in samples extracted from control and epileptic rats. In addition, the effect of SK channel antagonist UCL1684 and agonist NS309 on CA1 evoked population spikes was studied in hippocampal slices. Western blotting analysis showed a significant reduction in the expression of SK1 and SK2 channels at 10 days following status epilepticus (SE), but levels recovered at 1 month and at more than 2 months after SE. In contrast, a significant down-regulation of SK3 channels was detected after 10 days of SE. Analysis of gene expression by qPCR revealed a significant reduction of transcripts for SK2 (Kcnn1) and SK3 (Kcnn3) channels as early as 10 days following pilocarpine-induced SE and during the chronic phase of the pilocarpine model. Moreover, bath application of UCL1684 (100 nM for 15 min) induced a significant increase of the population spike amplitude and number of spikes in the hippocampal CA1 area of slices obtained control and chronic epileptic rats. This effect was obliterated by co-administration of UCL1684 with SK channel agonist NS309 (1 μM). Application of NS309 failed to modify population spikes in the CA1 area of slices taken from control and epileptic rats. These data indicate an abnormal expression of SK channels and a possible dysfunction of these channels in experimental MTLE. PMID:20553876

Voltage-independent inhibition of Ca(V)2.2 channels is delimited to a specific region of the membrane potential in rat SCG neurons.

PubMed

Vivas, Oscar; Arenas, Isabel; García, David E

2012-06-01

Neurotransmitters and hormones regulate Ca(V)2.2 channels through a voltage-independent pathway which is not well understood. It has been suggested that this voltage-independent inhibition is constant at all membrane voltages. However, changes in the percent of voltage-independent inhibition of Ca(V)2.2 have not been tested within a physiological voltage range. Here, we used a double-pulse protocol to isolate the voltage-independent inhibition of Ca(V)2.2 channels induced by noradrenaline in rat superior cervical ganglion neurons. To assess changes in the percent of the voltage-independent inhibition, the activation voltage of the channels was tested between -40 and +40 mV. We found that the percent of voltage-independent inhibition induced by noradrenaline changed with the activation voltage used. In addition, voltage-independent inhibition induced by oxo-M, a muscarinic agonist, exhibited the same dependence on activation voltage, which supports that this pattern is not exclusive for adrenergic activation. Our results suggested that voltage-independent inhibition of Ca(V)2.2 channels depends on the activation voltage of the channel in a physiological voltage range. This may have relevant implications in the understanding of the mechanism involved in voltage-independent inhibition.

KCNQ1, KCNE2, and Na+-Coupled Solute Transporters Form Reciprocally Regulating Complexes that Affect Neuronal Excitability

PubMed Central

Abbott, Geoffrey W.; Tai, Kwok-Keung; Neverisky, Daniel; Hansler, Alex; Hu, Zhaoyang; Roepke, Torsten K.; Lerner, Daniel J.; Chen, Qiuying; Liu, Li; Zupan, Bojana; Toth, Miklos; Haynes, Robin; Huang, Xiaoping; Demirbas, Didem; Buccafusca, Roberto; Gross, Steven S.; Kanda, Vikram A.; Berry, Gerard T.

2014-01-01

Na+-coupled solute transport is crucial for the uptake of nutrients and metabolic precursors, such as myo-inositol, an important osmolyte and precursor for various cell signaling molecules. Here, we found that various solute transporters and potassium channel subunits formed complexes and reciprocally regulated each other in vitro and in vivo. Global metabolite profiling revealed that mice lacking KCNE2, a K+ channel β subunit, showed a reduction in the myo-inositol concentration in cerebrospinal fluid (CSF) but not in serum. Increased behavorial responsiveness to stress and seizure susceptibility in Kcne2−/− mice were alleviated by injections of myo-inositol. Suspecting a defect in myo-inositol transport, we found that KCNE2 and KCNQ1, a voltage-gated potassium channel α subunit, colocalized and coimmunoprecipitated with SMIT1, a Na+-coupled myo-inositol transporter, in the choroid plexus epithelium. Heterologous coexpression demonstrated that myo-inositol transport by SMIT1 was augmented by coexpression of KCNQ1 but inhibited by coexpression of both KCNQ1 and KCNE2, which form a constitutively active, heteromeric K+ channel. SMIT1 and the related transporter SMIT2 were also inhibited by a constitutively active mutant form of KCNQ1. The activity of KCNQ1 and KCNQ1-KCNE2 were augmented by SMIT1 and the glucose transporter SGLT1, but suppressed by SMIT2. Channel-transporter signaling complexes may be a widespread mechanism to facilitate solute transport and electrochemical crosstalk. PMID:24595108

KCNQ1, KCNE2, and Na+-coupled solute transporters form reciprocally regulating complexes that affect neuronal excitability.

PubMed

Abbott, Geoffrey W; Tai, Kwok-Keung; Neverisky, Daniel L; Hansler, Alex; Hu, Zhaoyang; Roepke, Torsten K; Lerner, Daniel J; Chen, Qiuying; Liu, Li; Zupan, Bojana; Toth, Miklos; Haynes, Robin; Huang, Xiaoping; Demirbas, Didem; Buccafusca, Roberto; Gross, Steven S; Kanda, Vikram A; Berry, Gerard T

2014-03-04

Na(+)-coupled solute transport is crucial for the uptake of nutrients and metabolic precursors, such as myo-inositol, an important osmolyte and precursor for various cell signaling molecules. We found that various solute transporters and potassium channel subunits formed complexes and reciprocally regulated each other in vitro and in vivo. Global metabolite profiling revealed that mice lacking KCNE2, a K(+) channel β subunit, showed a reduction in myo-inositol concentration in cerebrospinal fluid (CSF) but not in serum. Increased behavioral responsiveness to stress and seizure susceptibility in Kcne2(-/-) mice were alleviated by injections of myo-inositol. Suspecting a defect in myo-inositol transport, we found that KCNE2 and KCNQ1, a voltage-gated potassium channel α subunit, colocalized and coimmunoprecipitated with SMIT1, a Na(+)-coupled myo-inositol transporter, in the choroid plexus epithelium. Heterologous coexpression demonstrated that myo-inositol transport by SMIT1 was augmented by coexpression of KCNQ1 but was inhibited by coexpression of both KCNQ1 and KCNE2, which form a constitutively active, heteromeric K(+) channel. SMIT1 and the related transporter SMIT2 were also inhibited by a constitutively active mutant form of KCNQ1. The activities of KCNQ1 and KCNQ1-KCNE2 were augmented by SMIT1 and the glucose transporter SGLT1 but were suppressed by SMIT2. Channel-transporter signaling complexes may be a widespread mechanism to facilitate solute transport and electrochemical crosstalk.

Functional and Molecular Evidence for Kv7 Channel Subtypes in Human Detrusor from Patients with and without Bladder Outflow Obstruction

PubMed Central

Svalø, Julie; Sheykhzade, Majid; Nordling, Jørgen; Matras, Christina; Bouchelouche, Pierre

2015-01-01

The aim of the study was to investigate whether Kv7 channels and their ancillary β-subunits, KCNE, are functionally expressed in the human urinary bladder. Kv7 channels were examined at the molecular level and by functional studies using RT-qPCR and myography, respectively. We found mRNA expression of KCNQ1, KCNQ3-KCNQ5 and KCNE1-5 in the human urinary bladder from patients with normal bladder function (n = 7) and in patients with bladder outflow obstruction (n = 3). Interestingly, a 3.4-fold up-regulation of KCNQ1 was observed in the latter. The Kv7 channel subtype selective modulators, ML277 (activator of Kv7.1 channels, 10 μM) and ML213 (activator of Kv7.2, Kv7.4, Kv7.4/7.5 and Kv7.5 channels, 10 μM), reduced the tone of 1 μM carbachol pre-constricted bladder strips. XE991 (blocker of Kv7.1–7.5 channels, 10 μM) had opposing effects as it increased contractions achieved with 20 mM KPSS. Furthermore, we investigated if there is interplay between Kv7 channels and β-adrenoceptors. Using cumulative additions of isoprenaline (β-adrenoceptor agonist) and forskolin (adenylyl cyclase activator) in combination with the Kv7 channel activator and blocker, retigabine and XE991, we did not find interplay between Kv7 channels and β-adrenoceptors in the human urinary bladder. The performed gene expression analysis combined with the organ bath studies imply that compounds that activate Kv7 channels could be useful for treatment of overactive bladder syndrome. PMID:25692982

Functional and molecular evidence for Kv7 channel subtypes in human detrusor from patients with and without bladder outflow obstruction.

PubMed

Svalø, Julie; Sheykhzade, Majid; Nordling, Jørgen; Matras, Christina; Bouchelouche, Pierre

2015-01-01

The aim of the study was to investigate whether Kv7 channels and their ancillary β-subunits, KCNE, are functionally expressed in the human urinary bladder. Kv7 channels were examined at the molecular level and by functional studies using RT-qPCR and myography, respectively. We found mRNA expression of KCNQ1, KCNQ3-KCNQ5 and KCNE1-5 in the human urinary bladder from patients with normal bladder function (n = 7) and in patients with bladder outflow obstruction (n = 3). Interestingly, a 3.4-fold up-regulation of KCNQ1 was observed in the latter. The Kv7 channel subtype selective modulators, ML277 (activator of Kv7.1 channels, 10 μM) and ML213 (activator of Kv7.2, Kv7.4, Kv7.4/7.5 and Kv7.5 channels, 10 μM), reduced the tone of 1 μM carbachol pre-constricted bladder strips. XE991 (blocker of Kv7.1-7.5 channels, 10 μM) had opposing effects as it increased contractions achieved with 20 mM KPSS. Furthermore, we investigated if there is interplay between Kv7 channels and β-adrenoceptors. Using cumulative additions of isoprenaline (β-adrenoceptor agonist) and forskolin (adenylyl cyclase activator) in combination with the Kv7 channel activator and blocker, retigabine and XE991, we did not find interplay between Kv7 channels and β-adrenoceptors in the human urinary bladder. The performed gene expression analysis combined with the organ bath studies imply that compounds that activate Kv7 channels could be useful for treatment of overactive bladder syndrome.

TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell Culture Model of the Corneal Epithelium.

PubMed

Martínez-Rendón, Jacqueline; Sánchez-Guzmán, Erika; Rueda, Angélica; González, James; Gulias-Cañizo, Rosario; Aquino-Jarquín, Guillermo; Castro-Muñozledo, Federico; García-Villegas, Refugio

2017-07-01

TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca 2+ imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. J. Cell. Physiol. 232: 1794-1807, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The Hv1 proton channel responds to mechanical stimuli.

PubMed

Pathak, Medha M; Tran, Truc; Hong, Liang; Joós, Béla; Morris, Catherine E; Tombola, Francesco

2016-11-01

The voltage-gated proton channel, Hv1, is expressed in tissues throughout the body and plays important roles in pH homeostasis and regulation of NADPH oxidase. Hv1 operates in membrane compartments that experience strong mechanical forces under physiological or pathological conditions. In microglia, for example, Hv1 activity is potentiated by cell swelling and causes an increase in brain damage after stroke. The channel complex consists of two proton-permeable voltage-sensing domains (VSDs) linked by a cytoplasmic coiled-coil domain. Here, we report that these VSDs directly respond to mechanical stimuli. We find that membrane stretch facilitates Hv1 channel opening by increasing the rate of activation and shifting the steady-state activation curve to less depolarized potentials. In the presence of a transmembrane pH gradient, membrane stretch alone opens the channel without the need for strong depolarizations. The effect of membrane stretch persists for several minutes after the mechanical stimulus is turned off, suggesting that the channel switches to a "facilitated" mode in which opening occurs more readily and then slowly reverts to the normal mode observed in the absence of membrane stretch. Conductance simulations with a six-state model recapitulate all the features of the channel's response to mechanical stimulation. Hv1 mechanosensitivity thus provides a mechanistic link between channel activation in microglia and brain damage after stroke. © 2016 Pathak et al.

