Ferritin protein nanocages use ion channels, catalytic sites, and nucleation channels to manage iron/oxygen chemistry.

PubMed

Theil, Elizabeth C

2011-04-01

The ferritin superfamily is composed of ancient, nanocage proteins with an internal cavity, 60% of total volume, that reversibly synthesize solid minerals of hydrated ferric oxide; the minerals are iron concentrates for cell nutrition as well as antioxidants due to ferrous and oxygen consumption during mineralization. The cages have multiple iron entry/exit channels, oxidoreductase enzyme sites, and, in eukaryotes, Fe(III)O nucleation channels with clustered exits that extend protein activity to include facilitated mineral growth. Ferritin protein cage differences include size, amino acid sequence, and location of the active sites, oxidant substrate and crystallinity of the iron mineral. Genetic regulation depends on iron and oxygen signals, which in animals includes direct ferrous signaling to RNA to release and to ubiquitin-ligases to degrade the protein repressors. Ferritin biosynthesis forms, with DNA, mRNA and the protein product, a feedback loop where the genetic signals are also protein substrates. The ferritin protein nanocages, which are required for normal iron homeostasis and are finding current use in the delivery of nanodrugs, novel nanomaterials, and nanocatalysts, are likely contributors to survival and success during the transition from anaerobic to aerobic life. Copyright © 2011. Published by Elsevier Ltd.

Ferritin Protein Nanocages Use Ion Channels, Catalytic Sites, and Nucleation Channels To Manage Iron/Oxygen Chemistry

PubMed Central

Theil, Elizabeth C.

2011-01-01

The ferritin superfamily is composed of ancient, nanocage proteins with an internal cavity, 60% of total volume, that reversibly synthesize solid minerals of hydrated ferric oxide; the minerals are iron concentrates for cell nutrition as well as antioxidants due to ferrous and oxygen consumption during mineralization. The cages have multiple iron entry/exit channels, oxidoreductase enzyme sites, and, in eukaryotes, Fe(III)O nucleation channels with clustered exits that extend protein activity to include facilitated mineral growth. Ferritin protein cage differences include size, amino acid sequence, and location of the active sites, oxidant substrate and crystallinity of the iron mineral. Genetic regulation depends on iron and oxygen signals, which in animals includes direct ferrous signaling to RNA to release and to ubiquitin-ligases to degrade the protein repressors. Ferritin biosynthesis forms, with DNA, mRNA and the protein product, a feedback loop where the genetic signals are also protein substrates. The ferritin protein nanocages, which are required for normal iron homeostasis and are finding current use in delivery of nanodrugs, novel nanomaterials, and nanocatalysts, are likely contributors to survival and success during the transition from anaerobic to aerobic life. PMID:21296609

Stream channel reference sites: An illustrated guide to field technique

Treesearch

Cheryl C Harrelson; C. L. Rawlins; John P. Potyondy

1994-01-01

This document is a guide to establishing permanent reference sites for gathering data about the physical characteristics of streams and rivers. The minimum procedure consists of the following: (1) select a site, (2) map the site and location, (3) measure the channel cross-section, (4) survey a longitudinal profile of the channel, (5) measure stream flow, (6) measure...

Riluzole activates TRPC5 channels independently of PLC activity

PubMed Central

Richter, Julia M; Schaefer, Michael; Hill, Kerstin

2014-01-01

BACKGROUND AND PURPOSE The transient receptor potential channel C5 (TRPC5) is a Ca2+-permeable cation channel, which is predominantly expressed in the brain. TRPC5 is activated in a PLC-dependent manner by, as yet, unidentified endogenous messengers. Recently, modulators of TRPC5, like Ca2+, pH and phospholipids, have been identified. However, the role of TRPC5 in vivo is only poorly understood. Novel specific modulators of TRPC5 might help to elucidate its function. EXPERIMENTAL APPROACH Novel modulators of TRPC5 were identified in a compound screening of approved drugs and natural compounds. The potency and selectivity of TRPC5-activating compounds were determined by fluorometric calcium imaging. The biophysical properties of channel activation by these compounds were analysed using electrophysiological measurements. KEY RESULTS Riluzole was identified as a novel activator of TRPC5 (EC50 9.2 ± 0.5 μM) and its mechanism of action was shown to be independent of G protein signalling and PLC activity. Riluzole-induced TRPC5 currents were potentiated by La3+ and, utilizing TRPC5 mutants that lack La3+ binding sites, it was confirmed that riluzole and La3+ activate TRPC5 by different mechanisms. Recordings of excised inside-out patches revealed a relatively direct effect of riluzole on TRPC5. CONCLUSIONS AND IMPLICATIONS Riluzole can activate TRPC5 heterologously expressed in HEK293 cells as well as those endogenously expressed in the U-87 glioblastoma cell line. Riluzole does not activate any other member of the TRPC family and could, therefore, despite its action on other ion channels, be a useful pharmacological tool for identifying TRPC5-specific currents in immortalized cell lines or in acutely isolated primary cells. PMID:24117252

Proton and non-proton activation of ASIC channels

PubMed Central

Gautschi, Ivan; van Bemmelen, Miguel Xavier; Schild, Laurent

2017-01-01

The Acid-Sensing Ion Channels (ASIC) exhibit a fast desensitizing current when activated by pH values below 7.0. By contrast, non-proton ligands are able to trigger sustained ASIC currents at physiological pHs. To analyze the functional basis of the ASIC desensitizing and sustained currents, we have used ASIC1a and ASIC2a mutants with a cysteine in the pore vestibule for covalent binding of different sulfhydryl reagents. We found that ASIC1a and ASIC2a exhibit two distinct currents, a proton-induced desensitizing current and a sustained current triggered by sulfhydryl reagents. These currents differ in their pH dependency, their sensitivity to the sulfhydryl reagents, their ionic selectivity and their relative magnitude. We propose a model for ASIC1 and ASIC2 activity where the channels can function in two distinct modes, a desensitizing mode and a sustained mode depending on the activating ligands. The pore vestibule of the channel represents a functional site for binding non-proton ligands to activate ASIC1 and ASIC2 at neutral pH and to prevent channel desensitization. PMID:28384246

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

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

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

Anesthetic sites and allosteric mechanisms of action on Cys-loop ligand-gated ion channels.

PubMed

Forman, Stuart A; Miller, Keith W

2011-02-01

The Cys-loop ligand-gated ion channel superfamily is a major group of neurotransmitter-activated receptors in the central and peripheral nervous system. The superfamily includes inhibitory receptors stimulated by γ-aminobutyric acid (GABA) and glycine and excitatory receptors stimulated by acetylcholine and serotonin. The first part of this review presents current evidence on the location of the anesthetic binding sites on these channels and the mechanism by which binding to these sites alters their function. The second part of the review addresses the basis for this selectivity, and the third part describes the predictive power of a quantitative allosteric model showing the actions of etomidate on γ-aminobutyric acid type A receptors (GABA(A)Rs). General anesthetics at clinical concentrations inhibit the excitatory receptors and enhance the inhibitory receptors. The location of general anesthetic binding sites on these receptors is being defined by photoactivable analogues of general anesthetics. The receptor studied most extensively is the muscle-type nicotinic acetylcholine receptor (nAChR), and progress is now being made with GABA(A)Rs. There are three categories of sites that are all in the transmembrane domain: 1) within a single subunit's four-helix bundle (intrasubunit site; halothane and etomidate on the δ subunit of AChRs); 2) between five subunits in the transmembrane conduction pore (channel lumen sites; etomidate and alcohols on nAChR); and 3) between two subunits (subunit interface sites; etomidate between the α1 and β2/3 subunits of the GABA(A)R). These binding sites function allosterically. Certain conformations of a receptor bind the anesthetic with greater affinity than others. Time-resolved photolabelling of some sites occurs within milliseconds of channel opening on the nAChR but not before. In GABA(A)Rs, electrophysiological data fit an allosteric model in which etomidate binds to and stabilizes the open state, increasing both the fraction

Atomic determinants of BK channel activation by polyunsaturated fatty acids

PubMed Central

Tian, Yutao; Aursnes, Marius; Hansen, Trond Vidar; Tungen, Jørn Eivind; Galpin, Jason D.; Leisle, Lilia; Ahern, Christopher A.; Xu, Rong; Heinemann, Stefan H.; Hoshi, Toshinori

2016-01-01

Docosahexaenoic acid (DHA), a polyunsaturated ω-3 fatty acid enriched in oily fish, contributes to better health by affecting multiple targets. Large-conductance Ca2+- and voltage-gated Slo1 BK channels are directly activated by nanomolar levels of DHA. We investigated DHA–channel interaction by manipulating both the fatty acid structure and the channel composition through the site-directed incorporation of unnatural amino acids. Electrophysiological measurements show that the para-group of a Tyr residue near the ion conduction pathway has a critical role. To robustly activate the channel, ionization must occur readily by a fatty acid for a good efficacy, and a long nonpolar acyl tail with a Z double bond present at the halfway position for a high affinity. The results suggest that DHA and the channel form an ion–dipole bond to promote opening and demonstrate the channel druggability. DHA, a marine-derived nutraceutical, represents a promising lead compound for rational drug design and discovery. PMID:27849612

Indoxacarb, Metaflumizone, and Other Sodium Channel Inhibitor Insecticides: Mechanism and Site of Action on Mammalian Voltage-Gated Sodium Channels

PubMed Central

von Stein, Richard T.; Silver, Kristopher S.; Soderlund, David M.

2013-01-01

Sodium channel inhibitor (SCI) insecticides were discovered almost four decades ago but have only recently yielded important commercial products (eg., indoxacarb and metaflumizone). SCI insecticides inhibit sodium channel function by binding selectively to slow-inactivated (non-conducting) sodium channel states. Characterization of the action of SCI insecticides on mammalian sodium channels using both biochemical and electrophysiological approaches demonstrates that they bind at or near a drug receptor site, the "local anesthetic (LA) receptor." This mechanism and site of action on sodium channels differentiates SCI insecticides from other insecticidal agents that act on sodium channels. However, SCI insecticides share a common mode of action with drugs currently under investigation as anticonvulsants and treatments for neuropathic pain. In this paper we summarize the development of the SCI insecticide class and the evidence that this structurally diverse group of compounds have a common mode of action on sodium channels. We then review research that has used site-directed mutagenesis and heterologous expression of cloned mammalian sodium channels in Xenopus laevis oocytes to further elucidate the site and mechanism of action of SCI insecticides. The results of these studies provide new insight into the mechanism of action of SCI insecticides on voltage-gated sodium channels, the location of the SCI insecticide receptor, and its relationship to the LA receptor that binds therapeutic SCI agents. PMID:24072940

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.

Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel.

PubMed

Malinowska, D H; Kupert, E Y; Bahinski, A; Sherry, A M; Cuppoletti, J

1995-01-01

cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.

Coupling interactions between voltage sensors of the sodium channel as revealed by site-specific measurements.

PubMed

Chanda, Baron; Asamoah, Osei Kwame; Bezanilla, Francisco

2004-03-01

The voltage-sensing S4 segments in the sodium channel undergo conformational rearrangements in response to changes in the electric field. However, it remains unclear whether these structures move independently or in a coordinated manner. Previously, site-directed fluorescence measurements were shown to track S4 transitions in each of the four domains. Here, using a similar technique, we provide direct evidence of coupling interactions between voltage sensors in the sodium channel. Pairwise interactions between S4s were evaluated by comparing site-specific conformational changes in the presence and absence of a gating perturbation in a distal domain. Reciprocity of effect, a fundamental property of thermodynamically coupled systems, was measured by generating converse mutants. The magnitude of a local gating perturbation induced by a remote S4 mutation depends on the coupling strength and the relative equilibrium positions of the two voltage sensors. In general, our data indicates that the movement of all four voltage sensors in the sodium channel are coupled to a varying extent. Moreover, a gating perturbation in S4-DI has the largest effect on the activation of S4-DIV and vice versa, demonstrating an energetic linkage between S4-DI and S4-DIV. This result suggests a physical mechanism by which the activation and inactivation process may be coupled in voltage-gated sodium channels. In addition, we propose that cooperative interactions between voltage sensors may be the mechanistic basis for the fast activation kinetics of the sodium channel.

Mechanism of allosteric activation of TMEM16A/ANO1 channels by a commonly used chloride channel blocker

PubMed Central

Ta, Chau M; Adomaviciene, Aiste; Rorsman, Nils J G; Garnett, Hannah

2016-01-01

Background and Purpose Calcium‐activated chloride channels (CaCCs) play varied physiological roles and constitute potential therapeutic targets for conditions such as asthma and hypertension. TMEM16A encodes a CaCC. CaCC pharmacology is restricted to compounds with relatively low potency and poorly defined selectivity. Anthracene‐9‐carboxylic acid (A9C), an inhibitor of various chloride channel types, exhibits complex effects on native CaCCs and cloned TMEM16A channels providing both activation and inhibition. The mechanisms underlying these effects are not fully defined. Experimental Approach Patch‐clamp electrophysiology in conjunction with concentration jump experiments was employed to define the mode of interaction of A9C with TMEM16A channels. Key Results In the presence of high intracellular Ca2+, A9C inhibited TMEM16A currents in a voltage‐dependent manner by entering the channel from the outside. A9C activation, revealed in the presence of submaximal intracellular Ca2+ concentrations, was also voltage‐dependent. The electric distance of A9C inhibiting and activating binding site was ~0.6 in each case. Inhibition occurred according to an open‐channel block mechanism. Activation was due to a dramatic leftward shift in the steady‐state activation curve and slowed deactivation kinetics. Extracellular A9C competed with extracellular Cl−, suggesting that A9C binds deep in the channel's pore to exert both inhibiting and activating effects. Conclusions and Implications A9C is an open TMEM16A channel blocker and gating modifier. These effects require A9C to bind to a region within the pore that is accessible from the extracellular side of the membrane. These data will aid the future drug design of compounds that selectively activate or inhibit TMEM16A channels. PMID:26562072

Site of anticonvulsant action on sodium channels: autoradiographic and electrophysiological studies in rat brain

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

Worley, P.F.; Baraban, J.M.

1987-05-01

The anticonvulsants phenytoin and carbamazepine interact allosterically with the batrachotoxin binding site of sodium channels. In the present study, we demonstrate an autoradiographic technique to localize the batrachotoxin binding site on sodium channels in rat brain using (/sup 3/H)batrachotoxinin-A 20-alpha-benzoate (BTX-B). Binding of (/sup 3/H)BTX-B to brain sections is dependent on potentiating allosteric interactions with scorpion venom and is displaced by BTX-B (Kd approximately 200 nM), aconitine, veratridine, and phenytoin with the same rank order of potencies as described in brain synaptosomes. The maximum number of (/sup 3/H)BTX-B binding sites in forebrain sections also agrees with biochemical determinations. Autoradiographic localizationsmore » indicate that (/sup 3/H)BTX-B binding sites are not restricted to cell bodies and axons but are present in synaptic zones throughout the brain. For example, a particularly dense concentration of these sites in the substantia nigra is associated with afferent terminals of the striatonigral projection. By contrast, myelinated structures possess much lower densities of binding sites. In addition, we present electrophysiological evidence that synaptic transmission, as opposed to axonal conduction, is preferentially sensitive to the action of aconitine and veratridine. Finally, the synaptic block produced by these sodium channel activators is inhibited by phenytoin and carbamazepine at therapeutic anticonvulsant concentrations.« less

Mefloquine inhibits voltage dependent Nav1.4 channel by overlapping the local anaesthetic binding site.

PubMed

Paiz-Candia, Bertin; Islas, Angel A; Sánchez-Solano, Alfredo; Mancilla-Simbro, Claudia; Scior, Thomas; Millan-PerezPeña, Lourdes; Salinas-Stefanon, Eduardo M

2017-02-05

Mefloquine constitutes a multitarget antimalaric that inhibits cation currents. However, the effect and the binding site of this compound on Na + channels is unknown. To address the mechanism of action of mefloquine, we employed two-electrode voltage clamp recordings on Xenopus laevis oocytes, site-directed mutagenesis of the rat Na + channel, and a combined in silico approach using Molecular Dynamics and docking protocols. We found that mefloquine: i) inhibited Na v 1.4 currents (IC 50 =60μM), ii) significantly delayed fast inactivation but did not affect recovery from inactivation, iii) markedly the shifted steady-state inactivation curve to more hyperpolarized potentials. The presence of the β1 subunit significantly reduced mefloquine potency, but the drug induced a significant frequency-independent rundown upon repetitive depolarisations. Computational and experimental results indicate that mefloquine overlaps the local anaesthetic binding site by docking at a hydrophobic cavity between domains DIII and DIV that communicates the local anaesthetic binding site with the selectivity filter. This is supported by the fact that mefloquine potency significantly decreased on mutant Na v 1.4 channel F1579A and significantly increased on K1237S channels. In silico this compound docked above F1579 forming stable π-π interactions with this residue. We provide structure-activity insights into how cationic amphiphilic compounds may exert inhibitory effects by docking between the local anaesthetic binding site and the selectivity filter of a mammalian Na + channel. Our proposed synergistic cycle of experimental and computational studies may be useful for elucidating binding sites of other drugs, thereby saving in vitro and in silico resources. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

External protons destabilize the activated voltage sensor in hERG channels.

PubMed

Shi, Yu Patrick; Cheng, Yen May; Van Slyke, Aaron C; Claydon, Tom W

2014-03-01

Extracellular acidosis shifts hERG channel activation to more depolarized potentials and accelerates channel deactivation; however, the mechanisms underlying these effects are unclear. External divalent cations, e.g., Ca(2+) and Cd(2+), mimic these effects and coordinate within a metal ion binding pocket composed of three acidic residues in hERG: D456 and D460 in S2 and D509 in S3. A common mechanism may underlie divalent cation and proton effects on hERG gating. Using two-electrode voltage clamp, we show proton sensitivity of hERG channel activation (pKa = 5.6), but not deactivation, was greatly reduced in the presence of Cd(2+) (0.1 mM), suggesting a common binding site for the Cd(2+) and proton effect on activation and separable effects of protons on activation and deactivation. Mutational analysis confirmed that D509 plays a critical role in the pH dependence of activation, as shown previously, and that cooperative actions involving D456 and D460 are also required. Importantly, neutralization of all three acidic residues abolished the proton-induced shift of activation, suggesting that the metal ion binding pocket alone accounts for the effects of protons on hERG channel activation. Voltage-clamp fluorimetry measurements demonstrated that protons shifted the voltage dependence of S4 movement to more depolarized potentials. The data indicate a site and mechanism of action for protons on hERG activation gating; protonation of D456, D460 and D509 disrupts interactions between these residues and S4 gating charges to destabilize the activated configuration of S4.

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.

Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels

PubMed Central

Shaya, David; Findeisen, Felix; Abderemane-Ali, Fayal; Arrigoni, Cristina; Wong, Stephanie; Nurva, Shailika Reddy; Loussouarn, Gildas; Minor, Daniel L.

2013-01-01

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail ‘neck’, are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the ‘outer ion’ site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies shows that this site forms a previously unknown determinant of CaV high affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily. PMID:24120938

Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels.

PubMed

Shaya, David; Findeisen, Felix; Abderemane-Ali, Fayal; Arrigoni, Cristina; Wong, Stephanie; Nurva, Shailika Reddy; Loussouarn, Gildas; Minor, Daniel L

2014-01-23

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail "neck", are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the "outer ion" site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of CaV high-affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily. © 2013. Published by Elsevier Ltd. All rights reserved.

Channeling by Proximity: The Catalytic Advantages of Active Site Colocalization Using Brownian Dynamics.

PubMed

Bauler, Patricia; Huber, Gary; Leyh, Thomas; McCammon, J Andrew

2010-05-06

Nature often colocalizes successive steps in a metabolic pathway. Such organization is predicted to increase the effective concentration of pathway intermediates near their recipient active sites and to enhance catalytic efficiency. Here, the pathway of a two-step reaction is modeled using a simple spherical approximation for the enzymes and substrate particles. Brownian dynamics are used to simulate the trajectory of a substrate particle as it diffuses between the active site zones of two different enzyme spheres. The results approximate distances for the most effective reaction pathways, indicating that the most effective reaction pathway is one in which the active sites are closely aligned. However, when the active sites are too close, the ability of the substrate to react with the first enzyme was hindered, suggesting that even the most efficient orientations can be improved for a system that is allowed to rotate or change orientation to optimize the likelihood of reaction at both sites.

Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites

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

Derebe, Mehabaw G.; Sauer, David B.; Zeng, Weizhong

2015-11-30

Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K{sup +} channel selectivity filters. Combined with single channel electrophysiology, we show that only themore » channel with four ion binding sites is K{sup +} selective, whereas those with two or three are nonselective and permeate Na{sup +} and K{sup +} equally well. These observations strongly suggest that the number of contiguous ion binding sites in a single file is the key determinant of the channel's selectivity properties and the presence of four sites in K{sup +} channels is essential for highly selective and efficient permeation of K{sup +} ions.« less

The amiodarone derivative KB130015 activates hERG1 potassium channels via a novel mechanism

PubMed Central

Gessner, Guido; Macianskiene, Regina; Starkus, John G.; Schönherr, Roland; Heinemann, Stefan H.

2010-01-01

Human ether à go-go related gene (hERG1) potassium channels underlie the repolarizing IKr current in the heart. Since they are targets of various drugs with cardiac side effects we tested whether the amiodarone derivative 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015) blocks hERG1 channels like its parent compound. Using patch-clamp and two-electrode voltage-clamp techniques we found that KB130015 blocks native and recombinant hERG1 channels at high voltages, but it activates them at low voltages. The activating effect has an apparent EC50 value of 12 μM and is brought about by an about 4-fold acceleration of activation kinetics and a shift in voltage-dependent activation by −16 mV. Channel activation was not use-dependent and was independent of inactivation gating. KB130015 presumably binds to the hERG1 pore from the cytosolic side and functionally competes with hERG1 block by amiodarone, E4031 (N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl] -4-piperidinyl] carbonyl] phenyl] methanesulfonamide dihydrochloride), and sertindole. Vice versa, amiodarone attenuates hERG1 activation by KB130015. Based on synergic channel activation by mallotoxin and KB130015 we conclude that the hERG1 pore contains at least two sites for activators that are functionally coupled among each other and to the cavity-blocker site. KB130015 and amiodarone may serve as lead structures for the identification of hERG1 pore-interacting drugs favoring channel activation vs. block. PMID:20097192

Intracellular zinc activates KCNQ channels by reducing their dependence on phosphatidylinositol 4,5-bisphosphate

PubMed Central

Gao, Haixia; Boillat, Aurélien; Huang, Dongyang; Liang, Ce; Peers, Chris

2017-01-01

M-type (Kv7, KCNQ) potassium channels are proteins that control the excitability of neurons and muscle cells. Many physiological and pathological mechanisms of excitation operate via the suppression of M channel activity or expression. Conversely, pharmacological augmentation of M channel activity is a recognized strategy for the treatment of hyperexcitability disorders such as pain and epilepsy. However, physiological mechanisms resulting in M channel potentiation are rare. Here we report that intracellular free zinc directly and reversibly augments the activity of recombinant and native M channels. This effect is mechanistically distinct from the known redox-dependent KCNQ channel potentiation. Interestingly, the effect of zinc cannot be attributed to a single histidine- or cysteine-containing zinc-binding site within KCNQ channels. Instead, zinc dramatically reduces KCNQ channel dependence on its obligatory physiological activator, phosphatidylinositol 4,5-bisphosphate (PIP2). We hypothesize that zinc facilitates interactions of the lipid-facing interface of a KCNQ protein with the inner leaflet of the plasma membrane in a way similar to that promoted by PIP2. Because zinc is increasingly recognized as a ubiquitous intracellular second messenger, this discovery might represent a hitherto unknown native pathway of M channel modulation and provide a fresh strategy for the design of M channel activators for therapeutic purposes. PMID:28716904

Intracellular zinc activates KCNQ channels by reducing their dependence on phosphatidylinositol 4,5-bisphosphate.

PubMed

Gao, Haixia; Boillat, Aurélien; Huang, Dongyang; Liang, Ce; Peers, Chris; Gamper, Nikita

2017-08-01

M-type (Kv7, KCNQ) potassium channels are proteins that control the excitability of neurons and muscle cells. Many physiological and pathological mechanisms of excitation operate via the suppression of M channel activity or expression. Conversely, pharmacological augmentation of M channel activity is a recognized strategy for the treatment of hyperexcitability disorders such as pain and epilepsy. However, physiological mechanisms resulting in M channel potentiation are rare. Here we report that intracellular free zinc directly and reversibly augments the activity of recombinant and native M channels. This effect is mechanistically distinct from the known redox-dependent KCNQ channel potentiation. Interestingly, the effect of zinc cannot be attributed to a single histidine- or cysteine-containing zinc-binding site within KCNQ channels. Instead, zinc dramatically reduces KCNQ channel dependence on its obligatory physiological activator, phosphatidylinositol 4,5-bisphosphate (PIP 2 ). We hypothesize that zinc facilitates interactions of the lipid-facing interface of a KCNQ protein with the inner leaflet of the plasma membrane in a way similar to that promoted by PIP 2 Because zinc is increasingly recognized as a ubiquitous intracellular second messenger, this discovery might represent a hitherto unknown native pathway of M channel modulation and provide a fresh strategy for the design of M channel activators for therapeutic purposes.

Influence of active site location on catalytic activity in de novo-designed zinc metalloenzymes.

PubMed

Zastrow, Melissa L; Pecoraro, Vincent L

2013-04-17

While metalloprotein design has now yielded a number of successful metal-bound and even catalytically active constructs, the question of where to put a metal site along a linear, repetitive sequence has not been thoroughly addressed. Often several possibilities in a given sequence may exist that would appear equivalent but may in fact differ for metal affinity, substrate access, or protein dynamics. We present a systematic variation of active site location for a hydrolytically active ZnHis3O site contained within a de novo-designed three-stranded coiled coil. We find that the maximal rate, substrate access, and metal-binding affinity are dependent on the selected position, while catalytic efficiency for p-nitrophenyl acetate hydrolysis can be retained regardless of the location of the active site. This achievement demonstrates how efficient, tailor-made enzymes which control rate, pKa, substrate and solvent access (and selectivity), and metal-binding affinity may be realized. These findings may be applied to the more advanced de novo design of constructs containing secondary interactions, such as hydrogen-bonding channels. We are now confident that changes to location for accommodating such channels can be achieved without location-dependent loss of catalytic efficiency. These findings bring us closer to our ultimate goal of incorporating the secondary interactions we believe will be necessary in order to improve both active site properties and the catalytic efficiency to be competitive with the native enzyme, carbonic anhydrase.

Locating the route of entry and binding sites of benzocaine and phenytoin in a bacterial voltage gated sodium channel.

PubMed

Martin, Lewis J; Corry, Ben

2014-07-01

Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites.

Locating the Route of Entry and Binding Sites of Benzocaine and Phenytoin in a Bacterial Voltage Gated Sodium Channel

PubMed Central

Martin, Lewis J.; Corry, Ben

2014-01-01

Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites. PMID:24992293

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.

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.

Permeation Mechanisms in the TMEM16B Calcium-Activated Chloride Channels

PubMed Central

2017-01-01

TMEM16A and TMEM16B encode for Ca2+-activated Cl− channels (CaCC) and are expressed in many cell types and play a relevant role in many physiological processes. Here, I performed a site-directed mutagenesis study to understand the molecular mechanisms of ion permeation of TMEM16B. I mutated two positive charged residues R573 and K540, respectively located at the entrance and inside the putative channel pore and I measured the properties of wild-type and mutant TMEM16B channels expressed in HEK-293 cells using whole-cell and excised inside-out patch clamp experiments. I found evidence that R573 and K540 control the ion permeability of TMEM16B depending both on which side of the membrane the ion substitution occurs and on the level of channel activation. Moreover, these residues contribute to control blockage or activation by permeant anions. Finally, R573 mutation abolishes the anomalous mole fraction effect observed in the presence of a permeable anion and it alters the apparent Ca2+-sensitivity of the channel. These findings indicate that residues facing the putative channel pore are responsible both for controlling the ion selectivity and the gating of the channel, providing an initial understanding of molecular mechanism of ion permeation in TMEM16B. PMID:28046119

Direct activation of the olfactory cyclic nucleotide-gated channel through modification of sulfhydryl groups by NO compounds.

PubMed

Broillet, M C; Firestein, S

1996-02-01

The activation of a cyclic nucleotide-gated channel is the final step in sensory transduction in olfaction. Normally, this channel is opened by the intracellular cyclic nucleotide second messenger cAMP or cGMP. However, in single channel recordings we found that donors of nitric oxide, a putative intercellular messenger, could directly activate the native olfactory neuron channel. Its action was independent of the presence of the normal ligand and did not involve the cyclic nucleotide binding site, suggesting an alternate site on the molecule that is critical in channel gating. The biochemical pathway appears to utilize nitric oxide in one of its alternate redox states, the nitrosonium ion, transnitrosylating a free sulfhydryl group belonging to a cysteine residue tentatively identified as being in the region linking the S6 transmembrane domain to the ligand binding domain.

Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels*

PubMed Central

Tang, Qiong-Yao; Larry, Trevor; Hendra, Kalen; Yamamoto, Erica; Bell, Jessica; Cui, Meng; Logothetis, Diomedes E.; Boland, Linda M.

2015-01-01

All vertebrate inwardly rectifying potassium (Kir) channels are activated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Logothetis, D. E., Petrou, V. I., Zhang, M., Mahajan, R., Meng, X. Y., Adney, S. K., Cui, M., and Baki, L. (2015) Annu. Rev. Physiol. 77, 81–104; Fürst, O., Mondou, B., and D'Avanzo, N. (2014) Front. Physiol. 4, 404–404). Structural components of a PIP2-binding site are conserved in vertebrate Kir channels but not in distantly related animals such as sponges and sea anemones. To expand our understanding of the structure-function relationships of PIP2 regulation of Kir channels, we studied AqKir, which was cloned from the marine sponge Amphimedon queenslandica, an animal that represents the phylogenetically oldest metazoans. A requirement for PIP2 in the maintenance of AqKir activity was examined in intact oocytes by activation of a co-expressed voltage-sensing phosphatase, application of wortmannin (at micromolar concentrations), and activation of a co-expressed muscarinic acetylcholine receptor. All three mechanisms to reduce the availability of PIP2 resulted in inhibition of AqKir current. However, time-dependent rundown of AqKir currents in inside-out patches could not be re-activated by direct application to the inside membrane surface of water-soluble dioctanoyl PIP2, and the current was incompletely re-activated by the more hydrophobic arachidonyl stearyl PIP2. When we introduced mutations to AqKir to restore two positive charges within the vertebrate PIP2-binding site, both forms of PIP2 strongly re-activated the mutant sponge channels in inside-out patches. Molecular dynamics simulations validate the additional hydrogen bonding potential of the sponge channel mutants. Thus, nature's mutations conferred a high affinity activation of vertebrate Kir channels by PIP2, and this is a more recent evolutionary development than the structures that explain ion channel selectivity and inward rectification. PMID:25957411

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

Activation of muscle nicotinic acetylcholine receptor channels by nicotinic and muscarinic agonists

PubMed Central

Akk, Gustav; Auerbach, Anthony

1999-01-01

The dose-response parameters of recombinant mouse adult neuromuscular acetylcholine receptor channels (nAChR) activated by carbamylcholine, nicotine, muscarine and oxotremorine were measured. Rate constants for agonist association and dissociation, and channel opening and closing, were estimated from single-channel kinetic analysis.The dissociation equilibrium constants were (mM): ACh (0.16)carbamylcholine (5.1)>oxotremorine M (0.6)>nicotine (0.5)>muscarine (0.15).Rat neuronal α4β2 nAChR can be activated by all of the agonists. However, detailed kinetic analysis was impossible because the recordings lacked clusters representing the activity of a single receptor complex. Thus, the number of channels in the patch was unknown and the activation rate constants could not be determined.Considering both receptor affinity and agonist efficacy, muscarine and oxotremorine are significant agonists of muscle-type nAChR. The results are discussed in terms of structure-function relationships at the nAChR transmitter binding site. PMID:10602325

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

S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK) channels

PubMed Central

Shipston, Michael J.

2014-01-01

Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation) represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs) and acyl thioesterases (APTs). S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signaling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease. PMID:25140154

Chloride channels are necessary for full platelet phosphatidylserine exposure and procoagulant activity.