Phosphatidic acid modulation of Kv channel voltage sensor function

PubMed Central

Hite, Richard K; Butterwick, Joel A; MacKinnon, Roderick

2014-01-01

Membrane phospholipids can function as potent regulators of ion channel function. This study uncovers and investigates the effect of phosphatidic acid on Kv channel gating. Using the method of reconstitution into planar lipid bilayers, in which protein and lipid components are defined and controlled, we characterize two effects of phosphatidic acid. The first is a non-specific electrostatic influence on activation mediated by electric charge density on the extracellular and intracellular membrane surfaces. The second is specific to the presence of a primary phosphate group, acts only through the intracellular membrane leaflet and depends on the presence of a particular arginine residue in the voltage sensor. Intracellular phosphatidic acid accounts for a nearly 50 mV shift in the midpoint of the activation curve in a direction consistent with stabilization of the voltage sensor's closed conformation. These findings support a novel mechanism of voltage sensor regulation by the signaling lipid phosphatidic acid. DOI: http://dx.doi.org/10.7554/eLife.04366.001 PMID:25285449

Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway

PubMed Central

Sandoval, Alejandro; Duran, Paz; Gandini, María A.; Andrade, Arturo; Almanza, Angélica; Kaja, Simon; Felix, Ricardo

2018-01-01

cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated CaV1.3L-type Ca2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant CaV1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the CaVα1 ion-conducting subunit of the CaV1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca2+ macroscopic currents and impair insulin release stimulated with high K+. In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for CaV1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the CaVα1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate CaV1.3 channels and contribute to regulate insulin secretion. PMID:28807144

Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway.

PubMed

Sandoval, Alejandro; Duran, Paz; Gandini, María A; Andrade, Arturo; Almanza, Angélica; Kaja, Simon; Felix, Ricardo

2017-09-01

cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca 2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated Ca V 1.3L-type Ca 2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant Ca V 1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the Ca V α 1 ion-conducting subunit of the Ca V 1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca 2+ macroscopic currents and impair insulin release stimulated with high K + . In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for Ca V 1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the Ca V α 1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate Ca V 1.3 channels and contribute to regulate insulin secretion. Copyright © 2017 Elsevier Ltd. All rights reserved.

Disturbed Processing of Contextual Information in HCN3 Channel Deficient Mice

PubMed Central

Stieglitz, Marc S.; Fenske, Stefanie; Hammelmann, Verena; Becirovic, Elvir; Schöttle, Verena; Delorme, James E.; Schöll-Weidinger, Martha; Mader, Robert; Deussing, Jan; Wolfer, David P.; Seeliger, Mathias W.; Albrecht, Urs; Wotjak, Carsten T.; Biel, Martin; Michalakis, Stylianos; Wahl-Schott, Christian

2018-01-01

Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) in the nervous system are implicated in a variety of neuronal functions including learning and memory, regulation of vigilance states and pain. Dysfunctions or genetic loss of these channels have been shown to cause human diseases such as epilepsy, depression, schizophrenia, and Parkinson's disease. The physiological functions of HCN1 and HCN2 channels in the nervous system have been analyzed using genetic knockout mouse models. By contrast, there are no such genetic studies for HCN3 channels so far. Here, we use a HCN3-deficient (HCN3−/−) mouse line, which has been previously generated in our group to examine the expression and function of this channel in the CNS. Specifically, we investigate the role of HCN3 channels for the regulation of circadian rhythm and for the determination of behavior. Contrary to previous suggestions we find that HCN3−/− mice show normal visual, photic, and non-photic circadian function. In addition, HCN3−/− mice are impaired in processing contextual information, which is characterized by attenuated long-term extinction of contextual fear and increased fear to a neutral context upon repeated exposure. PMID:29375299

Novel treatment strategies for smooth muscle disorders: Targeting Kv7 potassium channels.

PubMed

Haick, Jennifer M; Byron, Kenneth L

2016-09-01

Smooth muscle cells provide crucial contractile functions in visceral, vascular, and lung tissues. The contractile state of smooth muscle is largely determined by their electrical excitability, which is in turn influenced by the activity of potassium channels. The activity of potassium channels sustains smooth muscle cell membrane hyperpolarization, reducing cellular excitability and thereby promoting smooth muscle relaxation. Research over the past decade has indicated an important role for Kv7 (KCNQ) voltage-gated potassium channels in the regulation of the excitability of smooth muscle cells. Expression of multiple Kv7 channel subtypes has been demonstrated in smooth muscle cells from viscera (gastrointestinal, bladder, myometrial), from the systemic and pulmonary vasculature, and from the airways of the lung, from multiple species, including humans. A number of clinically used drugs, some of which were developed to target Kv7 channels in other tissues, have been found to exert robust effects on smooth muscle Kv7 channels. Functional studies have indicated that Kv7 channel activators and inhibitors have the ability to relax and contact smooth muscle preparations, respectively, suggesting a wide range of novel applications for the pharmacological tool set. This review summarizes recent findings regarding the physiological functions of Kv7 channels in smooth muscle, and highlights potential therapeutic applications based on pharmacological targeting of smooth muscle Kv7 channels throughout the body. Published by Elsevier Inc.

Touch, Tension, and Transduction – the Function and Regulation of Piezo Ion Channels

PubMed Central

Wu, Jason; Lewis, Amanda; Grandl, Jörg

2016-01-01

In 2010, two proteins, Piezo1 and Piezo2, were identified as the long-sought molecular carriers of an excitatory mechanically activated current found in many cells. This discovery has opened the floodgates for studying a vast number of mechanotransduction processes. Over the past six years, groundbreaking research has identified Piezos as ion channels that sense light touch, proprioception, and vascular blood flow, ruled out roles for Piezos in several other mechanotransduction processes, and revealed the basic structural and functional properties of the channel. Here, we review these findings and discuss the many aspects of Piezo function that remain mysterious, including how Piezos convert a variety of mechanical stimuli into channel activation and subsequent inactivation, and what molecules and mechanisms modulate Piezo function. PMID:27743844

Touch, Tension, and Transduction - The Function and Regulation of Piezo Ion Channels.

PubMed

Wu, Jason; Lewis, Amanda H; Grandl, Jörg

2017-01-01

In 2010, two proteins, Piezo1 and Piezo2, were identified as the long-sought molecular carriers of an excitatory mechanically activated current found in many cells. This discovery has opened the floodgates for studying a vast number of mechanotransduction processes. Over the past 6 years, groundbreaking research has identified Piezos as ion channels that sense light touch, proprioception, and vascular blood flow, ruled out roles for Piezos in several other mechanotransduction processes, and revealed the basic structural and functional properties of the channel. Here, we review these findings and discuss the many aspects of Piezo function that remain mysterious, including how Piezos convert a variety of mechanical stimuli into channel activation and subsequent inactivation, and what molecules and mechanisms modulate Piezo function. Copyright © 2016 Elsevier Ltd. All rights reserved.

The L‐type Ca2+ channel facilitates abnormal metabolic activity in the cTnI‐G203S mouse model of hypertrophic cardiomyopathy

PubMed Central

Viola, Helena; Johnstone, Victoria; Cserne Szappanos, Henrietta; Richman, Tara; Tsoutsman, Tatiana; Filipovska, Aleksandra; Semsarian, Christopher

2016-01-01

Key points Genetic mutations in cardiac troponin I (cTnI) are associated with development of hypertrophic cardiomyopathy characterized by myocyte remodelling, disorganization of cytoskeletal proteins and altered energy metabolism.The L‐type Ca2+ channel is the main route for calcium influx and is crucial to cardiac excitation and contraction. The channel also regulates mitochondrial function in the heart by a functional communication between the channel and mitochondria via the cytoskeletal network.We find that L‐type Ca2+ channel kinetics are altered in cTnI‐G203S cardiac myocytes and that activation of the channel causes a significantly greater increase in mitochondrial membrane potential and metabolic activity in cTnI‐G203S cardiac myocytes.These responses occur as a result of impaired communication between the L‐type Ca2+ channel and cytoskeletal protein F‐actin, involving decreased movement of actin–myosin and block of the mitochondrial voltage‐dependent anion channel, resulting in a ‘hypermetabolic’ mitochondrial state.We propose that L‐type Ca2+ channel antagonists, such as diltiazem, might be effective in reducing the cardiomyopathy by normalizing mitochondrial metabolic activity. Abstract Genetic mutations in cardiac troponin I (cTnI) account for 5% of families with hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy is associated with disorganization of cytoskeletal proteins and altered energy metabolism. The L‐type Ca2+ channel (ICa‐L) plays an important role in regulating mitochondrial function. This involves a functional communication between the channel and mitochondria via the cytoskeletal network. We investigate the role of ICa‐L in regulating mitochondrial function in 25‐ to 30‐week‐old cardiomyopathic mice expressing the human disease‐causing mutation Gly203Ser in cTnI (cTnI‐G203S). The inactivation rate of ICa‐L is significantly faster in cTnI‐G203S myocytes [cTnI‐G203S: τ1 = 40.68 ± 3.22, n = 10 vs. wild‐type (wt): τ1 = 59.05 ± 6.40, n = 6, P < 0.05]. Activation of ICa‐L caused a greater increase in mitochondrial membrane potential (Ψm, 29.19 ± 1.85%, n = 15 vs. wt: 18.84 ± 2.01%, n = 10, P < 0.05) and metabolic activity (24.40 ± 6.46%, n = 8 vs. wt: 9.98 ± 1.57%, n = 9, P < 0.05). The responses occurred because of impaired communication between ICa‐L and F‐actin, involving lack of dynamic movement of actin–myosin and block of the mitochondrial voltage‐dependent anion channel. Similar responses were observed in precardiomyopathic mice. ICa‐L antagonists nisoldipine and diltiazem decreased Ψm to basal levels. We conclude that the Gly203Ser mutation leads to impaired functional communication between ICa‐L and mitochondria, resulting in a ‘hypermetabolic’ state. This might contribute to development of cTnI‐G203S cardiomyopathy because the response is present in young precardiomyopathic mice. ICa‐L antagonists might be effective in reducing the cardiomyopathy by altering mitochondrial function. PMID:27062056

Two-pore channels (TPCs): Novel voltage-gated ion channels with pleiotropic functions

PubMed Central

Feijóo-Bandín, Sandra; García-Vence, María; García-Rúa, Vanessa; Roselló-Lletí, Esther; Portolés, Manuel; Rivera, Miguel; González-Juanatey, José Ramón; Lago, Francisca

2017-01-01

ABSTRACT Two-pore channels (TPC1-3) comprise a subfamily of the eukaryotic voltage-gated ion channels (VGICs) superfamily that are mainly expressed in acidic stores in plants and animals. TPCS are widespread across the animal kingdom, with primates, mice and rats lacking TPC3, and mainly act as Ca+ and Na+ channels, although it was also suggested that they could be permeable to other ions. Nowadays, TPCs have been related to the development of different diseases, including Parkinson´s disease, obesity or myocardial ischemia. Due to this, their study has raised the interest of the scientific community to try to understand their mechanism of action in order to be able to develop an efficient drug that could regulate TPCs activity. In this review, we will provide an updated view regarding TPCs structure, function and activation, as well as their role in different pathophysiological processes. PMID:27440385

Ubiquitin ligase Nedd4-2 modulates Kv1.3 current amplitude and ion channel protein targeting

PubMed Central

Velez, Patricio; Schwartz, Austin B.; Iyer, Subashini R.; Warrington, Anthony

2016-01-01

Voltage-dependent potassium channels (Kv) go beyond the stabilization of the resting potential and regulate biochemical pathways, regulate intracellular signaling, and detect energy homeostasis. Because targeted deletion and pharmacological block of the Kv1.3 channel protein produce marked changes in metabolism, resistance to diet-induced obesity, and changes in olfactory structure and function, this investigation explored Nedd4-2-mediated ubiquitination and degradation to regulate Kv1.3 channel density. Heterologous coexpression of Nedd4-2 ligase and Kv1.3 in HEK 293 cells reduced Kv1.3 current density without modulation of kinetic properties as measured by patch-clamp electrophysiology. Modulation of current density was dependent on ligase activity and was lost through point mutation of cysteine 938 in the catalytic site of the ligase (Nedd4-2CS). Incorporation of adaptor protein Grb10 relieved Nedd4-2-induced current suppression as did application of the proteasome inhibitor Mg-132. SDS-PAGE and immunoprecipitation strategies demonstrated a channel/adaptor/ligase signalplex. Pixel immunodensity was reduced for Kv1.3 in the presence of Nedd4-2, which was eliminated upon additional incorporation of Grb10. We confirmed Nedd4-2/Grb10 coimmunoprecipitation and observed an increased immunodensity for Nedd4-2 in the presence of Kv1.3 plus Grb10, regardless of whether the catalytic site was active. Kv1.3/Nedd4-2 were reciprocally coimmunoprecipated, whereby mutation of the COOH-terminal, SH3-recognition (493–498), or ubiquitination sites on Kv1.3 (lysines 467, 476, 498) retained coimmunoprecipitation, while the latter prevented the reduction in channel density. A model is presented for which an atypical interaction outside the canonical PY motif may permit channel/ligase interaction to lead to protein degradation and reduced current density, which can involve Nedd4-2/Grb10 interactions to disrupt Kv1.3 loss of current density. PMID:27146988

Renal intercalated cells and blood pressure regulation.