PubMed

Harper, M T; Poole, A W

2013-12-19

Platelets enhance thrombin generation at sites of vascular injury by exposing phosphatidylserine during necrosis-like cell death. Anoctamin 6 (Ano6) is required for Ca(2+)-dependent phosphatidylserine exposure and is defective in patients with Scott syndrome, a rare bleeding disorder. Ano6 may also form Cl(-) channels, though the role of Cl(-) fluxes in platelet procoagulant activity has not been explored. We found that Cl(-) channel blockers or removal of extracellular Cl(-) inhibited agonist-induced phosphatidylserine exposure. However, this was not due to direct inhibition of Ca(2+)-dependent scrambling since Ca(2+) ionophore-induced phosphatidylserine exposure was normal. This implies that the role of Ano6 in Ca(2+-)dependent PS exposure is likely to differ from any putative function of Ano6 as a Cl(-) channel. Instead, Cl(-) channel blockade inhibited agonist-induced Ca(2+) entry. Importantly, Cl(-) channel blockers also prevented agonist-induced membrane hyperpolarization, resulting in depolarization. We propose that Cl(-) entry through Cl(-) channels is required for this hyperpolarization, maintaining the driving force for Ca(2+) entry and triggering full phosphatidylserine exposure. This demonstrates a novel role for Cl(-) channels in controlling platelet death and procoagulant activity.

Direct demonstration of persistent Na+ channel activity in dendritic processes of mammalian cortical neurones

PubMed Central

Magistretti, Jacopo; Ragsdale, David S; Alonso, Angel

1999-01-01

Single Na+ channel activity was recorded in patch-clamp, cell-attached experiments performed on dendritic processes of acutely isolated principal neurones from rat entorhinal-cortex layer II. The distances of the recording sites from the soma ranged from ≈20 to ≈100 μm.Step depolarisations from holding potentials of −120 to −100 mV to test potentials of −60 to +10 mV elicited Na+ channel openings in all of the recorded patches (n= 16).In 10 patches, besides transient Na+ channel openings clustered within the first few milliseconds of the depolarising pulses, prolonged and/or late Na+ channel openings were also regularly observed. This ‘persistent’ Na+ channel activity produced net inward, persistent currents in ensemble-average traces, and remained stable over the entire duration of the experiments (≈9 to 30 min).Two of these patches contained <= 3 channels. In these cases, persistent Na+ channel openings could be attributed to the activity of one single channel.The voltage dependence of persistent-current amplitude in ensemble-average traces closely resembled that of whole-cell, persistent Na+ current expressed by the same neurones, and displayed the same characteristic low threshold of activation.Dendritic, persistent Na+ channel openings had relatively high single-channel conductance (≈20 pS), similar to what is observed for somatic, persistent Na+ channels.We conclude that a stable, persistent Na+ channel activity is expressed by proximal dendrites of entorhinal-cortex layer II principal neurones, and can contribute a significant low-threshold, persistent Na+ current to the dendritic processing of excitatory synaptic inputs. PMID:10601494

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

PubMed Central

Tilley, Drew C.; Eum, Kenneth S.; Fletcher-Taylor, Sebastian; Austin, Daniel C.; Dupré, Christophe; Patrón, Lilian A.; Garcia, Rita L.; Lam, Kit; Yarov-Yarovoy, Vladimir; Cohen, Bruce E.; Sack, Jon T.

2014-01-01

Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of Kv2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks Kv2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX–fluorophore conjugates colocalize with Kv2.1 clusters in live cells and are released from channels activated by voltage stimuli. Kv2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing Kv2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling. PMID:25331865

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

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

Ionizable Side Chains at Catalytic Active Sites of Enzymes

PubMed Central

Jimenez-Morales, David; Liang, Jie

2012-01-01

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

Site-Directed Spin Labeling Reveals Pentameric Ligand-Gated Ion Channel Gating Motions

PubMed Central

Dellisanti, Cosma D.; Ghosh, Borna; Hanson, Susan M.; Raspanti, James M.; Grant, Valerie A.; Diarra, Gaoussou M.; Schuh, Abby M.; Satyshur, Kenneth; Klug, Candice S.; Czajkowski, Cynthia

2013-01-01

Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitter, the channels desensitize (close). The flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, and 9) to the transmembrane channel domain (M2–M3 loop) are essential for coupling ligand binding to channel gating. Comparing the crystal structures of two bacterial pLGIC homologues, ELIC and the proton-activated GLIC, suggests channel gating is associated with rearrangements in these loops, but whether these motions accurately predict the motions in functional lipid-embedded pLGICs is unknown. Here, using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and functional GLIC channels reconstituted into liposomes, we examined if, and how far, the loops at the ECD/TMD gating interface move during proton-dependent gating transitions from the resting to desensitized state. Loop 9 moves ∼9 Å inward toward the channel lumen in response to proton-induced desensitization. Loop 9 motions were not observed when GLIC was in detergent micelles, suggesting detergent solubilization traps the protein in a nonactivatable state and lipids are required for functional gating transitions. Proton-induced desensitization immobilizes loop 2 with little change in position. Proton-induced motion of the M2–M3 loop was not observed, suggesting its conformation is nearly identical in closed and desensitized states. Our experimentally derived distance measurements of spin-labeled GLIC suggest ELIC is not a good model for the functional resting state of GLIC, and that the crystal structure of GLIC does not correspond to a desensitized state. These findings advance our

Glycine Hinges with Opposing Actions at the Acetylcholine Receptor-Channel Transmitter Binding SiteS⃞

PubMed Central

Purohit, Prasad

2011-01-01

The extent to which agonists activate synaptic receptor-channels depends on both the intrinsic tendency of the unliganded receptor to open and the amount of agonist binding energy realized in the channel-opening process. We examined mutations of the nicotinic acetylcholine receptor transmitter binding site (α subunit loop B) with regard to both of these parameters. αGly147 is an “activation” hinge where backbone flexibility maintains high values for intrinsic gating, the affinity of the resting conformation for agonists and net ligand binding energy. αGly153 is a “deactivation” hinge that maintains low values for these parameters. αTrp149 (between these two glycines) serves mainly to provide ligand binding energy for gating. We propose that a concerted motion of the two glycine hinges (plus other structural elements at the binding site) positions αTrp149 so that it provides physiologically optimal binding and gating function at the nerve-muscle synapse. PMID:21115636

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.

Shaking stack model of ion conduction through the Ca(2+)-activated K+ channel.

PubMed Central

Schumaker, M F

1992-01-01

Motivated by the results of Neyton and Miller (1988. J. Gen. Physiol. 92:549-586), suggesting that the Ca(2+)-activated K+ channel has four high affinity ion binding sites, we propose a physically attractive variant of the single-vacancy conduction mechanism for this channel. Simple analytical expressions for conductance, current, flux ratio exponent, and reversal potential under bi-ionic conditions are found. A set of conductance data are analyzed to determine a realistic range of parameter values. Using these, we find qualitative agreement with a variety of experimental results previously reported in the literature. The exquisite selectivity of the Ca(2+)-activated K+ channel may be explained as a consequence of the concerted motion of the "stack" in the proposed mechanism. PMID:1420923

Numbers of presynaptic Ca2+ channel clusters match those of functionally defined vesicular docking sites in single central synapses.

PubMed

Miki, Takafumi; Kaufmann, Walter A; Malagon, Gerardo; Gomez, Laura; Tabuchi, Katsuhiko; Watanabe, Masahiko; Shigemoto, Ryuichi; Marty, Alain

2017-06-27

Many central synapses contain a single presynaptic active zone and a single postsynaptic density. Vesicular release statistics at such "simple synapses" indicate that they contain a small complement of docking sites where vesicles repetitively dock and fuse. In this work, we investigate functional and morphological aspects of docking sites at simple synapses made between cerebellar parallel fibers and molecular layer interneurons. Using immunogold labeling of SDS-treated freeze-fracture replicas, we find that Ca v 2.1 channels form several clusters per active zone with about nine channels per cluster. The mean value and range of intersynaptic variation are similar for Ca v 2.1 cluster numbers and for functional estimates of docking-site numbers obtained from the maximum numbers of released vesicles per action potential. Both numbers grow in relation with synaptic size and decrease by a similar extent with age between 2 wk and 4 wk postnatal. Thus, the mean docking-site numbers were 3.15 at 2 wk (range: 1-10) and 2.03 at 4 wk (range: 1-4), whereas the mean numbers of Ca v 2.1 clusters were 2.84 at 2 wk (range: 1-8) and 2.37 at 4 wk (range: 1-5). These changes were accompanied by decreases of miniature current amplitude (from 93 pA to 56 pA), active-zone surface area (from 0.0427 μm 2 to 0.0234 μm 2 ), and initial success rate (from 0.609 to 0.353), indicating a tightening of synaptic transmission with development. Altogether, these results suggest a close correspondence between the number of functionally defined vesicular docking sites and that of clusters of voltage-gated calcium channels.

Mars Pathfinder Landing Site Workshop 2: Characteristics of the Ares Vallis Region and Field Trips in the Channeled Scabland, Washington

NASA Technical Reports Server (NTRS)

Golombek, M. P. (Editor); Edgett, K. S. (Editor); Rice, J. W. , Jr. (Editor)

1995-01-01

Mars Pathfinder will place a single lander on the surface of Mars on July 4, 1997, following a December 1996 launch. As a result of the very successful first Mars Pathfinder Landing Site Workshop, the project has selected the Ares Vallis outflow channel in Chryse Planitia as the landing site. This location is where a large catastrophic outflow channel debouches into the northern lowlands. A second workshop and series of field trips, entitled Mars Pathfinder Landing Site Workshop 2: Characteristics of the Ares Vallis Region and Field Trips in the Channeled Scabland, Washington, were held in Spokane and Moses Lake, Washington. The purpose of the workshop was to provide a focus for learning as much as possible about the Ares Vallis region on Mars before landing there. The rationale is that the more that can be learned about the general area prior to landing, the better scientists will be able interpret the observations made by the lander and rover and place them in the proper geologic context. The field trip included overflights and surface investigations of the Channeled Scabland (an Earth analog for the martian catastrophic outflow channels), focusing on areas particularly analogous to Ares Vallis and the landing site. The overflights were essential for placing the enormous erosional and depositional features of the Channeled Scabland into proper three-dimensional context. The field trips were a joint educational outreach activity involving K-12 science educators, Mars Pathfinder scientists and engineers, and interested scientists from the Mars scientific community. Part 1 of the technical report on this workshop includes a description of the Mars Pathfinder mission, abstracts accepted for presentation at the workshop, an introduction to the Channeled Scabland, and field trip guides for the overflight and two field trips. This part, Part 2, includes the program for the workshop, summaries of the workshop technical sessions, a summary of the field trips and ensuing

CNS sites activated by renal pelvic epithelial sodium channels (ENaCs) in response to hypertonic saline in awake rats.

PubMed

Goodwill, Vanessa S; Terrill, Christopher; Hopewood, Ian; Loewy, Arthur D; Knuepfer, Mark M

2017-05-01

In some patients, renal nerve denervation has been reported to be an effective treatment for essential hypertension. Considerable evidence suggests that afferent renal nerves (ARN) and sodium balance play important roles in the development and maintenance of high blood pressure. ARN are sensitive to sodium concentrations in the renal pelvis. To better understand the role of ARN, we infused isotonic or hypertonic NaCl (308 or 500mOsm) into the left renal pelvis of conscious rats for two 2hours while recording arterial pressure and heart rate. Subsequently, brain tissue was analyzed for immunohistochemical detection of the protein Fos, a marker for neuronal activation. Fos-immunoreactive neurons were identified in numerous sites in the forebrain and brainstem. These areas included the nucleus tractus solitarius (NTS), the lateral parabrachial nucleus, the paraventricular nucleus of the hypothalamus (PVH) and the supraoptic nucleus (SON). The most effective stimulus was 500mOsm NaCl. Activation of these sites was attenuated or prevented by administration of benzamil (1μM) or amiloride (10μM) into the renal pelvis concomitantly with hypertonic saline. In anesthetized rats, infusion of hypertonic saline but not isotonic saline into the renal pelvis elevated ARN activity and this increase was attenuated by simultaneous infusion of benzamil or amiloride. We propose that renal pelvic epithelial sodium channels (ENaCs) play a role in activation of ARN and, via central visceral afferent circuits, this system modulates fluid volume and peripheral blood pressure. These pathways may contribute to the development of hypertension. Copyright © 2016 Elsevier B.V. All rights reserved.

On the cellular site of two-pore channel TPC1 action in the Poaceae.

PubMed

Dadacz-Narloch, Beata; Kimura, Sachie; Kurusu, Takamitsu; Farmer, Edward E; Becker, Dirk; Kuchitsu, Kazuyuki; Hedrich, Rainer

2013-11-01

The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel. To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit. Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles. The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

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.

Mapping the Interaction Site for a β-Scorpion Toxin in the Pore Module of Domain III of Voltage-gated Na+ Channels*

PubMed Central

Zhang, Joel Z.; Yarov-Yarovoy, Vladimir; Scheuer, Todd; Karbat, Izhar; Cohen, Lior; Gordon, Dalia; Gurevitz, Michael; Catterall, William A.

2012-01-01

Activation of voltage-gated sodium (Nav) channels initiates and propagates action potentials in electrically excitable cells. β-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of NaV channels. CssIV stabilizes the voltage sensor in domain II in its activated state via a voltage-sensor trapping mechanism. Amino acid residues required for the action of CssIV have been identified in the S1-S2 and S3-S4 extracellular loops of domain II. The extracellular loops of domain III are also involved in toxin action, but individual amino acid residues have not been identified. We used site-directed mutagenesis and voltage clamp recording to investigate amino acid residues of domain III that are involved in CssIV action. In the IIISS2-S6 loop, five substitutions at four positions altered voltage-sensor trapping by CssIVE15A. Three substitutions (E1438A, D1445A, and D1445Y) markedly decreased voltage-sensor trapping, whereas the other two substitutions (N1436G and L1439A) increased voltage-sensor trapping. These bidirectional effects suggest that residues in IIISS2-S6 make both positive and negative interactions with CssIV. N1436G enhanced voltage-sensor trapping via increased binding affinity to the resting state, whereas L1439A increased voltage-sensor trapping efficacy. Based on these results, a three-dimensional model of the toxin-channel interaction was developed using the Rosetta modeling method. These data provide additional molecular insight into the voltage-sensor trapping mechanism of toxin action and define a three-point interaction site for β-scorpion toxins on NaV channels. Binding of α- and β-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites on NaV channels acts synergistically to modify channel gating and paralyze prey. PMID:22761417

Ionizable side chains at catalytic active sites of enzymes.

PubMed

Jimenez-Morales, David; Liang, Jie; Eisenberg, Bob

2012-05-01

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å(3). The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.

Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane.

PubMed

Oberhauser, A; Alvarez, O; Latorre, R

1988-07-01

Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(-7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites.

Single or Multiple Access Channels to the CYP450s Active Site? An Answer from Free Energy Simulations of the Human Aromatase Enzyme.

PubMed

Magistrato, Alessandra; Sgrignani, Jacopo; Krause, Rolf; Cavalli, Andrea

2017-05-04

Cytochromes P450 (CYP450s), in particular, CYP19A1 and CYP17A1, are key clinical targets of breast and prostate anticancer therapies, critical players in drug metabolism, and their overexpression in tumors is associated with drug resistance. In these enzymes, ligand (substrates, drugs) metabolism occurs in deeply buried active sites accessible only via several grueling channels, whose exact biological role remains unclear. Gaining direct insights on the mechanism by which ligands travel in and out is becoming increasingly important given that channels are involved in the modulation of binding/dissociation kinetics and the specificity of ligands toward a CYP450. This has profound implications for enzymatic efficiency and drug efficacy/toxicity. Here, by applying free energy methods, for a cumulative simulation time of 20 μs, we provide detailed atomistic characterization and free energy profiles of the entry/exit routes preferentially followed by a substrate (androstenedione) and a last-generation inhibitor (letrozole) to/from the catalytic site of CYP19A1 (the human aromatase (HA) enzyme), a key clinical target against breast cancer, studied here as prototypical CYP450. Despite the remarkably different size/shape/hydrophobicity of the ligands, two channels appear accessible to their entrance, while only one exit route appears to be preferential. Our study shows that the preferential paths may be conserved among different CYP450s. Moreover, our results highlight that, at least in the case of HA, ligand channeling is associated with large enzyme structural rearrangements. A wise choice of the computational method and very long simulations are, thus, required to obtain fully converged quantitative free energy profiles, which might be used to design novel biocatalysts or next-generation cytochrome inhibitors with an in silico tuned K m .

Slack, Slick, and Sodium-Activated Potassium Channels

PubMed Central

Kaczmarek, Leonard K.

2013-01-01

The Slack and Slick genes encode potassium channels that are very widely expressed in the central nervous system. These channels are activated by elevations in intracellular sodium, such as those that occur during trains of one or more action potentials, or following activation of nonselective cationic neurotransmitter receptors such as AMPA receptors. This review covers the cellular and molecular properties of Slack and Slick channels and compares them with findings on the properties of sodium-activated potassium currents (termed KNa currents) in native neurons. Human mutations in Slack channels produce extremely severe defects in learning and development, suggesting that KNa channels play a central role in neuronal plasticity and intellectual function. PMID:24319675

CNG channel subunit glycosylation regulates MMP-dependent changes in channel gating

PubMed Central

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

2013-01-01

Cyclic-nucleotide gated (CNG) channels are essential for phototransduction within retinal photoreceptors. We have demonstrated previously that enzymatic activity of matrix metalloproteinase-2 and -9, members of the MMP family of extracellular, Ca+2- and Zn+2-dependent proteases, enhances the ligand sensitivity of both rod (CNGA1 + CNGB1) and cone CNGA3 + CNGB3) CNG channels. Additionally, we have observed a decrease in maximal CNG channel current (IMAX) that begins late during MMP-directed gating changes. Here we demonstrate that CNG channels become non-conductive after prolonged MMP exposure. Concurrent with the loss of conductive channels is the increased relative contribution of channels exhibiting non-modified gating properties, suggesting the presence of a subpopulation of channels that are protected from MMP-induced gating effects. CNGA subunits are known to possess one extracellular core glycosylation site, located at one of two possible positions within the turret loop near the pore-forming region. Our results indicate that CNGA glycosylation can impede MMP-dependent modification of CNG channels. Furthermore, the relative position of the glycosylation site within the pore turret influences the extent of MMP-dependent proteolysis. Glycosylation at the site found in CNGA3 subunits was found to be protective, while glycosylation at the bovine CNGA1 site was not. Relocating the glycosylation site in CNGA1 to the position found in CNGA3 recapitulated CNGA3-like protection from MMP-dependent processing. Taken together, these data indicate that CNGA glycosylation may protect CNG channels from MMP-dependent proteolysis, consistent with MMP modification of channel function having a requirement for physical access to the extracellular face of the channel. PMID:24164424

The action of blocking agents applied to the inner face of Ca(2+)-activated K+ channels from human erythrocytes.

PubMed

Dunn, P M

1998-09-15

The actions of clotrimazole and cetiedil, two drugs known to inhibit the Gardos channel, have been studied on single intermediate conductance calcium-activated potassium (IKCa) channels in inside out patches from human red blood cells, and compared with those of TEA and Ba2+ applied to the cytoplasmic face of the membrane. TEA produced a fast block which was observed as a reduction in the amplitude of the single channel current. This effect was weakly voltage dependent with the fraction of the membrane potential sensed by TEA at its binding site (delta) of 0.18 and a Kd at 0 mV of 20.5 mM. Ba2+ was a very potent blocker of the channel, breaking the single channel activity up into bursts, inter-spersed with silent periods lasting several seconds. The effect of Ba2+ was very voltage sensitive, delta = 0.44, and a Kd at 0 mV of 0.15 microM. Clotrimazole applied to the inner face of the membrane at a concentration < or = 1 microM produced a slow block resulting in bursts of channel activity separated by quiescent periods lasting many seconds. The effect of clotrimazole was mimicked by a quaternary derivative UCL 1559, in keeping with an action at the cytoplasmic face of the channel. A high concentration of cetiedil (100 microM) produced only a weak block of the channel. The kinetics of this action were very slow, with burst and inter-burst intervals lasting several minutes. While inhibition of the Gardos channel by cetiedil is unlikely to involve an intracellular site of action, if clotrimazole is able to penetrate the membrane, part of its effect may result from binding to an intracellular site on the channel.

Interaction of a dinoflagellate neurotoxin with voltage-activated ion channels in a marine diatom.

PubMed

Kitchen, Sheila A; Bourdelais, Andrea J; Taylor, Alison R

2018-01-01

The potent neurotoxins produced by the harmful algal bloom species Karenia brevis are activators of sodium voltage-gated channels (VGC) in animals, resulting in altered channel kinetics and membrane hyperexcitability. Recent biophysical and genomic evidence supports widespread presence of homologous sodium (Na + ) and calcium (Ca 2+ ) permeable VGCs in unicellular algae, including marine phytoplankton. We therefore hypothesized that VGCs of these phytoplankton may be an allelopathic target for waterborne neurotoxins produced by K. brevis blooms that could lead to ion channel dysfunction and disruption of signaling in a similar manner to animal Na + VGCs. We examined the interaction of brevetoxin-3 (PbTx-3), a K. brevis neurotoxin, with the Na + /Ca 2+ VGC of the non-toxic diatom Odontella sinensi s using electrophysiology. Single electrode current- and voltage- clamp recordings from O. sinensis in the presence of PbTx-3 were used to examine the toxin's effect on voltage gated Na + /Ca 2+ currents. In silico analysis was used to identify the putative PbTx binding site in the diatoms. We identified Na + /Ca 2+ VCG homologs from the transcriptomes and genomes of 12 diatoms, including three transcripts from O. sinensis and aligned them with site-5 of Na + VGCs, previously identified as the PbTx binding site in animals. Up to 1 µM PbTx had no effect on diatom resting membrane potential or membrane excitability. The kinetics of fast inward Na + /Ca 2+ currents that underlie diatom action potentials were also unaffected. However, the peak inward current was inhibited by 33%, delayed outward current was inhibited by 25%, and reversal potential of the currents shifted positive, indicating a change in permeability of the underlying channels. Sequence analysis showed a lack of conservation of the PbTx binding site in diatom VGC homologs, many of which share molecular features more similar to single-domain bacterial Na + /Ca 2+ VGCs than the 4-domain eukaryote channels

An atypical CNG channel activated by a single cGMP molecule controls sperm chemotaxis.

PubMed

Bönigk, Wolfgang; Loogen, Astrid; Seifert, Reinhard; Kashikar, Nachiket; Klemm, Clementine; Krause, Eberhard; Hagen, Volker; Kremmer, Elisabeth; Strünker, Timo; Kaupp, U Benjamin

2009-10-27

Sperm of the sea urchin Arbacia punctulata can respond to a single molecule of chemoattractant released by an egg. The mechanism underlying this extreme sensitivity is unknown. Crucial signaling events in the response of A. punctulata sperm to chemoattractant include the rapid synthesis of the intracellular messenger guanosine 3',5'-monophosphate (cGMP) and the ensuing membrane hyperpolarization that results from the opening of potassium-selective cyclic nucleotide-gated (CNGK) channels. Here, we use calibrated photolysis of caged cGMP to show that approximately 45 cGMP molecules are generated during the response to a single molecule of chemoattractant. The CNGK channel can respond to such small cGMP changes because it is exquisitely sensitive to cGMP and activated in a noncooperative fashion. Like voltage-activated Ca(v) and Na(v) channels, the CNGK polypeptide consists of four homologous repeat sequences. Disabling each of the four cyclic nucleotide-binding sites through mutagenesis revealed that binding of a single cGMP molecule to repeat 3 is necessary and sufficient to activate the CNGK channel. Thus, CNGK has developed a mechanism of activation that is different from the activation of other CNG channels, which requires the cooperative binding of several ligands and operates in the micromolar rather than the nanomolar range.

SLITHER: a web server for generating contiguous conformations of substrate molecules entering into deep active sites of proteins or migrating through channels in membrane transporters.

PubMed

Lee, Po-Hsien; Kuo, Kuei-Ling; Chu, Pei-Ying; Liu, Eric M; Lin, Jung-Hsin

2009-07-01

Many proteins use a long channel to guide the substrate or ligand molecules into the well-defined active sites for catalytic reactions or for switching molecular states. In addition, substrates of membrane transporters can migrate to another side of cellular compartment by means of certain selective mechanisms. SLITHER (http://bioinfo.mc.ntu.edu.tw/slither/or http://slither.rcas.sinica.edu.tw/) is a web server that can generate contiguous conformations of a molecule along a curved tunnel inside a protein, and the binding free energy profile along the predicted channel pathway. SLITHER adopts an iterative docking scheme, which combines with a puddle-skimming procedure, i.e. repeatedly elevating the potential energies of the identified global minima, thereby determines the contiguous binding modes of substrates inside the protein. In contrast to some programs that are widely used to determine the geometric dimensions in the ion channels, SLITHER can be applied to predict whether a substrate molecule can crawl through an inner channel or a half-channel of proteins across surmountable energy barriers. Besides, SLITHER also provides the list of the pore-facing residues, which can be directly compared with many genetic diseases. Finally, the adjacent binding poses determined by SLITHER can also be used for fragment-based drug design.

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

Channel sialic acids limit hERG channel activity during the ventricular action potential.

PubMed

Norring, Sarah A; Ednie, Andrew R; Schwetz, Tara A; Du, Dongping; Yang, Hui; Bennett, Eric S

2013-02-01

Activity of human ether-a-go-go-related gene (hERG) 1 voltage-gated K(+) channels is responsible for portions of phase 2 and phase 3 repolarization of the human ventricular action potential. Here, we questioned whether and how physiologically and pathophysiologically relevant changes in surface N-glycosylation modified hERG channel function. Voltage-dependent hERG channel gating and activity were evaluated as expressed in a set of Chinese hamster ovary (CHO) cell lines under conditions of full glycosylation, no sialylation, no complex N-glycans, and following enzymatic deglycosylation of surface N-glycans. For each condition of reduced glycosylation, hERG channel steady-state activation and inactivation relationships were shifted linearly by significant depolarizing ∼9 and ∼18 mV, respectively. The hERG window current increased significantly by 50-150%, and the peak shifted by a depolarizing ∼10 mV. There was no significant change in maximum hERG current density. Deglycosylated channels were significantly more active (20-80%) than glycosylated controls during phases 2 and 3 of action potential clamp protocols. Simulations of hERG current and ventricular action potentials corroborated experimental data and predicted reduced sialylation leads to a 50-70-ms decrease in action potential duration. The data describe a novel mechanism by which hERG channel gating is modulated through physiologically and pathophysiologically relevant changes in N-glycosylation; reduced channel sialylation increases hERG channel activity during the action potential, thereby increasing the rate of action potential repolarization.

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.

Channel Transmission Loss Studies During Ephemeral Flow Events: ER-5-3 Channel and Cambric Ditch, Nevada Test Site, Nye County, Nevada

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

J.J. Miller; S.A. Mizell; R.H. French

2005-10-01

Transmission losses along ephemeral channels are an important, yet poorly understood, aspect of rainfall-runoff prediction. Losses occur as flow infiltrates channel bed, banks, and floodplains. Estimating transmission losses in arid environments is difficult because of the variability of surficial geomorphic characteristics and infiltration capacities of soils and near-surface low-permeability geologic layers (e.g., calcrete). Transmission losses in ephemeral channels are nonlinear functions of discharge and time (Lane, 1972), and vary spatially along the channel reach and with soil antecedent moisture conditions (Sharma and Murthy, 1994). Rainfall-runoff models used to estimate peak discharge and runoff volume for flood hazard assessment are notmore » designed specifically for ephemeral channels, where transmission loss can be significant because of the available storage volume in channel soils. Accuracy of the flow routing and rainfall-runoff models is dependent on the transmission loss estimate. Transmission loss rate is the most uncertain parameter in flow routing through ephemeral channels. This research, sponsored by the U.S. Department of Energy, National Nuclear Security Administration (DOE/NNSA) and conducted at the Nevada Test Site (NTS), is designed to improve understanding of the impact of transmission loss on ephemeral flood modeling and compare various methodologies for predicting runoff from rainfall events. Various applications of this research to DOE projects include more site-specific accuracy in runoff prediction; possible reduction in size of flood mitigation structures at the NTS; and a better understanding of expected infiltration from runoff losses into landfill covers. Two channel transmission loss field experiments were performed on the NTS between 2001 and 2003: the first was conducted in the ER-5-3 channel (Miller et al., 2003), between March and June 2001, and the second was conducted in the Cambric Ditch (Mizell et al., 2005), between

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.

The Barium Site in a Potassium Channel by X-Ray Crystallography

PubMed Central

Jiang, Youxing; MacKinnon, Roderick

2000-01-01

X-ray diffraction data were collected from frozen crystals (100°K) of the KcsA K+ channel equilibrated with solutions containing barium chloride. Difference electron density maps (Fbarium − Fnative, 5.0 Å resolution) show that Ba2+ resides at a single location within the selectivity filter. The Ba2+ blocking site corresponds to the internal aspect (adjacent to the central cavity) of the “inner ion” position where an alkali metal cation is found in the absence of the blocking Ba2+ ion. The location of Ba2+ with respect to Rb+ ions in the pore is in good agreement with the findings on the functional interaction of Ba2+ with K+ (and Rb+) in Ca2+-activated K+ channels (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549–567). Taken together, these structural and functional data imply that at physiological ion concentrations a third ion may interact with two ions in the selectivity filter, perhaps by entering from one side and displacing an ion on the opposite side. PMID:10694255

Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

PubMed

Maurel, C; Kado, R T; Guern, J; Chrispeels, M J

1995-07-03

The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS)

The probability of quantal secretion near a single calcium channel of an active zone.

PubMed Central

Bennett, M R; Farnell, L; Gibson, W G

2000-01-01

A Monte Carlo analysis has been made of calcium dynamics and quantal secretion at microdomains in which the calcium reaches very high concentrations over distances of <50 nm from a channel and for which calcium dynamics are dominated by diffusion. The kinetics of calcium ions in microdomains due to either the spontaneous or evoked opening of a calcium channel, both of which are stochastic events, are described in the presence of endogenous fixed and mobile buffers. Fluctuations in the number of calcium ions within 50 nm of a channel are considerable, with the standard deviation about half the mean. Within 10 nm of a channel these numbers of ions can give rise to calcium concentrations of the order of 100 microM. The temporal changes in free calcium and calcium bound to different affinity indicators in the volume of an entire varicosity or bouton following the opening of a single channel are also determined. A Monte Carlo analysis is also presented of how the dynamics of calcium ions at active zones, after the arrival of an action potential and the stochastic opening of a calcium channel, determine the probability of exocytosis from docked vesicles near the channel. The synaptic vesicles in active zones are found docked in a complex with their calcium-sensor associated proteins and a voltage-sensitive calcium channel, forming a secretory unit. The probability of quantal secretion from an isolated secretory unit has been determined for different distances of an open calcium channel from the calcium sensor within an individual unit: a threefold decrease in the probability of secretion of a quantum occurs with a doubling of the distance from 25 to 50 nm. The Monte Carlo analysis also shows that the probability of secretion of a quantum is most sensitive to the size of the single-channel current compared with its sensitivity to either the binding rates of the sites on the calcium-sensor protein or to the number of these sites that must bind a calcium ion to trigger

Molecular mechanism of ATP binding and ion channel activation in P2X receptors

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

Hattori, Motoyuki; Gouaux, Eric

P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure ofmore » the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.« less

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

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.

The Natural Plant Product Rottlerin Activates Kv7.1/KCNE1 Channels.

PubMed

Matschke, Veronika; Piccini, Ilaria; Schubert, Janina; Wrobel, Eva; Lang, Florian; Matschke, Johann; Amedonu, Elsie; Meuth, Sven G; Strünker, Timo; Strutz-Seebohm, Nathalie; Greber, Boris; Scherkenbeck, Jürgen; Seebohm, Guiscard

2016-01-01

Acquired as well as inherited channelopathies are disorders that are caused by altered ion channel function. A family of channels whose malfunction is associated with different channelopathies is the Kv7 K+ channel family; and restoration of normal Kv7 channel function by small molecule modulators is a promising approach for treatment of these often fatal diseases. Here, we show the modulation of Kv7 channels by the natural compound Rottlerin heterologously expressed in Xenopus laevis oocytes and on iPSC cardiomyocytes overexpressing Kv7.1 channels. We show that currents carried by Kv7.1 (EC50 = 1.48 μM), Kv7.1/KCNE1 (EC50 = 4.9 μM), and Kv7.4 (EC50 = 0.148 μM) are strongly enhanced by the compound, whereas Kv7.2, Kv7.2/Kv7.3, and Kv7.5 are not sensitive to Rottlerin. Studies on Kv7.1/KCNE1 mutants and in silico modelling indicate that Rottlerin binds to the R-L3-activator site. Rottlerin mediated activation of Kv7.1/KCNE1 channels might be a promising approach in long QT syndrome. As a proof of concept, we show that Rottlerin shortens cardiac repolarisation in iPSC-derived cardiomyocytes expressing Kv7.1. Rottlerin or an optimized derivative holds a potential as QT interval correcting drug. © 2016 The Author(s) Published by S. Karger AG, Basel.