PubMed

Wall, Susan M

2017-12-01

Type B and non-A, non-B intercalated cells are found within the connecting tubule and the cortical collecting duct. Of these cell types, type B intercalated cells are known to mediate Cl - absorption and HCO 3 - secretion largely through pendrin-dependent Cl - /HCO 3 - exchange. This exchange is stimulated by angiotensin II administration and is also stimulated in models of metabolic alkalosis, for instance after aldosterone or NaHCO 3 administration. In some rodent models, pendrin-mediated HCO 3 - secretion modulates acid-base balance. However, the role of pendrin in blood pressure regulation is likely of more physiological or clinical significance. Pendrin regulates blood pressure not only by mediating aldosterone-sensitive Cl - absorption, but also by modulating the aldosterone response for epithelial Na + channel (ENaC)-mediated Na + absorption. Pendrin regulates ENaC through changes in open channel of probability, channel surface density, and channels subunit total protein abundance. Thus, aldosterone stimulates ENaC activity through both direct and indirect effects, the latter occurring through its stimulation of pendrin expression and function. Therefore, pendrin contributes to the aldosterone pressor response. Pendrin may also modulate blood pressure in part through its action in the adrenal medulla, where it modulates the release of catecholamines, or through an indirect effect on vascular contractile force. This review describes how aldosterone and angiotensin II-induced signaling regulate pendrin and the contributory role of pendrin in distal nephron function and blood pressure.

Adenosine and adenine nucleotides as regulators of cerebral blood flow: roles of acidosis, cell swelling, and KATP channels.

PubMed

Phillis, John W

2004-01-01

A considerable volume of evidence implicates the purine adenosine in the regulation of cerebral blood flow during states such as hypotension, neural activation, hypoxia/ischemia, and hypercapnia/acidosis. The aim of this review is to describe developments in our understanding of the roles that adenosine and the adenine nucleotides play in cerebral blood flow control, with some comparisons to coronary blood flow. The first part of the review focuses on the categorization of receptors for adenosine (A1, A2A, A2B, and A3) and the adenine nucleotides, ATP and ADP (P2X and P2Y). Frequently used agonists and antagonists for these different receptors are mentioned. A description follows of the distribution of these different receptors in cerebral arterioles. The second part of the review initially deals with the literature on the release of adenosine and adenine nucleotides into the extracellular space of the brain, describing the various techniques used to make these measurements and assessing the pitfalls associated with their use. This is followed by a discussion of the factors affecting purine release, which include cell swelling and acidosis. The third section evaluates the role of smooth muscle potassium channels in controlling arteriolar diameter. There is evidence for an important role of KATP and KCa channels, but less is known about the contributions of voltage-dependent (KV) and inwardly rectifying (KIR) channels. This section ends with a discussion on the reported inhibitory effect of nitric oxide synthase inhibitors on the KATP channel and the consequences of such an action for the interpretation of much of the published work on nitric oxide as a regulator of cerebral blood flow. The fourth section evaluates the data supporting a role of adenosine and ATP in the regulation of cerebral blood flow during autoregulation, hypotension, neural activity, hypoxia/ ischemia, and hypercapnia. Studies using antagonists and potentiators of adenosine's actions have led to the conclusion that adenosine is involved in vascular flow control, matching metabolic activity to blood flow in all of these conditions, possibly with the exceptions of autoregulation at mean arterial blood pressures above approximately 60 mmHg. Evidence is presented for a major role of A2A, and a more limited role of A2B receptors, in balancing blood flow with metabolism. The primary effect of receptor occupancy is activation of KATP and KCa channels with smooth muscle relaxation and elevated blood flow rates. There are presently fewer data on ATP's participation in flow control, but recent evidence regarding glial cell control of cerebral arteriolar diameter suggests that this may be an important mechanism. The semi-final section, which briefly describes the evidence for a comparable role of adenosine in regulating coronary blood flow, is followed by a concluding statement reaffirming the importance of adenosine as a cerebral blood flow regulator.

Dams and water developments

Treesearch

Robert H. Schueneman

1979-01-01

The U.S. Army Corps of Engineers is involved in the construction and regulation of many activities relating to water resource development. Such activities include dams and reservoirs, channelization and erosion control on rivers and tributaries, and coastal works. These activities can result in an array of visual effects depending on the specific activity type and...

A mechanism regulating G protein-coupled receptor signaling that requires cycles of protein palmitoylation and depalmitoylation.

PubMed

Jia, Lixia; Chisari, Mariangela; Maktabi, Mohammad H; Sobieski, Courtney; Zhou, Hao; Konopko, Aaron M; Martin, Brent R; Mennerick, Steven J; Blumer, Kendall J

2014-02-28

Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K(+) (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.

TRP channels: sensors and transducers of gasotransmitter signals

PubMed Central

Takahashi, Nobuaki; Kozai, Daisuke; Mori, Yasuo

2012-01-01

The transient receptor potential (trp) gene superfamily encodes cation channels that act as multimodal sensors for a wide variety of stimuli from outside and inside the cell. Upon sensing, they transduce electrical and Ca2+ signals via their cation channel activities. These functional features of TRP channels allow the body to react and adapt to different forms of environmental changes. Indeed, members of one class of TRP channels have emerged as sensors of gaseous messenger molecules that control various cellular processes. Nitric oxide (NO), a vasoactive gaseous molecule, regulates TRP channels directly via cysteine (Cys) S-nitrosylation or indirectly via cyclic GMP (cGMP)/protein kinase G (PKG)-dependent phosphorylation. Recent studies have revealed that changes in the availability of molecular oxygen (O2) also control the activation of TRP channels. Anoxia induced by O2-glucose deprivation and severe hypoxia (1% O2) activates TRPM7 and TRPC6, respectively, whereas TRPA1 has recently been identified as a novel sensor of hyperoxia and mild hypoxia (15% O2) in vagal and sensory neurons. TRPA1 also detects other gaseous molecules such as hydrogen sulfide (H2S) and carbon dioxide (CO2). In this review, we focus on how signaling by gaseous molecules is sensed and integrated by TRP channels. PMID:22934072

Cell Surface Expression of Human Ether-a-go-go-related Gene (hERG) Channels Is Regulated by Caveolin-3 Protein via the Ubiquitin Ligase Nedd4-2*

PubMed Central

Guo, Jun; Wang, Tingzhong; Li, Xian; Shallow, Heidi; Yang, Tonghua; Li, Wentao; Xu, Jianmin; Fridman, Michael D.; Yang, Xiaolong; Zhang, Shetuan

2012-01-01

The human ether-a-go-go-related gene (hERG) encodes the rapidly activating delayed rectifier potassium channel (IKr) which plays an important role in cardiac repolarization. A reduction or increase in hERG current can cause long or short QT syndrome, respectively, leading to fatal cardiac arrhythmias. The channel density in the plasma membrane is a key determinant of the whole cell current amplitude. To gain insight into the molecular mechanisms for the regulation of hERG density at the plasma membrane, we used whole cell voltage clamp, Western blotting, and immunocytochemical methods to investigate the effects of an integral membrane protein, caveolin-3 (Cav3) on hERG expression levels. Our data demonstrate that Cav3, hERG, and ubiquitin-ligase Nedd4-2 interact with each other and form a complex. Expression of Cav3 thus enhances the hERG-Nedd4-2 interaction, leading to an increased ubiquitination and degradation of mature, plasma-membrane localized hERG channels. Disrupting Nedd4-2 interaction with hERG by mutations eliminates the effects of Cav3 on hERG channels. Knockdown of endogenous Cav3 or Nedd4-2 in cultured neonatal rat ventricular myocytes using siRNA led to an increase in native IKr. Our data demonstrate that hERG expression in the plasma membrane is regulated by Cav3 via Nedd4-2. These findings extend our understanding of the regulation of hERG channels and cardiac electrophysiology. PMID:22879586

Pharmacological actions of Uncaria alkaloids, rhynchophylline and isorhynchophylline.

PubMed

Shi, Jing-Shan; Yu, Jun-Xian; Chen, Xiu-Ping; Xu, Rui-Xia

2003-02-01

The pharmacological actions of Uncaria alkaloids, rhynchophylline and isorhynchophylline extracted from Uncaria rhynchophylla Miq Jacks were reviewed. The alkaloids mainly act on cardiovascular system and central nervous system including the hypotension, brachycardia, antiarrhythmia, and protection of cerebral ischemia and sedation. The active mechanisms were related to blocking of calcium channel, opening of potassium channel, and regulating of nerve transmitters transport and metabolism, etc.

A linkage analysis toolkit for studying allosteric networks in ion channels

PubMed Central

2013-01-01

A thermodynamic approach to studying allosterically regulated ion channels such as the large-conductance voltage- and Ca2+-dependent (BK) channel is presented, drawing from principles originally introduced to describe linkage phenomena in hemoglobin. In this paper, linkage between a principal channel component and secondary elements is derived from a four-state thermodynamic cycle. One set of parallel legs in the cycle describes the “work function,” or the free energy required to activate the principal component. The second are “lever operations” activating linked elements. The experimental embodiment of this linkage cycle is a plot of work function versus secondary force, whose asymptotes are a function of the parameters (displacements and interaction energies) of an allosteric network. Two essential work functions play a role in evaluating data from voltage-clamp experiments. The first is the conductance Hill energy WH[g], which is a “local” work function for pore activation, and is defined as kT times the Hill transform of the conductance (G-V) curve. The second is the electrical capacitance energy WC[q], representing “global” gating charge displacement, and is equal to the product of total gating charge per channel times the first moment (VM) of normalized capacitance (slope of Q-V curve). Plots of WH[g] and WC[q] versus voltage and Ca2+ potential can be used to measure thermodynamic parameters in a model-independent fashion of the core gating constituents (pore, voltage-sensor, and Ca2+-binding domain) of BK channel. The method is easily generalized for use in studying other allosterically regulated ion channels. The feasibility of performing linkage analysis from patch-clamp data were explored by simulating gating and ionic currents of a 17-particle model BK channel in response to a slow voltage ramp, which yielded interaction energies deviating from their given values in the range of 1.3 to 7.2%. PMID:23250867

Osteogenic and chondrogenic master genes expression is dependent on the Kir2.1 potassium channel through the bone morphogenetic protein pathway.