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

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

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

Interactions of divalent cations with calcium binding sites of BK channels reveal independent motions within the gating ring.

PubMed

Miranda, Pablo; Giraldez, Teresa; Holmgren, Miguel

2016-12-06

Large-conductance voltage- and calcium-activated K + (BK) channels are key physiological players in muscle, nerve, and endocrine function by integrating intracellular Ca 2+ and membrane voltage signals. The open probability of BK channels is regulated by the intracellular concentration of divalent cations sensed by a large structure in the BK channel called the "gating ring," which is formed by four tandems of regulator of conductance for K + (RCK1 and RCK2) domains. In contrast to Ca 2+ that binds to both RCK domains, Mg 2+ , Cd 2+ , or Ba 2+ interact preferentially with either one or the other. Interaction of cations with their binding sites causes molecular rearrangements of the gating ring, but how these motions occur remains elusive. We have assessed the separate contributions of each RCK domain to the cation-induced gating-ring structural rearrangements, using patch-clamp fluorometry. Here we show that Mg 2+ and Ba 2+ selectively induce structural movement of the RCK2 domain, whereas Cd 2+ causes motions of RCK1, in all cases substantially smaller than those elicited by Ca 2+ By combining divalent species interacting with unique sites, we demonstrate that RCK1 and RCK2 domains move independently when their specific binding sites are occupied. Moreover, binding of chemically distinct cations to both RCK domains is additive, emulating the effect of fully occupied Ca 2+ binding sites.

Fast activation of dihydropyridine-sensitive calcium channels of skeletal muscle. Multiple pathways of channel gating

PubMed Central

1996-01-01

Dihydropyridine (DHP) receptors of the transverse tubule membrane play two roles in excitation-contraction coupling in skeletal muscle: (a) they function as the voltage sensor which undergoes fast transition to control release of calcium from sarcoplasmic reticulum, and (b) they provide the conducting unit of a slowly activating L-type calcium channel. To understand this dual function of the DHP receptor, we studied the effect of depolarizing conditioning pulse on the activation kinetics of the skeletal muscle DHP-sensitive calcium channels reconstituted into lipid bilayer membranes. Activation of the incorporated calcium channel was imposed by depolarizing test pulses from a holding potential of -80 mV. The gating kinetics of the channel was studied with ensemble averages of repeated episodes. Based on a first latency analysis, two distinct classes of channel openings occurred after depolarization: most had delayed latencies, distributed with a mode of 70 ms (slow gating); a small number of openings had short first latencies, < 12 ms (fast gating). A depolarizing conditioning pulse to +20 mV placed 200 ms before the test pulse (-10 mV), led to a significant increase in the activation rate of the ensemble averaged-current; the time constant of activation went from tau m = 110 ms (reference) to tau m = 45 ms after conditioning. This enhanced activation by the conditioning pulse was due to the increase in frequency of fast open events, which was a steep function of the intermediate voltage and the interval between the conditioning pulse and the test pulse. Additional analysis demonstrated that fast gating is the property of the same individual channels that normally gate slowly and that the channels adopt this property after a sojourn in the open state. The rapid secondary activation seen after depolarizing prepulses is not compatible with a linear activation model for the calcium channel, but is highly consistent with a cyclical model. A six- state cyclical model is

Identification of the fatty acid activation site on human ClC-2.

PubMed

Cuppoletti, John; Tewari, Kirti P; Chakrabarti, Jayati; Malinowska, Danuta H

2017-06-01

Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl - channel activators. Molecular and cellular mechanisms underlying this activation were examined. Role of a four-amino acid PKA activation site, RGET 691 , of hClC-2 was investigated using wild-type (WT) and mutant (AGET, RGEA, and AGAA) hClC-2 expressed in 293EBNA cells as well as involvement of PKA, intracellular cAMP concentration ([cAMP] i ), EP 2 , or EP 4 receptor agonist activity. All fatty acids [lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid, and elaidic acid] caused significant rightward shifts in concentration-dependent Cl - current activation (increasing EC 50 s) with mutant compared with WT hClC-2 channels, without changing time and voltage dependence, current-voltage rectification, or methadone inhibition of the channel. As with lubiprostone, cobiprostone activation of hClC-2 occurred with PKA inhibitor (myristoylated protein kinase inhibitor) present or when using double PKA activation site (RRAA 655 /RGEA 691 ) mutant. Cobiprostone did not activate human CFTR. Fatty acids did not increase [cAMP] i in hClC-2/293EBNA or T84 cells. Using T84 CFTR knockdown cells, cobiprostone increased hClC-2 Cl - currents without increasing [cAMP] i, while PGE 2 and forskolin-IBMX increased both. Fatty acids were not agonists of EP 2 or EP 4 receptors. L-161,982, a supposed EP 4 -selective inhibitor, had no effect on lubiprostone-activated hClC-2 Cl - currents but significantly decreased T84 cell barrier function measured by transepithelial resistance and fluorescent dextran transepithelial movement. The present findings show that RGET 691 of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP] i , and EP 2 or EP 4 receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2. Copyright © 2017 the American Physiological Society.

Site-specific protein immobilization in a microfluidic chip channel via an IEF-gelation process.

PubMed

Shi, Mianhong; Peng, Youyuan; Yu, Shaoning; Liu, Baohong; Kong, Jilie

2007-05-01

A novel strategy for site-specific protein immobilization via combining chip IEF with low-temperature sol-gel technology, called IEF-GEL here, in the channel of a modified poly(methyl methacrylate) (PMMA) microfluidic chip is proposed in this work. The IEF-GEL process involves firstly IEF for homogeneously dissolved protein in PBS containing alumina sol and carrier ampholyte with prearranged pH gradient, and then gelation locally for protein encapsulation. The process and feasibility of proposed IEF-GEL were investigated by EOF measurements, fluorescence microscopic photography, Raman spectrum and further demonstrated by glucose oxidase (GOx) reactors integrated with end-column electrochemical detection. Site-controllable immobilization of protein was realized in a 30 mm long microfluidic chip channel by the strategy to create a approximately 1.7 mm concentrated FITC-BSA band, which leads to great improvement of the elute peak shape, accomplished with remarkably increased sensitivity, approximately 20 times higher than that without IEF-GEL treatment to GOx reactors. The kinetic response of GOx after IEF-GEL treatment was also investigated. The proposed system holds the advantages of IEF and low-temperature sol-gel technologies, i.e. concentrating the protein to be focused and retaining the biological activity for the gel-embedded protein, thus realizes site-specific immobilization of low-concentration protein at nL volume level.

Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator

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

Fitzpatrick, L.A.; Yasumoto, T.; Aurbach, G.D.

1989-01-01

Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivatesmore » a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release.« less

Strontium and barium in aqueous solution and a potassium channel binding site

NASA Astrophysics Data System (ADS)

Chaudhari, Mangesh I.; Rempe, Susan B.

2018-06-01

Ion hydration structure and free energy establish criteria for understanding selective ion binding in potassium (K+) ion channels and may be significant to understanding blocking mechanisms as well. Recently, we investigated the hydration properties of Ba2+, the most potent blocker of K+ channels among the simple metal ions. Here, we use a similar method of combining ab initio molecular dynamics simulations, statistical mechanical theory, and electronic structure calculations to probe the fundamental hydration properties of Sr2+, which does not block bacterial K+ channels. The radial distribution of water around Sr2+ suggests a stable 8-fold geometry in the local hydration environment, similar to Ba2+. While the predicted hydration free energy of -331.8 kcal/mol is comparable with the experimental result of -334 kcal/mol, the value is significantly more favorable than the -305 kcal/mol hydration free energy of Ba2+. When placed in the innermost K+ channel blocking site, the solvation free energies and lowest energy structures of both Sr2+ and Ba2+ are nearly unchanged compared with their respective hydration properties. This result suggests that the block is not attributable to ion trapping due to +2 charge, and differences in blocking behavior arise due to free energies associated with the exchange of water ligands for channel ligands instead of free energies of transfer from water to the binding site.

A human intermediate conductance calcium-activated potassium channel.

PubMed

Ishii, T M; Silvia, C; Hirschberg, B; Bond, C T; Adelman, J P; Maylie, J

1997-10-14

An intermediate conductance calcium-activated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is approximately 50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K0.5 = 0.3 microM). Although the K0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3. 5). Single-channel current amplitudes reflected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (Ki = 2.5 nM) and clotrimazole (Ki = 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 microM). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

Phosphorylation sites in the Hook domain of CaVβ subunits differentially modulate CaV1.2 channel function.

PubMed

Brunet, Sylvain; Emrick, Michelle A; Sadilek, Martin; Scheuer, Todd; Catterall, William A

2015-10-01

Regulation of L-type calcium current is critical for the development, function, and regulation of many cell types. Ca(V)1.2 channels that conduct L-type calcium currents are regulated by many protein kinases, but the sites of action of these kinases remain unknown in most cases. We combined mass spectrometry (LC-MS/MS) and whole-cell patch clamp techniques in order to identify sites of phosphorylation of Ca(V)β subunits in vivo and test the impact of mutations of those sites on Ca(V)1.2 channel function in vitro. Using the Ca(V)1.1 channel purified from rabbit skeletal muscle as a substrate for phosphoproteomic analysis, we found that Ser(193) and Thr(205) in the HOOK domain of Ca(V)β1a subunits were both phosphorylated in vivo. Ser(193) is located in a potential consensus sequence for casein kinase II, but it was not phosphorylated in vitro by that kinase. In contrast, Thr(205) is located in a consensus sequence for cAMP-dependent phosphorylation, and it was robustly phosphorylated in vitro by PKA. These two sites are conserved in multiple Ca(V)β subunit isoforms, including the principal Ca(V)β subunit of cardiac Ca(V)1.2 channels, Ca(V)β2b. In order to assess potential modulatory effects of phosphorylation at these sites separately from the effects of phosphorylation of the α11.2 subunit, we inserted phosphomimetic or phosphoinhibitory mutations in Ca(V)β2b and analyzed their effects on Ca(V)1.2 channel function in transfected nonmuscle cells. The phosphomimetic mutation Ca(V)β2b(S152E) decreased peak channel currents and shifted the voltage dependence of both activation and inactivation to more positive membrane potentials. The phosphoinhibitory mutation Ca(V)β2b(S152A) had opposite effects. There were no differences in peak Ca(V)1.2 currents or voltage dependence between the phosphomimetic mutation Ca(V)β2b(T164D) and the phosphoinhibitory mutation Ca(V)β2b(T164A). However, calcium-dependent inactivation was significantly increased for the

Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv7.1 channel

PubMed Central

Tobelaim, William Sam; Dvir, Meidan; Lebel, Guy; Cui, Meng; Buki, Tal; Peretz, Asher; Marom, Milit; Haitin, Yoni; Logothetis, Diomedes E.; Hirsch, Joel Alan; Attali, Bernard

2017-01-01

Voltage-gated potassium 7.1 (Kv7.1) channel and KCNE1 protein coassembly forms the slow potassium current IKS that repolarizes the cardiac action potential. The physiological importance of the IKS channel is underscored by the existence of mutations in human Kv7.1 and KCNE1 genes, which cause cardiac arrhythmias, such as the long-QT syndrome (LQT) and atrial fibrillation. The proximal Kv7.1 C terminus (CT) binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2), but the role of CaM in channel function is still unclear, and its possible interaction with PIP2 is unknown. Our recent crystallographic study showed that CaM embraces helices A and B with the apo C lobe and calcified N lobe, respectively. Here, we reveal the competition of PIP2 and the calcified CaM N lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor an LQT mutation. Protein pulldown, molecular docking, molecular dynamics simulations, and patch-clamp recordings indicate that residues K526 and K527 in Kv7.1 helix B form a critical site where CaM competes with PIP2 to stabilize the channel open state. Data indicate that both PIP2 and Ca2+-CaM perform the same function on IKS channel gating by producing a left shift in the voltage dependence of activation. The LQT mutant K526E revealed a severely impaired channel function with a right shift in the voltage dependence of activation, a reduced current density, and insensitivity to gating modulation by Ca2+-CaM. The results suggest that, after receptor-mediated PIP2 depletion and increased cytosolic Ca2+, calcified CaM N lobe interacts with helix B in place of PIP2 to limit excessive IKS current inhibition. PMID:28096388

Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv7.1 channel.

PubMed

Tobelaim, William Sam; Dvir, Meidan; Lebel, Guy; Cui, Meng; Buki, Tal; Peretz, Asher; Marom, Milit; Haitin, Yoni; Logothetis, Diomedes E; Hirsch, Joel Alan; Attali, Bernard

2017-01-31

Voltage-gated potassium 7.1 (Kv7.1) channel and KCNE1 protein coassembly forms the slow potassium current I KS that repolarizes the cardiac action potential. The physiological importance of the I KS channel is underscored by the existence of mutations in human Kv7.1 and KCNE1 genes, which cause cardiac arrhythmias, such as the long-QT syndrome (LQT) and atrial fibrillation. The proximal Kv7.1 C terminus (CT) binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP 2 ), but the role of CaM in channel function is still unclear, and its possible interaction with PIP 2 is unknown. Our recent crystallographic study showed that CaM embraces helices A and B with the apo C lobe and calcified N lobe, respectively. Here, we reveal the competition of PIP 2 and the calcified CaM N lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor an LQT mutation. Protein pulldown, molecular docking, molecular dynamics simulations, and patch-clamp recordings indicate that residues K526 and K527 in Kv7.1 helix B form a critical site where CaM competes with PIP 2 to stabilize the channel open state. Data indicate that both PIP 2 and Ca 2+ -CaM perform the same function on I KS channel gating by producing a left shift in the voltage dependence of activation. The LQT mutant K526E revealed a severely impaired channel function with a right shift in the voltage dependence of activation, a reduced current density, and insensitivity to gating modulation by Ca 2+ -CaM. The results suggest that, after receptor-mediated PIP 2 depletion and increased cytosolic Ca 2+ , calcified CaM N lobe interacts with helix B in place of PIP 2 to limit excessive I KS current inhibition.

A membrane-embedded pathway delivers general anesthetics to two interacting binding sites in the Gloeobacter violaceus ion channel.