PubMed

Pini, Jonathan; Giuliano, Serena; Matonti, Julia; Simkin, Dina; Rouleau, Matthieu; Bendahhou, Saïd

2018-05-29

Andersen's syndrome is a rare disorder affecting muscle, heart, and bone, that is associated with mutations leading to a loss of function of the inwardly rectifying K + channel Kir2.1. While the Kir2.1 function can be anticipated in excitable cells by controlling the electrical activity, its role in non-excitable cells remains to be investigated. Using Andersen's syndrome induced Pluripotent Stem cells, we investigated the cellular and molecular events during the osteoblastic and chondrogenic differentiation that are affected by the loss of the Ik1 current. We show that loss of Kir2.1 channel function impairs both osteoblastic and chondrogenic processes through the down regulation master gene expression. This down regulation is due to an impairment of the bone morphogenetic proteins signaling pathway through de-phosphorylation of the Smad proteins. Restoring Kir2.1 channel function in Andersen's syndrome cells rescued master genes expression, and restored normal osteoblasts and chondrocytes behavior. Our results show that Kir2.1-mediated activity controls endochondral and intramembranous ossification signaling pathways. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Fluorogenic Green-Inside Red-Outside (GIRO) Labeling Approach Reveals Adenylyl Cyclase-Dependent Control of BKα Surface Expression

PubMed Central

2015-01-01

The regulation of surface levels of protein is critical for proper cell function and influences properties including cell adhesion, ion channel contributions to current flux, and the sensitivity of surface receptors to ligands. Here we demonstrate a two-color labeling system in live cells using a single fluorogen activating peptide (FAP) based fusion tag, which enables the rapid and simultaneous quantification of surface and internal proteins. In the nervous system, BK channels can regulate neural excitability and neurotransmitter release, and the surface trafficking of BK channels can be modulated by signaling cascades and assembly with accessory proteins. Using this labeling approach, we examine the dynamics of BK channel surface expression in HEK293 cells. Surface pools of the pore-forming BKα subunit were stable, exhibiting a plasma membrane half-life of >10 h. Long-term activation of adenylyl cyclase by forskolin reduced BKα surface levels by 30%, an effect that could not be attributed to increased bulk endocytosis of plasma membrane proteins. This labeling approach is compatible with microscopic imaging and flow cytometry, providing a solid platform for examining protein trafficking in living cells. PMID:26301573

Regulation of Sodium Transport in the Inner Ear

PubMed Central

Kim, Sung Huhn; Marcus, Daniel C.

2011-01-01

Na+ concentrations in endolymph must be controlled to maintain hair cell function since the transduction channels of hair cells are cation-permeable, but not K+-selective. Flooding or fluctuations of the hair cell cytosol with Na+ would be expected to lead to cellular dysfunction, hearing loss and vertigo. This review briefly describes cellular mechanisms known to be responsible for Na+homeostasis in each compartment of the inner ear, including the cochlea, saccule, semicircular canals and endolymphatic sac. The influx of Na+into endolymph of each of the organs is likely via passive diffusion, but these pathways have not yet been identified or characterized. Na+ absorption is controlled by gate -keeper channels in the apical (endolymphatic) membrane of the transporting cells. Highly Na+-selective epithelial sodium channels (ENaC) control absorption by Reissner’s membrane, saccular extramacular epithelium, semicircular canal duct epithelium and endolymphatic sac. ENaC activity is controlled by a number of signal pathways, but most notably by genomic regulation of channel numbers in the membrane via glucocorticoid signaling. Nonselective cation channels in the apical membrane of outer sulcus epithelial cells and vestibular transitional cells mediate Na+ and parasensory K+ absorption. The K+-mediated transduction current in hair cells is also accompanied by a Na+ flux since the transduction channels are nonselective cation channels. Cation absorption by all of these cells is regulated by extracellular ATP via apical nonselective cation channels (P2X receptors). The heterogeneous population of epithelial cells in the endolymphatic sac is thought to have multiple absorptive pathways for Na+ with regulatory pathways that include glucocorticoids and purinergic agonists. PMID:21620939

Cysteine residues in the nucleotide binding domains regulate the conductance state of CFTR channels.

PubMed Central

Harrington, Melissa A; Kopito, Ron R

2002-01-01

Gating of cystic fibrosis transmembrane conductance regulator (CFTR) channels requires intermolecular or interdomain interactions, but the exact nature and physiological significance of those interactions remains uncertain. Subconductance states of the channel may result from alterations in interactions among domains, and studying mutant channels enriched for a single conductance type may elucidate those interactions. Analysis of CFTR channels in inside-out patches revealed that mutation of cysteine residues in NBD1 and NBD2 affects the frequency of channel opening to the full-size versus a 3-pS subconductance. Mutating cysteines in NBD1 resulted in channels that open almost exclusively to the 3-pS subconductance, while mutations of cysteines in NBD2 decreased the frequency of subconductance openings. Wild-type channels open to both size conductances and make fast transitions between them within a single open burst. Full-size and subconductance openings of both mutant and wild-type channels are similarly activated by ATP and phosphorylation. However, the different size conductances open very differently in the presence of a nonhydrolyzable ATP analog, with subconductance openings significantly shortened by ATPgammaS, while full-size channels are locked open. In wild-type channels, reducing conditions increase the frequency and decrease the open time of subconductance channels, while oxidizing conditions decrease the frequency of subconductance openings. In contrast, in the cysteine mutants studied, altering redox potential has little effect on gating of the subconductance. PMID:11867445

In vivo Expression of a Light-activatable Potassium Channel Using Unnatural Amino Acids

PubMed Central

Kang, Ji-Yong; Kawaguchi, Daichi; Coin, Irene; Xiang, Zheng; O’Leary, Dennis D. M.; Slesinger, Paul A.; Wang, Lei

2013-01-01

SUMMARY Optical control of protein function provides excellent spatial-temporal resolution for studying proteins in situ. Although light-sensitive exogenous proteins and ligands have been employed to manipulate neuronal activity, a method for optical control of neuronal proteins using unnatural amino acids (Uaa) in vivo is lacking. Here, we describe the genetic incorporation of a photoreactive Uaa into the pore of an inwardly-rectifying potassium channel Kir2.1. The Uaa occluded the pore, rendering the channel non-conducting, and upon brief light illumination, was released to permit outward K+ current. Expression of this photo-inducible inwardly rectifying potassium (PIRK) channel in rat hippocampal neurons created a light-activatable PIRK switch for suppressing neuronal firing. We also expressed PIRK channels in embryonic mouse neocortex in vivo and demonstrated a light-activated PIRK current in cortical neurons. The principles applied here to a potassium channel could be generally expanded to other proteins expressed in the brain to enable optical regulation. PMID:24139041

Diadenosine tetraphosphate-induced inhibition of ATP-sensitive K+ channels in patches excised from ventricular myocytes.

PubMed Central

Jovanovic, A.; Terzic, A.

1996-01-01

Diadenosine 5',5''-P1,P4-tetraphosphate (Ap4A) has been termed 'alarmone' due to its role in intracellular signaling during metabolic stress. It is not known whether Ap4A could modulate ATP-sensitive K+ (KATP) channels, a family of channels regulated by the metabolic status of a cell. We applied the single-channel patch-clamp technique to measure the effect of Ap4A on KATP channels. When applied to the intracellular side of patches, excised from guinea-pig ventricular myocytes, Ap4A inhibited KATP channel activity, in a reversible and concentration-dependent (half-maximal concentration approximately 17 microM) manner. We conclude that Ap4A, a naturally occurring diadenosine polyphosphate, is actually an inhibitor of the myocardial KATP channel. PMID:8789372

Artificial phosphorylation sites modulate the activity of a voltage-gated potassium channel

NASA Astrophysics Data System (ADS)

Ariyaratne, Amila; Zocchi, Giovanni

2015-03-01

The KvAP potassium channel is representative of a family of voltage-gated ion channels where the membrane potential is sensed by a transmembrane helix containing several positively charged arginines. Previous work by Wang and Zocchi [A. Wang and G. Zocchi, PLoS ONE 6, e18598 (2011), 10.1371/journal.pone.0018598] showed how a negatively charged polyelectrolyte attached in proximity to the voltage sensing element can bias the opening probability of the channel. Here we introduce three phosphorylation sites at the same location and show that the response curve of the channel shifts by about 20 mV upon phosphorylation, while other characteristics such as the single-channel conductance are unaffected. In summary, we construct an artificial phosphorylation site which confers allosteric regulation to the channel.

Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons.

PubMed

Li, Lin; Wu, Xiaomei; Yue, Hai-Yuan; Zhu, Yong-Chuan; Xu, Jianhua

2016-07-01

At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling, and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5-40 Hz) was slowed by acute inhibition of MLCK and down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as a common pathway contributing to the activity-dependent regulation of vesicle endocytosis at synapses. © 2016 International Society for Neurochemistry.

Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators

PubMed Central

Wang, Wei; Hong, Jeong S.; Rab, Andras; Sorscher, Eric J.; Kirk, Kevin L.

2016-01-01

W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3–5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein. PMID:27007499

The S4–S5 Linker Acts as a Signal Integrator for hERG K+ Channel Activation and Deactivation Gating

PubMed Central

Ng, Chai Ann; Perry, Matthew D.; Tan, Peter S.; Hill, Adam P.; Kuchel, Philip W.; Vandenberg, Jamie I.

2012-01-01

Human ether-à-go-go-related gene (hERG) K+ channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4–S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4–S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4–S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4–S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4–S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4–S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel. PMID:22359612

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

PubMed

MacDonald, Patrick E; De Marinis, Yang Zhang; Ramracheya, Reshma; Salehi, Albert; Ma, Xiaosong; Johnson, Paul R V; Cox, Roger; Eliasson, Lena; Rorsman, Patrik

2007-06-01

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

KV7 Channel Pharmacological Activation by the Novel Activator ML213: Role for Heteromeric KV7.4/KV7.5 Channels in Guinea Pig Detrusor Smooth Muscle Function.

PubMed

Provence, Aaron; Angoli, Damiano; Petkov, Georgi V

2018-01-01

Voltage-gated K V 7 channels (K V 7.1 to K V 7.5) are important regulators of the cell membrane potential in detrusor smooth muscle (DSM) of the urinary bladder. This study sought to further the current knowledge of K V 7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca 2+ imaging, amphotericin-B perforated patch-clamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound N -(2,4,6-trimethylphenyl)-bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of K V 7.2, K V 7.4, and K V 7.5 channels, to examine their physiologic roles in guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 µ M) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the K V 7 channel inhibitor XE991 (10 µ M). ML213 (0.1-30 µ M) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 µ M) decreased global intracellular Ca 2+ concentrations in Fura-2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 µ M) caused an increase in the amplitude of whole-cell K V 7 currents. Further, in current-clamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 µ M), consistent with ML213 activation of K V 7 channel currents. Preapplication of XE991 (10 µ M) not only depolarized the DSM cells, but also blocked ML213-induced hyperpolarization, confirming ML213 selectivity for K V 7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric K V 7.4/K V 7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive K V 7.4- and K V 7.5-containing channels are essential regulators of DSM excitability and contractility. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Direct and remote modulation of L-channels in chromaffin cells: distinct actions on alpha1C and alpha1D subunits?

PubMed

Baldelli, Pietro; Hernández-Guijo, Jesus Miguel; Carabelli, Valentina; Novara, Monica; Cesetti, Tiziana; Andrés-Mateos, Eva; Montiel, Carmen; Carbone, Emilio

2004-02-01

Understanding precisely the functioning of voltage-gated Ca2+ channels and their modulation by signaling molecules will help clarifying the Ca(2+)-dependent mechanisms controlling exocytosis in chromaffin cells. In recent years, we have learned more about the various pathways through which Ca2+ channels can be up- or down-modulated by hormones and neurotransmitters and how these changes may condition chromaffin cell activity and catecolamine release. Recently, the attention has been focused on the modulation of L-channels (CaV 1), which represent the major Ca2+ current component in rat and human chromaffin cells. L-channels are effectively inhibited by the released content of secretory granules or by applying mixtures of exogenous ATP, opioids, and adrenaline through the activation of receptor-coupled G proteins. This unusual inhibition persists in a wide range of potentials and results from a direct (membrane-delimited) interaction of G protein subunits with the L-channels co-localized in membrane microareas. Inhibition of L-channels can be reversed when the cAMP/PKA pathway is activated by membrane permeable cAMP analog or when cells are exposed to isoprenaline (remote action), suggesting the existence of parallel and opposite effects on L-channel gating by distinctly activated membrane autoreceptors. Here, the authors review the molecular components underlying these two opposing signaling pathways and present new evidence supporting the presence of two L-channel types in rat chromaffin cells (alpha1C and alpha1D), which open new interesting issues concerning Ca(2+)-channel modulation. In light of recent findings on the regulation of exocytosis by Ca(2+)-channel modulation, the authors explore the possible role of L-channels in the autocontrol of catecholamine release.

The roles of KCa, KATP, and KV channels in regulating cutaneous vasodilation and sweating during exercise in the heat.