PubMed

Arcario, Mark J; Mayne, Christopher G; Tajkhorshid, Emad

2017-06-09

General anesthetics exert their effects on the central nervous system by acting on ion channels, most notably pentameric ligand-gated ion channels. Although numerous studies have focused on pentameric ligand-gated ion channels, the details of anesthetic binding and channel modulation are still debated. A better understanding of the anesthetic mechanism of action is necessary for the development of safer and more efficacious drugs. Herein, we present a computational study identifying two anesthetic binding sites in the transmembrane domain of the Gloeobacter violaceus ligand-gated ion channel (GLIC) channel, characterize the putative binding pathway, and observe structural changes associated with channel function. Molecular simulations of desflurane reveal a binding pathway to GLIC via a membrane-embedded tunnel using an intrasubunit protein lumen as the conduit, an observation that explains the Meyer-Overton hypothesis, or why the lipophilicity of an anesthetic and its potency are generally proportional. Moreover, employing high concentrations of ligand led to the identification of a second transmembrane site (TM2) that inhibits dissociation of anesthetic from the TM1 site and is consistent with the high concentrations of anesthetics required to achieve clinical effects. Finally, asymmetric binding patterns of anesthetic to the channel were found to promote an iris-like conformational change that constricts and dehydrates the ion pore, creating a 13.5 kcal/mol barrier to ion translocation. Together with previous studies, the simulations presented herein demonstrate a novel anesthetic binding site in GLIC that is accessed through a membrane-embedded tunnel and interacts with a previously known site, resulting in conformational changes that produce a non-conductive state of the channel. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A human intermediate conductance calcium-activated potassium channel

PubMed Central

Ishii, Takahiro M.; Silvia, Christopher; Hirschberg, Birgit; Bond, Chris T.; Adelman, John P.; Maylie, James

1997-01-01

An intermediate conductance calcium-activated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is ≈50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K0.5 = 0.3 μM). Although the K0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3.5). Single-channel current amplitudes reflected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (Ki = 2.5 nM) and clotrimazole (Ki = 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 μM). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel. PMID:9326665

Strong activation of bile acid-sensitive ion channel (BASIC) by ursodeoxycholic acid

PubMed Central

Wiemuth, Dominik; Sahin, Hacer; Lefèvre, Cathérine M.T.; Wasmuth, Hermann E.; Gründer, Stefan

2013-01-01

Bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC gene family of unknown function. Rat BASIC (rBASIC) is inactive at rest. We have recently shown that cholangiocytes, the epithelial cells lining the bile ducts, are the main site of BASIC expression in the liver and identified bile acids, in particular hyo- and chenodeoxycholic acid, as agonists of rBASIC. Moreover, it seems that extracellular divalent cations stabilize the resting state of rBASIC, because removal of extracellular divalent cations opens the channel. In this addendum, we demonstrate that removal of extracellular divalent cations potentiates the activation of rBASIC by bile acids, suggesting an allosteric mechanism. Furthermore, we show that rBASIC is strongly activated by the anticholestatic bile acid ursodeoxycholic acid (UDCA), suggesting that BASIC might mediate part of the therapeutic effects of UDCA. PMID:23064163

Molecular Interactions between Tarantula Toxins and Low-Voltage-Activated Calcium Channels

PubMed Central

Salari, Autoosa; Vega, Benjamin S.; Milescu, Lorin S.; Milescu, Mirela

2016-01-01

Few gating-modifier toxins have been reported to target low-voltage-activated (LVA) calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Studies of voltage-gated potassium (Kv) channels have identified the S3b–S4 “paddle motif,” which moves at the protein-lipid interface to drive channel opening, as the target for these amphipathic neurotoxins. Voltage-gated calcium (Cav) channels contain four homologous voltage sensor domains, suggesting multiple toxin binding sites. We show here that the S3–S4 segments within Cav3.1 can be transplanted into Kv2.1 to examine their individual contributions to voltage sensing and pharmacology. With these results, we now have a more complete picture of the conserved nature of the paddle motif in all three major voltage-gated ion channel types (Kv, Nav, and Cav). When screened with tarantula toxins, the four paddle sequences display distinct toxin binding properties, demonstrating that gating-modifier toxins can bind to Cav channels in a domain specific fashion. Domain III was the most commonly and strongly targeted, and mutagenesis revealed an acidic residue that is important for toxin binding. We also measured the lipid partitioning strength of all toxins tested and observed a positive correlation with their inhibition of Cav3.1, suggesting a key role for membrane partitioning. PMID:27045173

Four Ca2+ Ions Activate TRPM2 Channels by Binding in Deep Crevices near the Pore but Intracellularly of the Gate

PubMed Central

Törőcsik, Beáta

2009-01-01

TRPM2 is a tetrameric Ca2+-permeable channel involved in immunocyte respiratory burst and in postischaemic neuronal death. In whole cells, TRPM2 activity requires intracellular ADP ribose (ADPR) and intra- or extracellular Ca2+, but the mechanism and the binding sites for Ca2+ activation remain unknown. Here we study TRPM2 gating in inside-out patches while directly controlling intracellular ligand concentrations. Concentration jump experiments at various voltages and Ca2+ dependence of steady-state single-channel gating kinetics provide unprecedented insight into the molecular mechanism of Ca2+ activation. In patches excised from Xenopus laevis oocytes expressing human TRPM2, coapplication of intracellular ADPR and Ca2+ activated ∼50-pS nonselective cation channels; K1/2 for ADPR was ∼1 µM at saturating Ca2+. Intracellular Ca2+ dependence of TRPM2 steady-state opening and closing rates (at saturating [ADPR] and low extracellular Ca2+) reveals that Ca2+ activation is a consequence of tighter binding of Ca2+ in the open rather than in the closed channel conformation. Four Ca2+ ions activate TRPM2 with a Monod-Wymann-Changeux mechanism: each binding event increases the open-closed equilibrium constant ∼33-fold, producing altogether 106-fold activation. Experiments in the presence of 1 mM of free Ca2+ on the extracellular side clearly show that closed channels do not sense extracellular Ca2+, but once channels have opened Ca2+ entering passively through the pore slows channel closure by keeping the “activating sites” saturated, despite rapid continuous Ca2+-free wash of the intracellular channel surface. This effect of extracellular Ca2+ on gating is gradually lost at progressively depolarized membrane potentials, where the driving force for Ca2+ influx is diminished. Thus, the activating sites lie intracellularly from the gate, but in a shielded crevice near the pore entrance. Our results suggest that in intact cells that contain micromolar ADPR a single

CASEIN KINASE-MEDIATED PHOSPHORYLATION OF SERINE 839 IS NECESSARY FOR BASOLATERAL LOCALIZATION OF THE Ca2+-ACTIVATED NON-SELECTIVE CATION CHANNEL TRPM4

PubMed Central

Cerda, Oscar; Cáceres, Mónica; Park, Kang-Sik; Leiva-Salcedo, Elías; Romero, Aníbal; Varela, Diego

2014-01-01

TRPM4 is a Ca2+-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction and allergic reactions. TRPM4 Ca2+ sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-biphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remain unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry), to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4. PMID:25231975

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

Large conductance Ca(2+)-activated K(+) channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects.

PubMed

Kirby, R W; Martelli, A; Calderone, V; McKay, N G; Lawson, K

2013-07-15

Large conductance calcium activated potassium channels (BKCa) are fundamental in the control of cellular excitability. Thus, compounds that activate BKCa channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BKCa channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BKCa channel α-subunit alone or α+β1-subunit complex. Channel activity was determined using a non-radioactive Rb(+) efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb(+) efflux both in cells expressing α-subunit alone or α+β1-subunits. Co-expression of the β1-subunit modified the Rb(+) efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC40 values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BKCa channel α+β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC40 values when tested on the BKCa α+β1-subunit expressing cells compared to BKCa α-subunit expressing cells. Further, the Emax values for BKPr4, BKVV and BKH1 were lower in the BKCa channel α+β1-subunit expressing cells. In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BKCa channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles. Copyright © 2013 Elsevier Ltd. All rights reserved.

Marte Valles site

NASA Technical Reports Server (NTRS)

Rice, Jim W.

1994-01-01

This site is located at 16 deg N, 177 deg W on the flood plains of Marte Valles, which is perhaps the youngest channel system on Mars. The young age of this channel warrants investigation because of climatic implications for fluvial activities in recent geologic time. The paucity of craters makes this an excellent site in terms of safety requirements. Some of the objectives stated previously for the Maja Valles region would also apply to this site (grab bag of rock types, etc.).

The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine*

PubMed Central

Syeda, Ruhma; Santos, Jose S.; Montal, Mauricio

2016-01-01

KCNQ (voltage-gated K+ channel family 7 (Kv7)) channels control cellular excitability and underlie the K+ current sensitive to muscarinic receptor signaling (the M current) in sympathetic neurons. Here we show that the novel anti-epileptic drug retigabine (RTG) modulates channel function of pore-only modules (PMs) of the human Kv7.2 and Kv7.3 homomeric channels and of Kv7.2/3 heteromeric channels by prolonging the residence time in the open state. In addition, the Kv7 channel PMs are shown to recapitulate the single-channel permeation and pharmacological specificity characteristics of the corresponding full-length proteins in their native cellular context. A mutation (W265L) in the reconstituted Kv7.3 PM renders the channel insensitive to RTG and favors the conductive conformation of the PM, in agreement to what is observed when the Kv7.3 mutant is heterologously expressed. On the basis of the new findings and homology models of the closed and open conformations of the Kv7.3 PM, we propose a structural mechanism for the gating of the Kv7.3 PM and for the site of action of RTG as a Kv7.2/Kv7.3 K+ current activator. The results validate the modular design of human Kv channels and highlight the PM as a high-fidelity target for drug screening of Kv channels. PMID:26627826

Common Anesthetic-binding Site for Inhibition of Pentameric Ligand-gated Ion Channels.

PubMed

Kinde, Monica N; Bu, Weiming; Chen, Qiang; Xu, Yan; Eckenhoff, Roderic G; Tang, Pei

2016-03-01

Identifying functionally relevant anesthetic-binding sites in pentameric ligand-gated ion channels (pLGICs) is an important step toward understanding the molecular mechanisms underlying anesthetic action. The anesthetic propofol is known to inhibit cation-conducting pLGICs, including a prokaryotic pLGIC from Erwinia chrysanthemi (ELIC), but the sites responsible for functional inhibition remain undetermined. We photolabeled ELIC with a light-activated derivative of propofol (AziPm) and performed fluorine-19 nuclear magnetic resonance experiments to support propofol binding to a transmembrane domain (TMD) intrasubunit pocket. To differentiate sites responsible for propofol inhibition from those that are functionally irrelevant, we made an ELIC-γ-aminobutyric acid receptor (GABAAR) chimera that replaced the ELIC-TMD with the α1β3GABAAR-TMD and compared functional responses of ELIC-GABAAR and ELIC with propofol modulations. Photolabeling showed multiple AziPm-binding sites in the extracellular domain (ECD) but only one site in the TMD with labeled residues M265 and F308 in the resting state of ELIC. Notably, this TMD site is an intrasubunit pocket that overlaps with binding sites for anesthetics, including propofol, found previously in other pLGICs. Fluorine-19 nuclear magnetic resonance experiments supported propofol binding to this TMD intrasubunit pocket only in the absence of agonist. Functional measurements of ELIC-GABAAR showed propofol potentiation of the agonist-elicited current instead of inhibition observed on ELIC. The distinctly different responses of ELIC and ELIC-GABAAR to propofol support the functional relevance of propofol binding to the TMD. Combining the newly identified TMD intrasubunit pocket in ELIC with equivalent TMD anesthetic sites found previously in other cationic pLGICs, we propose this TMD pocket as a common site for anesthetic inhibition of pLGICs.

Copper and protons directly activate the zinc-activated channel.

PubMed

Trattnig, Sarah M; Gasiorek, Agnes; Deeb, Tarek Z; Ortiz, Eydith J Comenencia; Moss, Stephen J; Jensen, Anders A; Davies, Paul A

2016-03-01

The zinc-activated channel (ZAC) is a cationic ion channel belonging to the superfamily of Cys-loop receptors, which consists of pentameric ligand-gated ion channels. ZAC is the least understood member of this family so in the present study we sought to characterize the properties of this channel further. We demonstrate that not only zinc (Zn(2+)) but also copper (Cu(2+)) and protons (H(+)) are agonists of ZAC, displaying potencies and efficacies in the rank orders of H(+)>Cu(2+)>Zn(2+) and H(+)>Zn(2+)>Cu(2+), respectively. The responses elicited by Zn(2+), Cu(2+) and H(+) through ZAC are all characterized by low degrees of desensitization. In contrast, currents evoked by high concentrations of the three agonists comprise distinctly different activation and decay components, with transitions to and from an open state being significantly faster for H(+) than for the two metal ions. The permeabilities of ZAC for Na(+) and K(+) relative to Cs(+) are indistinguishable, whereas replacing all of extracellular Na(+) and K(+) with the divalent cations Ca(2+) or Mg(2+) results in complete elimination of Zn(2+)-activated currents at both negative and positive holding potentials. This indicates that ZAC is non-selectively permeable to monovalent cations, whereas Ca(2+) and Mg(2+) inhibit the channel. In conclusion, this is the first report of a Cys-loop receptor being gated by Zn(2+), Cu(2+) and H(+). ZAC could be an important mediator of some of the wide range of physiological functions regulated by or involving Zn(2+), Cu(2+) and H(+). Copyright © 2016 Elsevier Inc. All rights reserved.

Active integrated filters for RF-photonic channelizers.

PubMed

El Nagdi, Amr; Liu, Ke; LaFave, Tim P; Hunt, Louis R; Ramakrishna, Viswanath; Dabkowski, Mieczyslaw; MacFarlane, Duncan L; Christensen, Marc P

2011-01-01

A theoretical study of RF-photonic channelizers using four architectures formed by active integrated filters with tunable gains is presented. The integrated filters are enabled by two- and four-port nano-photonic couplers (NPCs). Lossless and three individual manufacturing cases with high transmission, high reflection, and symmetric couplers are assumed in the work. NPCs behavior is dependent upon the phenomenon of frustrated total internal reflection. Experimentally, photonic channelizers are fabricated in one single semiconductor chip on multi-quantum well epitaxial InP wafers using conventional microelectronics processing techniques. A state space modeling approach is used to derive the transfer functions and analyze the stability of these filters. The ability of adapting using the gains is demonstrated. Our simulation results indicate that the characteristic bandpass and notch filter responses of each structure are the basis of channelizer architectures, and optical gain may be used to adjust filter parameters to obtain a desired frequency magnitude response, especially in the range of 1-5 GHz for the chip with a coupler separation of ∼9 mm. Preliminarily, the measurement of spectral response shows enhancement of quality factor by using higher optical gains. The present compact active filters on an InP-based integrated photonic circuit hold the potential for a variety of channelizer applications. Compared to a pure RF channelizer, photonic channelizers may perform both channelization and down-conversion in an optical domain.

Parallel Evolution of Sperm Hyper-Activation Ca2+ Channels

PubMed Central

Phadnis, Nitin

2017-01-01

Abstract Sperm hyper-activation is a dramatic change in sperm behavior where mature sperm burst into a final sprint in the race to the egg. The mechanism of sperm hyper-activation in many metazoans, including humans, consists of a jolt of Ca2+ into the sperm flagellum via CatSper ion channels. Surprisingly, all nine CatSper genes have been independently lost in several animal lineages. In Drosophila, sperm hyper-activation is performed through the cooption of the polycystic kidney disease 2 (pkd2) Ca2+ channel. The parallels between CatSpers in primates and pkd2 in Drosophila provide a unique opportunity to examine the molecular evolution of the sperm hyper-activation machinery in two independent, nonhomologous calcium channels separated by > 500 million years of divergence. Here, we use a comprehensive phylogenomic approach to investigate the selective pressures on these sperm hyper-activation channels. First, we find that the entire CatSper complex evolves rapidly under recurrent positive selection in primates. Second, we find that pkd2 has parallel patterns of adaptive evolution in Drosophila. Third, we show that this adaptive evolution of pkd2 is driven by its role in sperm hyper-activation. These patterns of selection suggest that the evolution of the sperm hyper-activation machinery is driven by sexual conflict with antagonistic ligands that modulate channel activity. Together, our results add sperm hyper-activation channels to the class of fast evolving reproductive proteins and provide insights into the mechanisms used by the sexes to manipulate sperm behavior. PMID:28810709

Results of gamma activity traverses made in process tube channels and VSR channels at 105-KW

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

Greene, M.C. Jr.

1955-02-03

Activity traverses were made at Hanford reactor 105-KW in VSR channels 23, 29, 37, 46, 55, 63 and 69 and in process channels 4669, 4569, 4668, 4670 and 4769. These traverses were made during cleaning operations to assist in the location of any contaminated material in these channels and again after completion of the cleaning operations to determine if all of the contaminated material was removed. Upon completion of the cleaning of the VSR channels and process channels activity traverses were made in all of the channels. The results of these traverses show that no detectable amounts of contaminated materialmore » were present in any of these channels. The traverses made in the VSR channels all show a large peak in the lower part of the pile indicating that the metal in the lower part of the pile received as much as five times the integrated exposure received by the metal in the upper half of the pile. 15 figs.« less

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

Electrostatic channeling in P. falciparum DHFR-TS: Brownian dynamics and Smoluchowski modeling.

PubMed

Metzger, Vincent T; Eun, Changsun; Kekenes-Huskey, Peter M; Huber, Gary; McCammon, J Andrew

2014-11-18

We perform Brownian dynamics simulations and Smoluchowski continuum modeling of the bifunctional Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (P. falciparum DHFR-TS) with the objective of understanding the electrostatic channeling of dihydrofolate generated at the TS active site to the DHFR active site. The results of Brownian dynamics simulations and Smoluchowski continuum modeling suggest that compared to Leishmania major DHFR-TS, P. falciparum DHFR-TS has a lower but significant electrostatic-mediated channeling efficiency (?15-25%) at physiological pH (7.0) and ionic strength (150 mM). We also find that removing the electric charges from key basic residues located between the DHFR and TS active sites significantly reduces the channeling efficiency of P. falciparum DHFR-TS. Although several protozoan DHFR-TS enzymes are known to have similar tertiary and quaternary structure, subtle differences in structure, active-site geometry, and charge distribution appear to influence both electrostatic-mediated and proximity-based substrate channeling.

Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity.

PubMed

de San Martin, Javier Zorrilla; Jalil, Abdelali; Trigo, Federico F

2015-12-01

Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABA(A)Rs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABA(A) autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca(2+) photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl(-)](i), autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30-150 GABA(A) channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Na(v)-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABA(A) autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity. © 2015 Zorrilla de San Martin et al.

Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

PubMed Central

Zorrilla de San Martin, Javier; Jalil, Abdelali

2015-01-01

Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABAARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABAA autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca2+ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl−]i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABAA channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Nav-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABAA autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity. PMID:26621773

Effect of chloride channel inhibitors on cytosolic Ca2+ levels and Ca2+-activated K+ (Gardos) channel activity in human red blood cells.

PubMed

Kucherenko, Yuliya V; Wagner-Britz, Lisa; Bernhardt, Ingolf; Lang, Florian

2013-04-01

DIDS, NPPB, tannic acid (TA) and AO1 are widely used inhibitors of Cl(-) channels. Some Cl(-) channel inhibitors (NPPB, DIDS, niflumic acid) were shown to affect phosphatidylserine (PS) scrambling and, thus, the life span of human red blood cells (hRBCs). Since a number of publications suggest Ca(2+) dependence of PS scrambling, we explored whether inhibitors of Cl(-) channels (DIDS, NPPB) or of Ca(2+)-activated Cl(-) channels (DIDS, NPPB, TA, AO1) modified intracellular free Ca(2+) concentration ([Ca(2+)]i) and activity of Ca(2+)-activated K(+) (Gardos) channel in hRBCs. According to Fluo-3 fluorescence in flow cytometry, a short treatment (15 min, +37 °C) with Cl(-) channels inhibitors decreased [Ca(2+)]i in the following order: TA > AO1 > DIDS > NPPB. According to forward scatter, the decrease of [Ca(2+)]i was accompanied by a slight but significant increase in cell volume following DIDS, NPPB and AO1 treatments. TA treatment resulted in cell shrinkage. According to whole-cell patch-clamp experiments, TA activated and NPPB and AO1 inhibited Gardos channels. The Cl(-) channel blockers further modified the alterations of [Ca(2+)]i following ATP depletion (glucose deprivation, iodoacetic acid, 6-inosine), oxidative stress (1 mM t-BHP) and treatment with Ca(2+) ionophore ionomycin (1 μM). The ability of the Cl(-) channel inhibitors to modulate PS scrambling did not correlate with their influence on [Ca(2+)]i as TA and AO1 had a particularly strong decreasing effect on [Ca(2+)]i but at the same time enhanced PS exposure. In conclusion, Cl(-) channel inhibitors affect Gardos channels, influence Ca(2+) homeostasis and induce PS exposure of hRBCs by Ca(2+)-independent mechanisms.

Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block

PubMed Central

Fujiwara, Yuichiro; Arrigoni, Cristina; Domigan, Courtney; Ferrara, Giuseppina; Pantoja, Carlos; Thiel, Gerhard; Moroni, Anna; Minor, Daniel L.

2009-01-01

Background Understanding the interactions between ion channels and blockers remains an important goal that has implications for delineating the basic mechanisms of ion channel function and for the discovery and development of ion channel directed drugs. Methodology/Principal Findings We used genetic selection methods to probe the interaction of two ion channel blockers, barium and amantadine, with the miniature viral potassium channel Kcv. Selection for Kcv mutants that were resistant to either blocker identified a mutant bearing multiple changes that was resistant to both. Implementation of a PCR shuffling and backcrossing procedure uncovered that the blocker resistance could be attributed to a single change, T63S, at a position that is likely to form the binding site for the inner ion in the selectivity filter (site 4). A combination of electrophysiological and biochemical assays revealed a distinct difference in the ability of the mutant channel to interact with the blockers. Studies of the analogous mutation in the mammalian inward rectifier Kir2.1 show that the T→S mutation affects barium block as well as the stability of the conductive state. Comparison of the effects of similar barium resistant mutations in Kcv and Kir2.1 shows that neighboring amino acids in the Kcv selectivity filter affect blocker binding. Conclusions/Significance The data support the idea that permeant ions have an integral role in stabilizing potassium channel structure, suggest that both barium and amantadine act at a similar site, and demonstrate how genetic selections can be used to map blocker binding sites and reveal mechanistic features. PMID:19834614

Parallel Evolution of Sperm Hyper-Activation Ca2+ Channels.

PubMed

Cooper, Jacob C; Phadnis, Nitin

2017-07-01

Sperm hyper-activation is a dramatic change in sperm behavior where mature sperm burst into a final sprint in the race to the egg. The mechanism of sperm hyper-activation in many metazoans, including humans, consists of a jolt of Ca2+ into the sperm flagellum via CatSper ion channels. Surprisingly, all nine CatSper genes have been independently lost in several animal lineages. In Drosophila, sperm hyper-activation is performed through the cooption of the polycystic kidney disease 2 (pkd2) Ca2+ channel. The parallels between CatSpers in primates and pkd2 in Drosophila provide a unique opportunity to examine the molecular evolution of the sperm hyper-activation machinery in two independent, nonhomologous calcium channels separated by > 500 million years of divergence. Here, we use a comprehensive phylogenomic approach to investigate the selective pressures on these sperm hyper-activation channels. First, we find that the entire CatSper complex evolves rapidly under recurrent positive selection in primates. Second, we find that pkd2 has parallel patterns of adaptive evolution in Drosophila. Third, we show that this adaptive evolution of pkd2 is driven by its role in sperm hyper-activation. These patterns of selection suggest that the evolution of the sperm hyper-activation machinery is driven by sexual conflict with antagonistic ligands that modulate channel activity. Together, our results add sperm hyper-activation channels to the class of fast evolving reproductive proteins and provide insights into the mechanisms used by the sexes to manipulate sperm behavior. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Expression, purification and functional reconstitution of slack sodium-activated potassium channels.

PubMed

Yan, Yangyang; Yang, Youshan; Bian, Shumin; Sigworth, Fred J

2012-11-01

The slack (slo2.2) gene codes for a potassium-channel α-subunit of the 6TM voltage-gated channel family. Expression of slack results in Na(+)-activated potassium channel activity in various cell types. We describe the purification and reconstitution of Slack protein and show that the Slack α-subunit alone is sufficient for potassium channel activity activated by sodium ions as assayed in planar bilayer membranes and in membrane vesicles.

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

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

External pH effects on the depolarization-activated K channels in guard cell protoplasts of Vicia faba

PubMed Central

1994-01-01

Previous studies reveal that the pH of the apoplastic solution in the guard cell walls may vary between 7.2 and 5.1 in closed and open stomata, respectively. During these aperture and pH changes, massive K+ fluxes cross the cellular plasma membrane driving the osmotic turgor and volume changes of guard cells. Therefore, we examined the effect of extracellular pH on the depolarization-activated K channels (KD channels), which constitute the K+ efflux pathway, in the plasma membrane of Vicia faba guard cell protoplasts. We used patch clamp, both in whole cells as well as in excised outside-out membrane patches. Approximately 500 KD channels, at least, could be activated by depolarization in one protoplast (density: approximately 0.6 micron-2). Acidification from ph 8.1 to 4.4 decreased markedly the whole-cell conductance, GK, of the KD channels, shifted its voltage dependence, GK- EM, to the right on the voltage axis, slowed the rate of activation and increased the rate of deactivation, whereas the single channel conductance was not affected significantly. Based on the GK-EM shifts, the estimated average negative surface charge spacing near the KD channel is 39 A. To quantify the effects of protons on the rates of transitions between the hypothesized conformational states of the channels, we fitted the experimental macroscopic steady state conductance-voltage relationship and the voltage dependence of time constants of activation and deactivation, simultaneously, with a sequential three-state model CCO. In terms of this model, protonation affects the voltage-dependent properties via a decrease in localized, rather than homogeneous, surface charge sensed by the gating moieties. In terms of either the CO or CCO model, the protonation of a site with a pKa of 4.8 decreases the voltage-independent number of channels, N, that are available for activation by depolarization. PMID:8035163

The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine.

PubMed

Syeda, Ruhma; Santos, Jose S; Montal, Mauricio

2016-02-05

KCNQ (voltage-gated K(+) channel family 7 (Kv7)) channels control cellular excitability and underlie the K(+) current sensitive to muscarinic receptor signaling (the M current) in sympathetic neurons. Here we show that the novel anti-epileptic drug retigabine (RTG) modulates channel function of pore-only modules (PMs) of the human Kv7.2 and Kv7.3 homomeric channels and of Kv7.2/3 heteromeric channels by prolonging the residence time in the open state. In addition, the Kv7 channel PMs are shown to recapitulate the single-channel permeation and pharmacological specificity characteristics of the corresponding full-length proteins in their native cellular context. A mutation (W265L) in the reconstituted Kv7.3 PM renders the channel insensitive to RTG and favors the conductive conformation of the PM, in agreement to what is observed when the Kv7.3 mutant is heterologously expressed. On the basis of the new findings and homology models of the closed and open conformations of the Kv7.3 PM, we propose a structural mechanism for the gating of the Kv7.3 PM and for the site of action of RTG as a Kv7.2/Kv7.3 K(+) current activator. The results validate the modular design of human Kv channels and highlight the PM as a high-fidelity target for drug screening of Kv channels. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of the pH sensor and activation by chemical modification of the ClC-2G Cl- channel.

PubMed

Stroffekova, K; Kupert, E Y; Malinowska, D H; Cuppoletti, J

1998-10-01

Rabbit and human ClC-2G Cl- channels are voltage sensitive and activated by protein kinase A and low extracellular pH. The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2G Cl- channel and to determine which amino acid residues play a role in this acid activation. Channel open probability (Po) at +/-80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H+ concentration (that is, extracellular pH, pHtrans), with an apparent acidic dissociation constant of pH 4.95 +/- 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increased Po threefold at pHtrans 7.4, at which the channel normally exhibits low Po. With extracellular pH reduction (protonation) or amidation, increased Po was due to a significant increase in open time constants and a significant decrease in closed time constants of the channel gating, and this effect was insensitive to applied voltage. With the use of site-directed mutagenesis, the extracellular region EELE (amino acids 416-419) was identified as the pH sensor and amino acid Glu-419 was found to play the key or predominant role in activation of the ClC-2G Cl- channel by extracellular acid.

Thermally activated TRP channels: molecular sensors for temperature detection.

PubMed

Castillo, Karen; Diaz-Franulic, Ignacio; Canan, Jonathan; Gonzalez-Nilo, Fernando; Latorre, Ramon

2018-01-24

Temperature sensing is one of the oldest capabilities of living organisms, and is essential for sustaining life, because failure to avoid extreme noxious temperatures can result in tissue damage or death. A subset of members of the transient receptor potential (TRP) ion channel family is finely tuned to detect temperatures ranging from extreme cold to noxious heat, giving rise to thermoTRP channels. Structural and functional experiments have shown that thermoTRP channels are allosteric proteins, containing different domains that sense changes in temperature, among other stimuli, triggering pore opening. Although temperature-dependence is well characterized in thermoTRP channels, the molecular nature of temperature-sensing elements remains unknown. Importantly, thermoTRP channels are involved in pain sensation, related to pathological conditions. Here, we provide an overview of thermoTRP channel activation. We also discuss the structural and functional evidence supporting the existence of an intrinsic temperature sensor in this class of channels, and we explore the basic thermodynamic principles for channel activation. Finally, we give a view of their role in painful pathophysiological conditions.

Remediation System Evaluation, Comm. Bay/South Tacoma Channel, Well 12A Superfund Site

EPA Pesticide Factsheets

The first operable unit (OU1) of the Commencement Bay/South Tacoma Channel Superfund Site addressessoil and groundwater contamination associated with the Time Oil property that was first discovered inpublic supply Well 12A in 1981.

Lubiprostone: a chloride channel activator.

PubMed

Lacy, Brian E; Levy, L Campbell

2007-04-01

In January 2006 the Food and Drug Administration approved lubiprostone for the treatment of chronic constipation in men and women aged 18 and over. Lubiprostone is categorized as a prostone, a bicyclic fatty acid metabolite of prostaglandin E1. Lubiprostone activates a specific chloride channel (ClC-2) in the gastrointestinal (GI) tract to enhance intestinal fluid secretion, which increases GI transit and improves symptoms of constipation. This article reviews the role of chloride channels in the GI tract, describes the structure, function, and pharmacokinetics of lubiprostone, and discusses clinically important data on this new medication.

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.

Synergistic activation of G protein-gated inwardly rectifying potassium channels by cholesterol and PI(4,5)P2.

PubMed

Bukiya, Anna N; Rosenhouse-Dantsker, Avia

2017-07-01

G-protein gated inwardly rectifying potassium (GIRK or Kir3) channels play a major role in the control of the heart rate, and require the membrane phospholipid phosphatidylinositol-bis-phosphate (PI(4,5)P 2 ) for activation. Recently, we have shown that the activity of the heterotetrameric Kir3.1/Kir3.4 channel that underlies atrial K ACh currents was enhanced by cholesterol. Similarly, the activities of both the Kir3.4 homomer and its active pore mutant Kir3.4* (Kir3.4_S143T) were also enhanced by cholesterol. Here we employ planar lipid bilayers to investigate the crosstalk between PI(4,5)P 2 and cholesterol, and demonstrate that these two lipids act synergistically to activate Kir3.4* currents. Further studies using the Xenopus oocytes heterologous expression system suggest that PI(4,5)P 2 and cholesterol act via distinct binding sites. Whereas PI(4,5)P 2 binds to the cytosolic domain of the channel, the putative binding region of cholesterol is located at the center of the transmembrane domain overlapping the central glycine hinge region of the channel. Together, our data suggest that changes in the levels of two key membrane lipids - cholesterol and PI(4,5)P 2 - could act in concert to provide fine-tuning of Kir3 channel function. Copyright © 2017 Elsevier B.V. All rights reserved.

Phloretin differentially inhibits volume-sensitive and cyclic AMP-activated, but not Ca-activated, Cl− channels

PubMed Central

Fan, Hai-Tian; Morishima, Shigeru; Kida, Hajime; Okada, Yasunobu

2001-01-01

Some phenol derivatives are known to block volume-sensitive Cl− channels. However, effects on the channel of the bisphenol phloretin, which is a known blocker of glucose uniport and anion antiport, have not been examined. In the present study, we investigated the effects of phloretin on volume-sensitive Cl− channels in comparison with cyclic AMP-activated CFTR Cl− channels and Ca2+-activated Cl− channels using the whole-cell patch-clamp technique.Extracellular application of phloretin (over 10 μM) voltage-independently, and in a concentration-dependent manner (IC50 ∼30 μM), inhibited the Cl− current activated by a hypotonic challenge in human epithelial T84, Intestine 407 cells and mouse mammary C127/CFTR cells.In contrast, at 30 μM phloretin failed to inhibit cyclic AMP-activated Cl− currents in T84 and C127/CFTR cells. Higher concentrations (over 100 μM) of phloretin, however, partially inhibited the CFTR Cl− currents in a voltage-dependent manner.At 30 and 300 μM, phloretin showed no inhibitory effect on Ca2+-dependent Cl− currents induced by ionomycin in T84 cells.It is concluded that phloretin preferentially blocks volume-sensitive Cl− channels at low concentrations (below 100 μM) and also inhibits cyclic AMP-activated Cl− channels at higher concentrations, whereas phloretin does not inhibit Ca2+-activated Cl− channels in epithelial cells. PMID:11487521

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 mechanism underlying capsaicin binding and activation of TRPV1 ion channel

PubMed Central

Cheng, Wei; Yang, Wei; Yu, Peilin; Song, Zhenzhen; Yarov-Yarovoy, Vladimir; Zheng, Jie

2015-01-01

Capsaicin bestows spiciness by activating TRPV1 channel with exquisite potency and selectivity. Capsaicin-bound channel structure was previously resolved by cryo-EM at 4.2-to-4.5 Å resolution, however important details required for mechanistic understandings are unavailable: capsaicin was registered as a small electron density, reflecting neither its chemical structure nor specific ligand-channel interactions. We obtained the missing atomic-level details by iterative computation, which were confirmed by systematic site-specific functional tests. We observed that the bound capsaicin takes “tail-up, head-down” configurations. The vanillyl and amide groups form specific interactions to anchor its bound position, while the aliphatic tail may sample a range of conformations, making it invisible in cryo-EM images. Capsaicin stabilizes the open state by “pull-and-contact” interactions between the vanillyl group and the S4-S5 linker. Our study provided a structural mechanism for the agonistic function of capsaicin and its analogs, and demonstrated an effective approach to obtain atomic level information from cryo-EM structures. PMID:26053297

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.