PubMed

Louie, Jeffrey C; Fujii, Naoto; Meade, Robert D; McNeely, Brendan D; Kenny, Glen P

2017-05-01

We recently showed the varying roles of Ca 2+ -activated (K Ca ), ATP-sensitive (K ATP ), and voltage-gated (K V ) K + channels in regulating cholinergic cutaneous vasodilation and sweating in normothermic conditions. However, it is unclear whether the respective contributions of these K + channels remain intact during dynamic exercise in the heat. Eleven young (23 ± 4 yr) men completed a 30-min exercise bout at a fixed rate of metabolic heat production (400 W) followed by a 40-min recovery period in the heat (35°C, 20% relative humidity). Cutaneous vascular conductance (CVC) and local sweat rate were assessed at four forearm skin sites perfused via intradermal microdialysis with: 1 ) lactated Ringer solution (control); 2 ) 50 mM tetraethylammonium (nonspecific K Ca channel blocker); 3 ) 5 mM glybenclamide (selective K ATP channel blocker); or 4 ) 10 mM 4-aminopyridine (nonspecific K V channel blocker). Responses were compared at baseline and at 10-min intervals during and following exercise. K Ca channel inhibition resulted in greater CVC versus control at end exercise ( P = 0.04) and 10 and 20 min into recovery (both P < 0.01). K ATP channel blockade attenuated CVC compared with control during baseline ( P = 0.04), exercise (all P ≤ 0.04), and 10 min into recovery ( P = 0.02). No differences in CVC were observed with K V channel inhibition during baseline ( P = 0.15), exercise (all P ≥ 0.06), or recovery (all P ≥ 0.14). With the exception of K V channel inhibition augmenting sweating during baseline ( P = 0.04), responses were similar to control with all K + channel blockers during each time period (all P ≥ 0.07). We demonstrated that K Ca and K ATP channels contribute to the regulation of cutaneous vasodilation during rest and/or exercise and recovery in the heat. Copyright © 2017 the American Physiological Society.

TRPV1 channels and the progesterone receptor Sig-1R interact to regulate pain.

PubMed

Ortíz-Rentería, Miguel; Juárez-Contreras, Rebeca; González-Ramírez, Ricardo; Islas, León D; Sierra-Ramírez, Félix; Llorente, Itzel; Simon, Sidney A; Hiriart, Marcia; Rosenbaum, Tamara; Morales-Lázaro, Sara L

2018-02-13

The Transient Receptor Potential Vanilloid 1 (TRPV1) ion channel is expressed in nociceptors where, when activated by chemical or thermal stimuli, it functions as an important transducer of painful and itch-related stimuli. Although the interaction of TRPV1 with proteins that regulate its function has been previously explored, their modulation by chaperones has not been elucidated, as is the case for other mammalian TRP channels. Here we show that TRPV1 physically interacts with the Sigma 1 Receptor (Sig-1R), a chaperone that binds progesterone, an antagonist of Sig-1R and an important neurosteroid associated to the modulation of pain. Antagonism of Sig-1R by progesterone results in the down-regulation of TRPV1 expression in the plasma membrane of sensory neurons and, consequently, a decrease in capsaicin-induced nociceptive responses. This is observed both in males treated with a synthetic antagonist of Sig-1R and in pregnant females where progesterone levels are elevated. This constitutes a previously undescribed mechanism by which TRPV1-dependent nociception and pain can be regulated.

Altering intracellular pH reveals the kinetic basis of intraburst gating in the CFTR Cl− channel

PubMed Central

Xu, Weiyi; Sheppard, David N.

2017-01-01

Key points The cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF), forms a gated pathway for chloride movement regulated by intracellular ATP.To understand better CFTR function, we investigated the regulation of channel openings by intracellular pH.We found that short‐lived channel closures during channel openings represent subtle changes in the structure of CFTR that are regulated by intracellular pH, in part, at ATP‐binding site 1 formed by the nucleotide‐binding domains.Our results provide a framework for future studies to understand better the regulation of channel openings, the dysfunction of CFTR in CF and the action of drugs that repair CFTR gating defects. Abstract Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP‐gated Cl− channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP‐binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single‐channel activity of human CFTR at different intracellular pH (pHi) values. When compared with the control (pHi 7.3), acidifying pHi to 6.3 or alkalinizing pHi to 8.3 and 8.8 caused small reductions in the open‐time constant (τo) of wild‐type CFTR. By contrast, the fast closed‐time constant (τcf), which describes the short‐lived closures that interrupt open bursts, was greatly increased at pHi 5.8 and 6.3. To analyse intraburst kinetics, we used linear three‐state gating schemes. All data were satisfactorily modelled by the C1 ↔ O ↔ C2 kinetic scheme. Changing the intracellular ATP concentration was without effect on τo, τcf and their responses to pHi changes. However, mutations that disrupt the interaction of ATP with ATP‐binding site 1, including K464A, D572N and the CF‐associated mutation G1349D all abolished the prolongation of τcf at pHi 6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP‐dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP‐binding site 1 modulates intraburst gating. PMID:27779763

Sodium-dependent potassium channels of a Slack-like subtype contribute to the slow afterhyperpolarization in lamprey spinal neurons

PubMed Central

Wallén, Peter; Robertson, Brita; Cangiano, Lorenzo; Löw, Peter; Bhattacharjee, Arin; Kaczmarek, Leonard K; Grillner, Sten

2007-01-01

The slow afterhyperpolarization (sAHP) following the action potential is the main determinant of spike frequency regulation. The sAHP after single action potentials in neurons of the lamprey locomotor network is largely due to calcium-dependent K+ channels (80%), activated by calcium entering the cell during the spike. The residual (20%) component becomes prominent during high level activity (50% of the sAHP). It is not Ca2+ dependent, has a reversal potential like that of potassium, and is not affected by chloride injection. It is not due to rapid activation of Na+/K+-ATPase. This non-KCa-sAHP is reduced markedly in amplitude when sodium ions are replaced by lithium ions, and is thus sodium dependent. Quinidine also blocks this sAHP component, further indicating an involvement of sodium-dependent potassium channels (KNa). Modulators tested do not influence the KNa-sAHP amplitude. Immunofluorescence labelling with an anti-Slack antibody revealed distinct immunoreactivity of medium-sized and large neurons in the grey matter of the lamprey spinal cord, suggesting the presence of a Slack-like subtype of KNa channel. The results strongly indicate that a KNa potassium current contributes importantly to the sAHP and thereby to neuronal frequency regulation during high level burst activity as during locomotion. This is, to our knowledge, the first demonstration of a functional role for the Slack gene in contributing to the slow AHP. PMID:17884929

TMEM16A is associated with voltage-gated calcium channels in mouse retina and its function is disrupted upon mutation of the auxiliary α2δ4 subunit

PubMed Central

Caputo, Antonella; Piano, Ilaria; Demontis, Gian Carlo; Bacchi, Niccolò; Casarosa, Simona; Santina, Luca Della; Gargini, Claudia

2015-01-01

Photoreceptors rely upon highly specialized synapses to efficiently transmit signals to multiple postsynaptic targets. Calcium influx in the presynaptic terminal is mediated by voltage-gated calcium channels (VGCC). This event triggers neurotransmitter release, but also gates calcium-activated chloride channels (TMEM), which in turn regulate VGCC activity. In order to investigate the relationship between VGCC and TMEM channels, we analyzed the retina of wild type (WT) and Cacna2d4 mutant mice, in which the VGCC auxiliary α2δ4 subunit carries a nonsense mutation, disrupting the normal channel function. Synaptic terminals of mutant photoreceptors are disarranged and synaptic proteins as well as TMEM16A channels lose their characteristic localization. In parallel, calcium-activated chloride currents are impaired in rods, despite unaltered TMEM16A protein levels. Co-immunoprecipitation revealed the interaction between VGCC and TMEM16A channels in the retina. Heterologous expression of these channels in tsA-201 cells showed that TMEM16A associates with the CaV1.4 subunit, and the association persists upon expression of the mutant α2δ4 subunit. Collectively, our experiments show association between TMEM16A and the α1 subunit of VGCC. Close proximity of these channels allows optimal function of the photoreceptor synaptic terminal under physiological conditions, but also makes TMEM16A channels susceptible to changes occurring to calcium channels. PMID:26557056

The role of KATP channels in cerebral ischemic stroke and diabetes

PubMed Central

Szeto, Vivian; Chen, Nai-hong; Sun, Hong-shuo; Feng, Zhong-ping

2018-01-01

ATP-sensitive potassium (KATP) channels are ubiquitously expressed on the plasma membrane of cells in multiple organs, including the heart, pancreas and brain. KATP channels play important roles in controlling and regulating cellular functions in response to metabolic state, which are inhibited by ATP and activated by Mg-ADP, allowing the cell to couple cellular metabolic state (ATP/ADP ratio) to electrical activity of the cell membrane. KATP channels mediate insulin secretion in pancreatic islet beta cells, and controlling vascular tone. Under pathophysiological conditions, KATP channels play cytoprotective role in cardiac myocytes and neurons during ischemia and/or hypoxia. KATP channel is a hetero-octameric complex, consisting of four pore-forming Kir6.x and four regulatory sulfonylurea receptor SURx subunits. These subunits are differentially expressed in various cell types, thus determining the sensitivity of the cells to specific channel modifiers. Sulfonylurea class of antidiabetic drugs blocks KATP channels, which are neuroprotective in stroke, can be one of the high stoke risk factors for diabetic patients. In this review, we discussed the potential effects of KATP channel blockers when used under pathological conditions related to diabetics and cerebral ischemic stroke. PMID:29671418

Analysis of the K+ current in human CD4+ T lymphocytes in hypercholesterolemic state.

PubMed

Somodi, Sándor; Balajthy, András; Szilágyi, Orsolya; Pethő, Zoltán; Harangi, Mariann; Paragh, György; Panyi, György; Hajdu, Péter

2013-01-01

Atherosclerosis involves immune mechanisms: T lymphocytes are found in atherosclerotic plaques, suggesting their activation during atherogenesis. The predominant voltage-gated potassium channel of T cells, Kv1.3 is a key regulator of the Ca(2+)-dependent activation pathway. In the present experiments we studied the proliferation capacity and functional changes of Kv1.3 channels in T cells from healthy and hypercholestaeremic patients. By means of CFSE-assay (carboxyfluorescein succinimidyl ester) we showed that spontaneous activation rate of lymphocytes in hypercholesterolemia was elevated and the antiCD3/antiCD28 co-stimulation was less effective as compared to the healthy group. Using whole-cell patch-clamping we obtained that the activation and deactivation kinetics of Kv1.3 channels were faster in hypercholesterolemic state but no change in other parameters of Kv1.3 were found (inactivation kinetics, steady-state activation, expression level). We suppose that incorporation of oxLDL species via its raft-rupturing effect can modify proliferative rate of T cells as well as the gating of Kv1.3 channels. Copyright © 2013 Elsevier Inc. All rights reserved.

Coronary arterial BK channel dysfunction exacerbates ischemia/reperfusion-induced myocardial injury in diabetic mice.

PubMed

Lu, Tong; Jiang, Bin; Wang, Xiao-Li; Lee, Hon-Chi

2016-09-01

The large conductance Ca(2+)-activated K(+) (BK) channels, abundantly expressed in coronary artery smooth muscle cells (SMCs), play a pivotal role in regulating coronary circulation. A large body of evidence indicates that coronary arterial BK channel function is diminished in both type 1 and type 2 diabetes. However, the consequence of coronary BK channel dysfunction in diabetes is not clear. We hypothesized that impaired coronary BK channel function exacerbates myocardial ischemia/reperfusion (I/R) injury in streptozotocin-induced diabetic mice. Combining patch-clamp techniques and cellular biological approaches, we found that diabetes facilitated the colocalization of angiotensin II (Ang II) type 1 receptors and BK channel α-subunits (BK-α), but not BK channel β1-subunits (BK-β1), in the caveolae of coronary SMCs. This caveolar compartmentation in vascular SMCs not only enhanced Ang II-mediated inhibition of BK-α but also produced a physical disassociation between BK-α and BK-β1, leading to increased infarct size in diabetic hearts. Most importantly, genetic ablation of caveolae integrity or pharmacological activation of coronary BK channels protected the cardiac function of diabetic mice from experimental I/R injury in both in vivo and ex vivo preparations. Our results demonstrate a vascular ionic mechanism underlying the poor outcome of myocardial injury in diabetes. Hence, activation of coronary BK channels may serve as a therapeutic target for cardiovascular complications of diabetes.

Imaging Large Cohorts of Single Ion Channels and Their Activity

PubMed Central

Hiersemenzel, Katia; Brown, Euan R.; Duncan, Rory R.

2013-01-01

As calcium is the most important signaling molecule in neurons and secretory cells, amongst many other cell types, it follows that an understanding of calcium channels and their regulation of exocytosis is of vital importance. Calcium imaging using calcium dyes such as Fluo3, or FRET-based dyes that have been used widely has provided invaluable information, which combined with modeling has estimated the subtypes of channels responsible for triggering the exocytotic machinery as well as inferences about the relative distances away from vesicle fusion sites these molecules adopt. Importantly, new super-resolution microscopy techniques, combined with novel Ca2+ indicators and imaginative imaging approaches can now define directly the nano-scale locations of very large cohorts of single channel molecules in relation to single vesicles. With combinations of these techniques the activity of individual channels can be visualized and quantified using novel Ca2+ indicators. Fluorescently labeled specific channel toxins can also be used to localize endogenous assembled channel tetramers. Fluorescence lifetime imaging microscopy and other single-photon-resolution spectroscopic approaches offer the possibility to quantify protein–protein interactions between populations of channels and the SNARE protein machinery for the first time. Together with simultaneous electrophysiology, this battery of quantitative imaging techniques has the potential to provide unprecedented detail describing the locations, dynamic behaviors, interactions, and conductance activities of many thousands of channel molecules and vesicles in living cells. PMID:24027557

Pacemaker rate and depolarization block in nigral dopamine neurons: a somatic sodium channel balancing act

PubMed Central

Tucker, Kristal R.; Huertas, Marco A.; Horn, John P.; Canavier, Carmen C.; Levitan, Edwin S.

2012-01-01

Midbrain dopamine (DA) neurons are slow intrinsic pacemakers that undergo depolarization (DP) block upon moderate stimulation. Understanding DP block is important because it has been correlated with the clinical efficacy of chronic antipsychotic drug treatment. Here we describe how voltage-gated sodium (NaV) channels regulate DP block and pacemaker activity in DA neurons of the substantia nigra using rat brain slices. The distribution, density and gating of NaV currents were manipulated by blocking native channels with tetrodotoxin and by creating virtual channels and anti-channels with dynamic clamp. Although action potentials initiate in the axon initial segment (AIS) and NaV channels are distributed in multiple dendrites, selective reduction of NaV channel activity in the soma was sufficient to decrease pacemaker frequency and increase susceptibility to DP block. Conversely, increasing somatic NaV current density raised pacemaker frequency and lowered susceptibility to DP block. Finally, when NaV currents were restricted to the soma, pacemaker activity occurred at abnormally high rates due to excessive local subthreshold NaV current. Together with computational simulations, these data show that both the slow pacemaker rate and the sensitivity to DP block that characterizes DA neurons result from the low density of somatic NaV channels. More generally, we conclude that the somatodendritic distribution of NaV channels is a major determinant of repetitive spiking frequency. PMID:23077037

Control of somatic membrane potential in nociceptive neurons and its implications for peripheral nociceptive transmission

PubMed Central

Du, Xiaona; Hao, Han; Gigout, Sylvain; Huang, Dongyang; Yang, Yuehui; Li, Li; Wang, Caixue; Sundt, Danielle; Jaffe, David B.; Zhang, Hailin; Gamper, Nikita

2014-01-01

Peripheral sensory ganglia contain somata of afferent fibres conveying somatosensory inputs to the central nervous system. Growing evidence suggests that the somatic/perisomatic region of sensory neurons can influence peripheral sensory transmission. Control of resting membrane potential (Erest) is an important mechanism regulating excitability, but surprisingly little is known about how Erest is regulated in sensory neuron somata or how changes in somatic/perisomatic Erest affect peripheral sensory transmission. We first evaluated the influence of several major ion channels on Erest in cultured small-diameter, mostly capsaicin-sensitive (presumed nociceptive) dorsal root ganglion (DRG) neurons. The strongest and most prevalent effect on Erest was achieved by modulating M channels, K2P and 4-aminopiridine-sensitive KV channels, while hyperpolarization-activated cyclic nucleotide-gated, voltage-gated Na+, and T-type Ca2+ channels to a lesser extent also contributed to Erest. Second, we investigated how varying somatic/perisomatic membrane potential, by manipulating ion channels of sensory neurons within the DRG, affected peripheral nociceptive transmission in vivo. Acute focal application of M or KATP channel enhancers or a hyperpolarization-activated cyclic nucleotide-gated channel blocker to L5 DRG in vivo significantly alleviated pain induced by hind paw injection of bradykinin. Finally, we show with computational modelling how somatic/perisomatic hyperpolarization, in concert with the low-pass filtering properties of the t-junction within the DRG, can interfere with action potential propagation. Our study deciphers a complement of ion channels that sets the somatic Erest of nociceptive neurons and provides strong evidence for a robust filtering role of the somatic and perisomatic compartments of peripheral nociceptive neuron. PMID:25168672

Effect of K+ATP channel and adenosine receptor blockade during rest and exercise in congestive heart failure.

PubMed

Traverse, Jay H; Chen, YingJie; Hou, MingXiao; Li, Yunfang; Bache, Robert J

2007-06-08

K(+)(ATP) channels are important metabolic regulators of coronary blood flow (CBF) that are activated in the setting of reduced levels of ATP or perfusion pressure. In the normal heart, blockade of K(+)(ATP) channels results in a approximately 20% reduction in resting CBF but does not impair the increase in CBF that occurs during exercise. In contrast, adenosine receptor blockade fails to alter CBF or myocardial oxygen consumption (MVO(2)) in the normal heart but contributes to the increase in CBF during exercise when vascular K(+)(ATP) channels are blocked. Congestive heart failure (CHF) is associated with a decrease in CBF that is matched to a decrease in MVO(2) suggesting downregulation of myocardial energy utilization. Because myocardial ATP levels and coronary perfusion pressure are reduced in CHF, this study was undertaken to examine the role of K(+)(ATP) channels and adenosine in dogs with pacing-induced CHF. Myocardial blood flow (MBF) and MVO(2) were measured during rest and treadmill exercise before and after K(+)(ATP) channel blockade with glibenclamide (50 microg/kg/min ic) or adenosine receptor blockade with 8-phenyltheophylline (8-PT; 5 mg/kg iv). Inhibition of K(+)(ATP) channels resulted in a decrease in CBF and MVO(2) at rest and during exercise without a change in the relationship between CBF and MVO(2). In contrast, adenosine receptor blockade caused a significant increase in CBF that occurred secondary to an increase of MVO(2). These findings demonstrate that coronary K(+)(ATP) channel activity contribute to the regulation of resting MBF in CHF, and that endogenous adenosine may act to inhibit MVO(2) in the failing heart.

Cellular mechanisms underlying the inhibitory effect of flufenamic acid on chloride secretion in human intestinal epithelial cells.

PubMed

Pongkorpsakol, Pawin; Yimnual, Chantapol; Chatsudthipong, Varanuj; Rukachaisirikul, Vatcharin; Muanprasat, Chatchai

2017-06-01

Intestinal Cl - secretion is involved in the pathogenesis of secretory diarrheas including cholera. We recently demonstrated that flufenamic acid (FFA) suppressed Vibrio cholerae El Tor variant-induced intestinal fluid secretion via mechanisms involving AMPK activation and NF-κB-suppression. The present study aimed to investigate the effect of FFA on transepithelial Cl - secretion in human intestinal epithelial (T84) cells. FFA inhibited cAMP-dependent Cl - secretion in T84 cell monolayers with IC 50 of ∼8 μM. Other fenamate drugs including tolfenamic acid, meclofenamic acid and mefenamic acid exhibited the same effect albeit with lower potency. FFA also inhibited activities of CFTR, a cAMP-activated apical Cl - channel, and KCNQ1/KCNE3, a cAMP-activated basolateral K + channel. Mechanisms of CFTR inhibition by FFA did not involve activation of its negative regulators. Interestingly, FFA inhibited Ca 2+ -dependent Cl - secretion with IC 50 of ∼10 μM. FFA inhibited activities of Ca 2+ -activated Cl - channels and K Ca 3.1, a Ca 2+ -activated basolateral K + channels, but had no effect on activities of Na + -K + -Cl - cotransporters and Na + -K + ATPases. These results indicate that FFA inhibits both cAMP and Ca 2+ -dependent Cl - secretion by suppressing activities of both apical Cl - channels and basolateral K + channels. FFA and other fenamate drugs may be useful in the treatment of secretory diarrheas. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Involvement of WNK1-mediated potassium channels in the sexual dimorphism of blood pressure.

PubMed

Yu, Guofeng; Cheng, Mengting; Wang, Wei; Zhao, Rong; Liu, Zhen

2017-04-01

Potassium homeostasis plays an essential role in the control of blood pressure. It is unknown, however, whether potassium balance is involved in the gender-associated blood pressure differences. We therefore investigated the possible mechanism of sexual dimorphism in blood pressure regulation by measuring the blood pressure, plasma potassium, renal actions of potassium channels and upstream regulator in male and female mice. Here we found that female mice exhibited lower blood pressure and higher plasma K + level as compared to male littermates. Western blot analyses of mouse kidney extract revealed a significant decrease in renal outer medullary potassium (ROMK) channel expression, while large-conductance Ca 2+ -activated K + (BK) channel and Na-K-2Cl cotransporter (NKCC2) as well as the upstream regulator with-no-lysine kinase 1 (WNK1) enhanced in female mice under normal condition. Surprisingly, both dietary K + loading and K + depletion eliminated the differences in plasma K + and blood pressure between females and males, and the differences of renal K + channels and WNK1 also attenuated in both groups of mice. These findings indicated the existence of a close correlation between K + homeostasis and sex-associated blood pressure. Moreover, the differential regulation of ROMK, BK-α and NKCC2 between female and male mice, at least, were partly mediated via WNK1 pathway, which may contribute to the sexual dimorphism of plasma K + and blood pressure control. Copyright © 2017 Elsevier Inc. All rights reserved.

Studies on the System Regulating Proton Movement across the Chloroplast Envelope 1

PubMed Central

Peters, Jeanne S.; Berkowitz, Gerald A.

1991-01-01

Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H+ movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg2+ causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg2+ was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg2+ and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and H+ fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg2+ modulated) H+ flux linked to monovalent cation antiport, and ATPase-dependent H+ efflux. PMID:16668116

Iron overload causes endolysosomal deficits modulated by NAADP-regulated 2-pore channels and RAB7A

PubMed Central

Fernández, Belén; Fdez, Elena; Gómez-Suaga, Patricia; Gil, Fernando; Molina-Villalba, Isabel; Ferrer, Isidro; Patel, Sandip; Churchill, Grant C.; Hilfiker, Sabine

2016-01-01

ABSTRACT Various neurodegenerative disorders are associated with increased brain iron content. Iron is known to cause oxidative stress, which concomitantly promotes cell death. Whereas endolysosomes are known to serve as intracellular iron storage organelles, the consequences of increased iron on endolysosomal functioning, and effects on cell viability upon modulation of endolysosomal iron release remain largely unknown. Here, we show that increasing intracellular iron causes endolysosomal alterations associated with impaired autophagic clearance of intracellular protein aggregates, increased cytosolic oxidative stress and increased cell death. These effects are subject to regulation by NAADP, a potent second messenger reported to target endolysosomal TPCNs (2-pore channels). Consistent with endolysosomal iron storage, cytosolic iron levels are modulated by NAADP, and increased cytosolic iron is detected when overexpressing active, but not inactive TPCNs, indicating that these channels can modulate endolysosomal iron release. Cell death triggered by altered intralysosomal iron handling is abrogated in the presence of an NAADP antagonist or when inhibiting RAB7A activity. Taken together, our results suggest that increased endolysosomal iron causes cell death associated with increased cytosolic oxidative stress as well as autophagic impairments, and these effects are subject to modulation by endolysosomal ion channel activity in a RAB7A-dependent manner. These data highlight alternative therapeutic strategies for neurodegenerative disorders associated with increased intracellular iron load. PMID:27383256

Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking.

PubMed

Andersen, Martin N; Hefting, Louise L; Steffensen, Annette B; Schmitt, Nicole; Olesen, Søren-Peter; Olsen, Jesper V; Lundby, Alicia; Rasmussen, Hanne B

2015-11-15

The potassium channel Kv7.1 plays critical physiological roles in both heart and epithelial tissues. In heart, Kv7.1 and the accessory subunit KCNE1 forms the slowly activating delayed-rectifier potassium current current, which is enhanced by protein kinase A (PKA)-mediated phosphorylation. The observed current increase requires both phosphorylation of Kv7.1 and the presence of KCNE1. However, PKA also stimulates Kv7.1 currents in epithelial tissues, such as colon, where the channel does not coassemble with KCNE1. Here, we demonstrate that PKA activity significantly impacts the subcellular localization of Kv7.1 in Madin-Darby canine kidney cells. While PKA inhibition reduced the fraction of channels at the cell surface, PKA activation increased it. We show that PKA inhibition led to intracellular accumulation of Kv7.1 in late endosomes/lysosomes. By mass spectroscopy we identified eight phosphorylated residues on Kv7.1, however, none appeared to play a role in the observed response. Instead, we found that PKA acted by regulating endocytic trafficking involving the ubiquitin ligase Nedd4-2. We show that a Nedd4-2-resistant Kv7.1-mutant displayed significantly reduced intracellular accumulation upon PKA inhibition. Similar effects were observed upon siRNA knockdown of Nedd4-2. However, although Nedd4-2 is known to regulate Kv7.1 by ubiquitylation, biochemical analyses demonstrated that PKA did not influence the amount of Nedd4-2 bound to Kv7.1 or the ubiquitylation level of the channel. This suggests that PKA influences Nedd4-2-dependent Kv7.1 transport though a different molecular mechanism. In summary, we identify a novel mechanism whereby PKA can increase Kv7.1 current levels, namely by regulating Nedd4-2-dependent Kv7.1 transport. Copyright © 2015 the American Physiological Society.

Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

PubMed Central

Andersen, Martin N.; Hefting, Louise L.; Steffensen, Annette B.; Schmitt, Nicole; Olesen, Søren-Peter; Olsen, Jesper V.; Lundby, Alicia

2015-01-01

The potassium channel Kv7.1 plays critical physiological roles in both heart and epithelial tissues. In heart, Kv7.1 and the accessory subunit KCNE1 forms the slowly activating delayed-rectifier potassium current current, which is enhanced by protein kinase A (PKA)-mediated phosphorylation. The observed current increase requires both phosphorylation of Kv7.1 and the presence of KCNE1. However, PKA also stimulates Kv7.1 currents in epithelial tissues, such as colon, where the channel does not coassemble with KCNE1. Here, we demonstrate that PKA activity significantly impacts the subcellular localization of Kv7.1 in Madin-Darby canine kidney cells. While PKA inhibition reduced the fraction of channels at the cell surface, PKA activation increased it. We show that PKA inhibition led to intracellular accumulation of Kv7.1 in late endosomes/lysosomes. By mass spectroscopy we identified eight phosphorylated residues on Kv7.1, however, none appeared to play a role in the observed response. Instead, we found that PKA acted by regulating endocytic trafficking involving the ubiquitin ligase Nedd4-2. We show that a Nedd4-2-resistant Kv7.1-mutant displayed significantly reduced intracellular accumulation upon PKA inhibition. Similar effects were observed upon siRNA knockdown of Nedd4-2. However, although Nedd4-2 is known to regulate Kv7.1 by ubiquitylation, biochemical analyses demonstrated that PKA did not influence the amount of Nedd4-2 bound to Kv7.1 or the ubiquitylation level of the channel. This suggests that PKA influences Nedd4-2-dependent Kv7.1 transport though a different molecular mechanism. In summary, we identify a novel mechanism whereby PKA can increase Kv7.1 current levels, namely by regulating Nedd4-2-dependent Kv7.1 transport. PMID:26405101

Drug development targeting the glycogen synthase kinase-3beta (GSK-3beta)-mediated signal transduction pathway: the role of GSK-3beta in the maintenance of steady-state levels of insulin receptor signaling molecules and Na(v)1.7 sodium channel in adrenal chromaffin cells.

PubMed

Nemoto, Takayuki; Yanagita, Toshihiko; Kanai, Tasuku; Wada, Akihiko

2009-02-01

Glycogen synthase kinase-3 (GSK-3) is constitutively active in nonstimulated cells, where the majority of its substrates undergo inactivation/proteolysis by phosphorylation. Extracellular stimuli (e.g., insulin) catalyze inhibitory Ser(9)-phosphorylation of GSK-3beta, turning on signaling and causing other biological consequences otherwise constitutively suppressed by GSK-3beta. Regulated and dysregulated activities of GSK-3beta are pivotal to health, disease, and therapeutics (e.g., insulin resistance, neurodegeneration, tumorigenesis, inflammation); however, the underlying mechanisms of multifunctional GSK-3beta remain elusive. In cultured bovine adrenal chromaffin cells, 1) constitutive and negatively-regulated activities of GSK-3beta up- and down-regulated insulin receptor, insulin receptor substrate-1 (IRS-1), IRS-2, and Akt levels via controlling proteasomal degradation and protein synthesis; 2) nicotinic receptor/protein kinase C-alpha (PKC-alpha)/extracellular signal-regulated kinase (ERK) pathway up-regulated IRS-1 and IRS-2 levels, enhancing insulin-induced the phosphoinositide 3-kinase (PI3K)/Akt/GSK-3beta pathway; 3) inhibition of calcineurin by cyclosporin A or FK506 down-regulated IRS-2 level, attenuating insulin-like growth factor-I (IGF-I)-induced ERK and GSK-3beta pathways; and 4) insulin, IGF-I or therapeutics (e.g., lithium) up-regulated the voltage-dependent Na(v)1.7 sodium channel.

An endogenous RNA transcript antisense to CNG(alpha)1 cation channel mRNA.

PubMed

Cheng, Chin-Hung; Yew, David Tai-Wai; Kwan, Hiu-Yee; Zhou, Qing; Huang, Yu; Liu, Yong; Chan, Wing-Yee; Yao, Xiaoqiang

2002-10-01

CNG channels are cyclic nucleotide-gated Ca(2+)-permeable channels that are suggested to be involved in the activity-dependent alterations of synaptic strength that are thought to underlie information storage in the CNS. In this study, we isolated an endogenous RNA transcript antisense to CNG(alpha)1 mRNA. This transcript was capable of down-regulating the expression of sense CNG(alpha)1 in the Xenopus oocyte expression system. RT-PCR, Northern blot, and in situ hybridization analyses showed that the transcript was coexpressed with CNG(alpha)1 mRNA in many regions of human brain, notably in those regions that were involved in long-term potentiation and long-term depression, such as hippocampal CA1 and CA3, dentate gyrus, and cerebellar Purkinje layer. Comparison of expression patterns between adult and fetal cerebral cortex revealed that there were concurrent developmental changes in the expression levels of anti-CNG1 and CNG(alpha)1. Treatment of human glioma cell T98 with thyroid hormone T(3) caused a significant increase in anti-CNG1 expression and a parallel decrease in sense CNG(alpha)1 expression. These data suggest that the suppression of CNG(alpha)1 expression by anti-CNG1 may play an important role in neuronal functions, especially in synaptic plasticity and cortical development. Endogenous antisense RNA-mediated regulation may represent a new mechanism through which the activity of ion channels can be regulated in the human CNS.

Swelling and Eicosanoid Metabolites Differentially Gate TRPV4 Channels in Retinal Neurons and Glia

PubMed Central

Ryskamp, Daniel A.; Jo, Andrew O.; Frye, Amber M.; Vazquez-Chona, Felix; MacAulay, Nanna; Thoreson, Wallace B.

2014-01-01

Activity-dependent shifts in ionic concentrations and water that accompany neuronal and glial activity can generate osmotic forces with biological consequences for brain physiology. Active regulation of osmotic gradients and cellular volume requires volume-sensitive ion channels. In the vertebrate retina, critical support to volume regulation is provided by Müller astroglia, but the identity of their osmosensor is unknown. Here, we identify TRPV4 channels as transducers of mouse Müller cell volume increases into physiological responses. Hypotonic stimuli induced sustained [Ca2+]i elevations that were inhibited by TRPV4 antagonists and absent in TRPV4−/− Müller cells. Glial TRPV4 signals were phospholipase A2- and cytochrome P450-dependent, characterized by slow-onset and Ca2+ waves, and, in excess, were sufficient to induce reactive gliosis. In contrast, neurons responded to TRPV4 agonists and swelling with fast, inactivating Ca2+ signals that were independent of phospholipase A2. Our results support a model whereby swelling and proinflammatory signals associated with arachidonic acid metabolites differentially gate TRPV4 in retinal neurons and glia, with potentially significant consequences for normal and pathological retinal function. PMID:25411497

Molecular Dynamics Simulations of KirBac1.1 Mutants Reveal Global Gating Changes of Kir Channels.

PubMed

Linder, Tobias; Wang, Shizhen; Zangerl-Plessl, Eva-Maria; Nichols, Colin G; Stary-Weinzinger, Anna

2015-04-27

Prokaryotic inwardly rectifying (KirBac) potassium channels are homologous to mammalian Kir channels. Their activity is controlled by dynamical conformational changes that regulate ion flow through a central pore. Understanding the dynamical rearrangements of Kir channels during gating requires high-resolution structure information from channels crystallized in different conformations and insight into the transition steps, which are difficult to access experimentally. In this study, we use MD simulations on wild type KirBac1.1 and an activatory mutant to investigate activation gating of KirBac channels. Full atomistic MD simulations revealed that introducing glutamate in position 143 causes significant widening at the helix bundle crossing gate, enabling water flux into the cavity. Further, global rearrangements including a twisting motion as well as local rearrangements at the subunit interface in the cytoplasmic domain were observed. These structural rearrangements are similar to recently reported KirBac3.1 crystal structures in closed and open conformation, suggesting that our simulations capture major conformational changes during KirBac1.1 opening. In addition, an important role of protein-lipid interactions during gating was observed. Slide-helix and C-linker interactions with lipids were strengthened during activation gating.

78 FR 56609 - Drawbridge Operation Regulations; Reynolds Channel, Lawrence, NY

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-13

... Regulations; Reynolds Channel, Lawrence, NY AGENCY: Coast Guard, DHS. ACTION: Notice canceling temporary... Beach Bridge, mile 0.4, across Reynolds Channel, at Lawrence, New York. The owner of the bridge, Nassau... published a temporary deviation entitled ``Drawbridge Operation Regulations; Reynolds Channel, Lawrence, NY...

LE135, a retinoid acid receptor antagonist, produces pain through direct activation of TRP channels

PubMed Central

Yin, Shijin; Luo, Jialie; Qian, Aihua; Yu, Weihua; Hu, Hongzhen

2014-01-01

Background and PurposeRetinoids, through their activation of retinoic acid receptors (RARs) and retinoid X receptors, regulate diverse cellular processes, and pharmacological intervention in their actions has been successful in the treatment of skin disorders and cancers. Despite the many beneficial effects, administration of retinoids causes irritating side effects with unknown mechanisms. Here, we demonstrate that LE135 [4-(7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid], a selective antagonist of RARβ, is a potent activator of the capsaicin (TRPV1) and wasabi (TRPA1) receptors, two critical pain-initiating cation channels. Experimental ApproachWe performed to investigate the excitatory effects of LE135 on TRPV1 and TRPA1 channels expressed in HEK293T cells and in dorsal root ganglia neurons with calcium imaging and patch-clamp recordings. We also used site-directed mutagenesis of the channels to determine the structural basis of LE135-induced activation of TRPV1 and TRPA1 channels and behavioural testing to examine if pharmacological inhibition and genetic deletion of the channels affected LE135-evoked pain-related behaviours. Key ResultsLE135 activated both the capsaicin receptor (TRPV1) and the allyl isothiocyanate receptor (TRPA1) heterologously expressed in HEK293T cells and endogenously expressed by sensory nociceptors. Mutations disrupting the capsaicin-binding site attenuated LE135 activation of TRPV1 channels and a single mutation (K170R) eliminated TRPA1 activity evoked by LE135. Intraplantar injection of LE135 evoked pain-related behaviours. Both TRPV1 and TRPA1 channels were involved in LE135-elicited pain-related responses, as shown by pharmacological and genetic ablation studies. Conclusions and ImplicationsThis blocker of retinoid acid signalling also exerted non-genomic effects through activating the pain-initiating TRPV1 and TRPA1 channels. PMID:24308840

LE135, a retinoid acid receptor antagonist, produces pain through direct activation of TRP channels.

PubMed

Yin, Shijin; Luo, Jialie; Qian, Aihua; Yu, Weihua; Hu, Hongzhen

2014-03-01

Retinoids, through their activation of retinoic acid receptors (RARs) and retinoid X receptors, regulate diverse cellular processes, and pharmacological intervention in their actions has been successful in the treatment of skin disorders and cancers. Despite the many beneficial effects, administration of retinoids causes irritating side effects with unknown mechanisms. Here, we demonstrate that LE135 [4-(7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid], a selective antagonist of RARβ , is a potent activator of the capsaicin (TRPV1) and wasabi (TRPA1) receptors, two critical pain-initiating cation channels. We performed to investigate the excitatory effects of LE135 on TRPV1 and TRPA1 channels expressed in HEK293T cells and in dorsal root ganglia neurons with calcium imaging and patch-clamp recordings. We also used site-directed mutagenesis of the channels to determine the structural basis of LE135-induced activation of TRPV1 and TRPA1 channels and behavioural testing to examine if pharmacological inhibition and genetic deletion of the channels affected LE135-evoked pain-related behaviours. LE135 activated both the capsaicin receptor (TRPV1) and the allyl isothiocyanate receptor (TRPA1) heterologously expressed in HEK293T cells and endogenously expressed by sensory nociceptors. Mutations disrupting the capsaicin-binding site attenuated LE135 activation of TRPV1 channels and a single mutation (K170R) eliminated TRPA1 activity evoked by LE135. Intraplantar injection of LE135 evoked pain-related behaviours. Both TRPV1 and TRPA1 channels were involved in LE135-elicited pain-related responses, as shown by pharmacological and genetic ablation studies. This blocker of retinoid acid signalling also exerted non-genomic effects through activating the pain-initiating TRPV1 and TRPA1 channels. © 2013 The British Pharmacological Society.

Voltage-Gated Potassium Channels Kv1.3--Potentially New Molecular Target in Cancer Diagnostics and Therapy.

PubMed

Teisseyre, Andrzej; Gąsiorowska, Justyna; Michalak, Krystyna

2015-01-01

Voltage-gated potassium channels, Kv1.3, which were discovered in 1984, are integral membrane proteins which are activated ("open") upon change of the cell membrane potential, enabling a passive flux of potassium ions across the cell membrane. The channels are expressed in many different tissues, both normal and cancer. Since 2005 it has been known that the channels are expressed not only in the plasma membrane, but also in the inner mitochondrial membrane. The activity of Kv1.3 channels plays an important role, among others, in setting the cell resting membrane potential, cell proliferation, apoptosis and volume regulation. For some years, these channels have been considered a potentially new molecular target in both the diagnostics and therapy of some cancer diseases. This review article focuses on: 1) changes of expression of the channels in cancer disorders with special regard to correlations between the channels' expression and stage of the disease, 2) influence of inhibitors of Kv1.3 channels on proliferation and apoptosis of cancer cells, 3) possible future applications of Kv1.3 channels' inhibitors in therapy of some cancer diseases. In the last section, the results of studies performed in our Laboratory of Bioelectricity on the influence of selected biologically active plant-derived compounds from the groups of flavonoids and stilbenes and their natural and synthetic derivatives on the activity of Kv1.3 channels in normal and cancer cells are reviewed. A possible application of some compounds from these groups to support therapy of cancer diseases, such as breast, colon and lymph node cancer, and melanoma or chronic lymphocytic leukemia (B-CLL), is announced.

xCT expression reduces the early cell cycle requirement for calcium signaling

PubMed Central

Lastro, Michele; Kourtidis, Antonis; Farley, Kate; Conklin, Douglas S.

2009-01-01

Calcium has long been recognized as an important regulator of cell cycle transitions although the mechanisms are largely unknown. A functional genomic screen has identified genes involved in the regulation of early cell cycle progression by calcium. These genes when overexpressed confer the ability to bypass the G1/S arrest induced by Ca2+- channel antagonists in mouse fibroblasts. Overexpression of the cystine-glutamate exchanger, xCT, had the greatest ability to evade calcium antagonist-induced cell cycle arrest. xCT carries out the rate limiting step of glutathione synthesis in many cell types and is responsible for the uptake of cystine in most human cancer cell lines. Functional analysis indicates that the cystine uptake activity of xCT overcomes the G1/S arrest induced by Ca2+- channel antagonists by bypassing the requirement for calcium signaling. Since cells overexpressing xCT were found to have increased levels and activity of the AP-1 transcription factor in G1, redox stimulation of AP-1 activity accounts for the observed growth of these cells in the presence of calcium channel antagonists. These results suggest that reduced calcium signaling impairs AP-1 activation and that xCT expression may directly affect cell proliferation. PMID:18054200

Mechanical sensitivity of Piezo1 ion channels can be tuned by cellular membrane tension

PubMed Central

Lewis, Amanda H; Grandl, Jörg

2015-01-01

Piezo1 ion channels mediate the conversion of mechanical forces into electrical signals and are critical for responsiveness to touch in metazoans. The apparent mechanical sensitivity of Piezo1 varies substantially across cellular environments, stimulating methods and protocols, raising the fundamental questions of what precise physical stimulus activates the channel and how its stimulus sensitivity is regulated. Here, we measured Piezo1 currents evoked by membrane stretch in three patch configurations, while simultaneously visualizing and measuring membrane geometry. Building on this approach, we developed protocols to minimize resting membrane curvature and tension prior to probing Piezo1 activity. We find that Piezo1 responds to lateral membrane tension with exquisite sensitivity as compared to other mechanically activated channels and that resting tension can drive channel inactivation, thereby tuning overall mechanical sensitivity of Piezo1. Our results explain how Piezo1 can function efficiently and with adaptable sensitivity as a sensor of mechanical stimulation in diverse cellular contexts. DOI: http://dx.doi.org/10.7554/eLife.12088.001 PMID:26646186

Activation and Regulation of Purinergic P2X Receptor Channels

PubMed Central

Coddou, Claudio; Yan, Zonghe; Obsil, Tomas; Huidobro-Toro, J. Pablo

2011-01-01

Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions. PMID:21737531

Calcium Homeostasis and Cone Signaling Are Regulated by Interactions between Calcium Stores and Plasma Membrane Ion Channels

PubMed Central

Bartoletti, Theodore M.; Huang, Wei; Akopian, Abram; Thoreson, Wallace B.; Krizaj, David

2009-01-01

Calcium is a messenger ion that controls all aspects of cone photoreceptor function, including synaptic release. The dynamic range of the cone output extends beyond the activation threshold for voltage-operated calcium entry, suggesting another calcium influx mechanism operates in cones hyperpolarized by light. We have used optical imaging and whole-cell voltage clamp to measure the contribution of store-operated Ca2+ entry (SOCE) to Ca2+ homeostasis and its role in regulation of neurotransmission at cone synapses. Mn2+ quenching of Fura-2 revealed sustained divalent cation entry in hyperpolarized cones. Ca2+ influx into cone inner segments was potentiated by hyperpolarization, facilitated by depletion of intracellular Ca2+ stores, unaffected by pharmacological manipulation of voltage-operated or cyclic nucleotide-gated Ca2+ channels and suppressed by lanthanides, 2-APB, MRS 1845 and SKF 96365. However, cation influx through store-operated channels crossed the threshold for activation of voltage-operated Ca2+ entry in a subset of cones, indicating that the operating range of inner segment signals is set by interactions between store- and voltage-operated Ca2+ channels. Exposure to MRS 1845 resulted in ∼40% reduction of light-evoked postsynaptic currents in photopic horizontal cells without affecting the light responses or voltage-operated Ca2+ currents in simultaneously recorded cones. The spatial pattern of store-operated calcium entry in cones matched immunolocalization of the store-operated sensor STIM1. These findings show that store-operated channels regulate spatial and temporal properties of Ca2+ homeostasis in vertebrate cones and demonstrate their role in generation of sustained excitatory signals across the first retinal synapse. PMID:19696927

Myosin light chain kinase controls voltage-dependent calcium channels in vascular smooth muscle.

PubMed

Martinsen, A; Schakman, O; Yerna, X; Dessy, C; Morel, N

2014-07-01

The Ca(2+)-dependent kinase myosin light chain kinase (MLCK) is the activator of smooth muscle contraction. In addition, it has been reported to be involved in Ca(2+) channel regulation in cultured cells, and we previously showed that the MLCK inhibitor ML-7 decreases arginine vasopressin (AVP)-induced Ca(2+) influx in rat aorta. This study was designed to investigate whether MLCK is involved in Ca(2+) regulation in resistance artery smooth muscle cell, which plays a major role in the control of blood pressure. As ML compounds were shown to have off-target effects, MLCK was downregulated by transfection with a small interfering RNA targeting MLCK (MLCK-siRNA) in rat small resistance mesenteric artery (RMA) and in the rat embryonic aortic cell line A7r5. Noradrenaline-induced contraction and Ca(2+) signal were significantly depressed in MLCK-siRNA compared to scramble-siRNA-transfected RMA. Contraction and Ca(2+) signal induced by high KCl and voltage-activated Ca(2+) current were also significantly decreased in MLCK-siRNA-transfected RMA, suggesting that MLCK depletion modifies voltage-operated Ca(2+) channels. KCl- and AVP-induced Ca(2+) signals and voltage-activated Ca(2+) current were decreased in MLCK-depleted A7r5 cells. Eventually, real-time quantitative PCR analysis indicated that in A7r5, MLCK controlled mRNA expression of CaV1.2 (L-type) and CaV3.1 (T-type) voltage-dependent Ca(2+) channels. Our results suggest that MLCK controls the transcription of voltage-dependent Ca(2+) channels in vascular smooth muscle cells.

Cytosolic Na+ Controls an Epithelial Na+ Channel Via the Go Guanine Nucleotide-Binding Regulatory Protein

NASA Astrophysics Data System (ADS)

Komwatana, P.; Dinudom, A.; Young, J. A.; Cook, D. I.

1996-07-01

In tight Na+-absorbing epithelial cells, the rate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-β -S, pertussis toxin, and antibodies against the α -subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-.

Functional reconstitution of the voltage-regulated sodium channel purified from electroplax of Electrophorus electricus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosenberg, R.L.

1985-01-01

The voltage-regulated NA channel is responsible for the depolarization of the excitable cell membrane during the normal action potential. This research has focused on the functional properties of the Na channel, purified from detergent extracts of electroplax membranes of the electric eel, and reconstituted into vesicles of defined phospholipid. These properties were assessed by measuring neurotoxin-modulated ion flux into the reconstituted membrane vesicles and by recording the single-channel currents of the purified channel by the patch-clamp method. The binding of tritiated tetrodotoxin (TTX) was employed as a marker for the purification of the channel. Two high-resolution fractionation steps, based onmore » molecular charge and protein size, were used to obtain a preparation that is 80% homogeneous for a large peptide of 270,000 daltons. Radiotracer /sup 22/Na/sup +/ influx into the vesicles was stimulated by veratridine and by batrachotoxin (BTX) at concentrations of 100 ..mu..M and 5 ..mu..M, respectively. The stimulation by BTX was greater than that by veratridine, and can be as much as 16-fold over control influx levels. The stimulated influx is blocked by TTX with a K/sub i/ of 35 nM, and by local anesthetics in the normal pharmacological range. Large multilamellar vesicles prepared with a freeze-thaw step are suitable for single-channel recording techniques. When excised patches of the reconstituted membranes were voltage-clamped in the absence of activating neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties similar to those from native membranes of nerve and muscle. These results indicate that the protein purified on the basis of TTX binding is a functional Na channel possessing these functional domains: the ion-selective channel, the voltage sensors controlling activation and inactivation, and the sites of action of TTX, alkaloid neurotoxins, and local anesthetics.« less