α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels

PubMed Central

Li, Peiyao; Miao, Yong; Dani, Adish; Vig, Monika

2016-01-01

Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP–deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP–depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1. PMID:27335124

Activation of single heteromeric GABAA receptor ion channels by full and partial agonists

PubMed Central

Mortensen, Martin; Kristiansen, Uffe; Ebert, Bjarke; Frølund, Bente; Krogsgaard-Larsen, Povl; Smart, Trevor G

2004-01-01

The linkage between agonist binding and the activation of a GABAA receptor ion channel is yet to be resolved. This aspect was examined on human recombinant α1β2γ2S GABAA receptors expressed in human embryonic kidney cells using the following series of receptor agonists: GABA, isoguvacine, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), isonipecotic acid, piperidine-4-sulphonic acid (P4S), imidazole-4-acetic acid (IAA), 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL) and 5-(4-piperidyl)-3-isoxazolol (4-PIOL). Whole-cell concentration–response curves enabled the agonists to be categorized into four classes based upon their maximum responses. Single channel analyses revealed that the channel conductance of 25–27 pS was unaffected by the agonists. However, two open states were resolved from the open period distributions with mean open times reduced 5-fold by the weakest partial agonists. Using saturating agonist concentrations, estimates of the channel shutting rate, α, ranged from 200 to 600 s−1. The shut period distributions were described by three or four components and for the weakest partial agonists, the interburst shut periods increased whilst the mean burst durations and longest burst lengths were reduced relative to the full agonists. From the burst analyses, the opening rates for channel activation, β, and the total dissociation rates, k−1, for the agonists leaving the receptor were estimated. The agonist efficacies were larger for the full agonists (E ∼7−9) compared to the weak partial agonists (∼0.4–0.6). Overall, changes in agonist efficacy largely determined the different agonist profiles with contributions from the agonist affinities and the degree of receptor desensitization. From this we conclude that GABAA receptor activation does not occur in a switch-like manner since the agonist recognition sites are flexible, accommodating diverse agonist structures which differentially influence the opening and shutting rates of the ion

Casein kinase-mediated phosphorylation of serine 839 is necessary for basolateral localization of the Ca²⁺-activated non-selective cation channel TRPM4.

PubMed

Cerda, Oscar; Cáceres, Mónica; Park, Kang-Sik; Leiva-Salcedo, Elías; Romero, Aníbal; Varela, Diego; Trimmer, James S; Stutzin, Andrés

2015-08-01

Transient receptor potential melastatin-like 4 (TRPM4) is a Ca(2+)-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction, and allergic reactions. TRPM4 Ca(2+) sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-bisphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remains unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry) to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4.

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.

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.

Modulation of Cardiac Ryanodine Receptor Channels by Alkaline Earth Cations

PubMed Central

Diaz-Sylvester, Paula L.; Porta, Maura; Copello, Julio A.

2011-01-01

Cardiac ryanodine receptor (RyR2) function is modulated by Ca2+ and Mg2+. To better characterize Ca2+ and Mg2+ binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M2+: Mg2+, Ca2+, Sr2+, Ba2+) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M2+ binding to high affinity activating sites at the cytosolic channel surface, specific for Ca2+ or Sr2+. This activation was interfered by Mg2+ and Ba2+ acting at low affinity M2+-unspecific binding sites. When testing the effects of luminal M2+ as current carriers, all M2+ increased maximal RyR2 open probability (compared to Cs+), suggesting the existence of low affinity activating M2+-unspecific sites at the luminal surface. Responses to M2+ vary from channel to channel (heterogeneity). However, with luminal Ba2+or Mg2+, RyR2 were less sensitive to cytosolic Ca2+ and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca2+or Sr2+). Kinetics of RyR2 with mixtures of luminal Ba2+/Ca2+ and additive action of luminal plus cytosolic Ba2+ or Mg2+ suggest luminal M2+ differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca2+/Sr2+-specific sites, which stabilize high Po mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca2+ activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M2+ binding sites (specific for Ca2+ and unspecific for Ca2+/Mg2+) that dynamically modulate channel activity and gating status, depending on SR voltage. PMID:22039534

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

Recent Experience Using Active Love Wave Techniques to Characterize Seismographic Station Sites

NASA Astrophysics Data System (ADS)

Martin, A. J.; Yong, A.; Salomone, L.

2014-12-01

Active-source Love waves recorded by the multi-channel analysis of surface wave (MASLW) technique were recently analyzed in two site characterization projects. Between 2010 and 2011, the 2009 American Recovery and Reinvestment Act (ARRA) funded GEOVision to conduct geophysical investigations at 189 seismographic stations—185 in California and 4 in the Central Eastern U.S. (CEUS). The original project plan was to utilize active and passive Rayleigh wave-based techniques to obtain shear-wave velocity (VS) profiles to a minimum depth of 30 m and the time-averaged VS of the upper 30 meters (VS30). Early in the investigation it became evident that Rayleigh wave techniques, such as multi-channel analysis of surface waves (MASRW), were not effective at characterizing all sites. Shear-wave seismic refraction and MASLW techniques were therefore applied. The MASLW technique was deployed at a total of 38 sites, in addition to other methods, and used as the primary technique to characterize 22 sites, 5 of which were also characterized using Rayleigh wave techniques. In 2012, the Electric Power Research Institute funded characterization of 33 CEUS station sites. Based on experience from the ARRA investigation, both MASRW and MASLW data were acquired by GEOVision at 24 CEUS sites—the remaining 9 sites and 2 overlapping sites were characterized by University of Texas, Austin. Of the 24 sites characterized by GEOVision, 16 were characterized using MASLW data, 4 using both MASLW and MASRW data and 4 using MASRW data. Love wave techniques were often found to perform better, or at least yield phase velocity data that could be more readily modeled using the fundamental mode assumption, at shallow rock sites, sites with steep velocity gradients, and, sites with a thin, low velocity, surficial soil layer overlying stiffer sediments. These types of velocity structure often excite dominant higher modes in Rayleigh wave data, but not in Love wave data. At such sites, it may be possible

Molecular Basis of Cardiac Delayed Rectifier Potassium Channel Function and Pharmacology.

PubMed

Wu, Wei; Sanguinetti, Michael C

2016-06-01

Human cardiomyocytes express 3 distinct types of delayed rectifier potassium channels. Human ether-a-go-go-related gene (hERG) channels conduct the rapidly activating current IKr; KCNQ1/KCNE1 channels conduct the slowly activating current IKs; and Kv1.5 channels conduct an ultrarapid activating current IKur. Here the authors provide a general overview of the mechanistic and structural basis of ion selectivity, gating, and pharmacology of the 3 types of cardiac delayed rectifier potassium ion channels. Most blockers bind to S6 residues that line the central cavity of the channel, whereas activators interact with the channel at 4 symmetric binding sites outside the cavity. Copyright © 2016 Elsevier Inc. All rights reserved.

Irreversible temperature gating in trpv1 sheds light on channel activation.

PubMed

Sánchez-Moreno, Ana; Guevara-Hernández, Eduardo; Contreras-Cervera, Ricardo; Rangel-Yescas, Gisela; Ladrón-de-Guevara, Ernesto; Rosenbaum, Tamara; Islas, León D

2018-06-05

Temperature-activated TRP channels or thermoTRPs are among the only proteins that can directly convert temperature changes into changes in channel open probability. In spite of a wealth of functional and structural information, the mechanism of temperature activation remains unknown. We have carefully characterized the repeated activation of TRPV1 by thermal stimuli and discovered a previously unknown inactivation process, which is irreversible. We propose that this form of gating in TRPV1 channels is a consequence of the heat absorption process that leads to channel opening. © 2018, Sánchez-Moreno et al.

Irreversible temperature gating in trpv1 sheds light on channel activation

PubMed Central

Sánchez-Moreno, Ana; Guevara-Hernández, Eduardo; Contreras-Cervera, Ricardo; Rangel-Yescas, Gisela; Ladrón-de-Guevara, Ernesto; Rosenbaum, Tamara

2018-01-01

Temperature-activated TRP channels or thermoTRPs are among the only proteins that can directly convert temperature changes into changes in channel open probability. In spite of a wealth of functional and structural information, the mechanism of temperature activation remains unknown. We have carefully characterized the repeated activation of TRPV1 by thermal stimuli and discovered a previously unknown inactivation process, which is irreversible. We propose that this form of gating in TRPV1 channels is a consequence of the heat absorption process that leads to channel opening. PMID:29869983

[Mechanism of action of neurotoxins acting on the inactivation of voltage-gated sodium channels].

PubMed

Benoit, E

1998-01-01

This review focuses on the mechanism(s) of action of neurotoxins acting on the inactivation of voltage-gated Na channels. Na channels are transmembrane proteins which are fundamental for cellular communication. These proteins form pores in the plasma membrane allowing passive ionic movements to occur. Their opening and closing are controlled by gating systems which depend on both membrane potential and time. Na channels have three functional properties, mainly studied using electrophysiological and biochemical techniques, to ensure their role in the generation and propagation of action potentials: 1) a highly selectivity for Na ions, 2) a rapid opening ("activation"), responsible for the depolarizing phase of the action potential, and 3) a late closing ("inactivation") involved in the repolarizing phase of the action potential. As an essential protein for membrane excitability, the Na channel is the specific target of a number of vegetal and animal toxins which, by binding to the channel, alter its activity by affecting one or more of its properties. At least six toxin receptor sites have been identified on the neuronal Na channel on the basis of binding studies. However, only toxins interacting with four of these sites (sites 2, 3, 5 et 6) produce alterations of channel inactivation. The maximal percentage of Na channels modified by the binding of neurotoxins to sites 2 (batrachotoxin and some alkaloids), 3 (alpha-scorpion and sea anemone toxins), 5 (brevetoxins and ciguatoxins) et 6 (delta-conotoxins) is different according to the site considered. However, in all cases, these channels do not inactivate. Moreover, Na channels modified by toxins which bind to sites 2, 5 and 6 activate at membrane potentials more negative than do unmodified channels. The physiological consequences of Na channel modifications, induced by the binding of neurotoxins to sites 2, 3, 5 and 6, are (i) an inhibition of cellular excitability due to an important membrane depolarization (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.

Targeting mechanisms of high voltage-activated Ca2+ channels.

PubMed

Herlitze, Stefan; Xie, Mian; Han, Jing; Hümmer, Alexander; Melnik-Martinez, Katya V; Moreno, Rosa L; Mark, Melanie D

2003-12-01

Functional voltage-dependent Ca2+ channel complexes are assembled by three to four subunits: alpha1, beta, alpha2delta subunits (C. Leveque et al., 1994, J. Biol Chem. 269, 6306-6312; M. W. McEnery et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88, 11095-11099) and at least in muscle cells also y subunits (B. M. Curtis and W. A. Catterall, 1984, Biochemistry 23, 2113-2118). Ca2+ channels mediate the voltage-dependent Ca2+ influx in subcellular compartments, triggering such diverse processes as neurotransmitter release, dendritic action potentials, excitation-contraction, and excitation-transcription coupling. The targeting of biophysically defined Ca2+ channel complexes to the correct subcellular structures is, thus, critical to proper cell and physiological functioning. Despite their importance, surprisingly little is known about the targeting mechanisms by which Ca2+ channel complexes are transported to their site of function. Here we summarize what we know about the targeting of Ca2+ channel complexes through the cell to the plasma membrane and subcellular structures.

Chloride channels as tools for developing selective insecticides.

PubMed

Bloomquist, Jeffrey R

2003-12-01

Ligand-gated chloride channels underlie inhibition in excitable membranes and are proven target sites for insecticides. The gamma-aminobutyric acid (GABA(1)) receptor/chloride ionophore complex is the primary site of action for a number of currently used insecticides, such as lindane, endosulfan, and fipronil. These compounds act as antagonists by stabilizing nonconducting conformations of the chloride channel. Blockage of the GABA-gated chloride channel reduces neuronal inhibition, which leads to hyperexcitation of the central nervous system, convulsions, and death. We recently investigated the mode of action of the silphinenes, plant-derived natural compounds that structurally resemble picrotoxinin. These materials antagonize the action of GABA on insect neurons and block GABA-mediated chloride uptake into mouse brain synaptoneurosomes in a noncompetitive manner. In mammals, avermectins have a blocking action on the GABA-gated chloride channel consistent with a coarse tremor, whereas at longer times and higher concentrations, activation of the channel suppresses neuronal activity. Invertebrates display ataxia, paralysis, and death as the predominant signs of poisoning, with a glutamate-gated chloride channel playing a major role. Additional target sites for the avermectins or other chloride channel-directed compounds might include receptors gated by histamine, serotonin, or acetylcholine.The voltage-sensitive chloride channels form another large gene family of chloride channels. Voltage-dependent chloride channels are involved in a number of physiological processes including: maintenance of electrical excitability, chloride ion secretion and resorption, intravesicular acidification, and cell volume regulation. A subset of these channels is affected by convulsants and insecticides in mammals, although the role they play in acute lethality in insects is unclear. Given the wide range of functions that they mediate, these channels are also potential targets for

Channeling Children's Energy through Vocabulary Activities

ERIC Educational Resources Information Center

Schindler, Andrea

2006-01-01

In this article, the author shares vocabulary development activities for young learners. These activities channel students' energy and make learning more effective and fun. The author stresses the importance of giving young learners a good language-learning experience, and the challenges of teaching young learners who are not literate in their L1.…

TRPV3 channels mediate strontium-induced mouse egg activation

PubMed Central

Carvacho, Ingrid; Lee, Hoi Chang; Fissore, Rafael A.; Clapham, David E.

2014-01-01

SUMMARY In mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a Transient Receptor Potential (TRP) ion channel current activated by TRP agonists that is absent in TrpV3−/− eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII), the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, as TrpV3−/− eggs failed to permeate Sr2+ or undergo strontium-induced activation. We propose that TRPV3 is the major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation. PMID:24316078

Control of rectification and permeation by two distinct sites after the second transmembrane region in Kir2.1 K+ channel

PubMed Central

Kubo, Yoshihiro; Murata, Yoshimichi

2001-01-01

The rectification property of the inward rectifier K+ channel is chiefly due to the block of outward current by cytoplasmic Mg2+ and polyamines. In the cloned inward rectifier K+ channel Kir2.1 (IRK1), Asp172 in the second transmembrane region (M2) and Glu224 in the putative cytoplasmic region after M2 are reported to be critical for the sensitivity to these blockers. However, the difference in the inward rectification properties between Kir2.1 and a very weak inward rectifier sWIRK could not be explained by differences at these two sites. Following sequence comparison of Kir2.1 and sWIRK, we focused this study on Glu299 located in the centre of the putative cytoplasmic region after M2. Single-point mutants of Kir2.1 (Glu224Gly and Glu299Ser) and a double-point mutant (Glu224Gly-Glu299Ser) were made and expressed in Xenopus oocytes or in HEK293T cells. Their electrophysiological properties were compared with those of wild-type (WT) Kir2.1 and the following observations were made. (a) Glu299Ser showed a weaker inward rectification, a slower activation upon hyperpolarization, a slower decay of the outward current upon depolarization, a lower sensitivity to block by cytoplasmic spermine and a smaller single-channel conductance than WT. (b) The features of Glu224Gly were similar to those of Glu299Ser. (c) In the double mutant (Glu224Gly-Glu299Ser), the differences from WT described above were more prominent. These results demonstrate that Glu299 as well as Glu224 control rectification and permeation, and suggest the possibility that the two sites contribute to the inner vestibule of the channel pore. The slowing down of the on- and off-blocking processes by mutation of these sites implies that Glu224 and Glu299 function to facilitate the entry (and exit) of spermine to (and from) the blocking site. PMID:11251047

Elucidation of pyrethroid and DDT receptor sites in the voltage-gated sodium channel.

PubMed

Zhorov, Boris S; Dong, Ke

2017-05-01

DDT and pyrethroid insecticides were among the earliest neurotoxins identified to act on voltage-gated sodium channels. In the 1960s, equipped with, at the time, new voltage-clamp techniques, Professor Narahashi and associates provided the initial evidence that DDT and allethrin (the first commercial pyrethroid insecticide) caused prolonged flow of sodium currents in lobster and squid giant axons. Over the next several decades, continued efforts by Prof. Narahashi's group as well as other laboratories led to a comprehensive understanding of the mechanism of action of DDT and pyrethroids on sodium channels. Fast forward to the 1990s, genetic, pharmacological and toxicological data all further confirmed voltage-gated sodium channels as the primary targets of DDT and pyrethroid insecticides. Modifications of the gating kinetics of sodium channels by these insecticides result in repetitive firing and/or membrane depolarization in the nervous system. This mini-review focuses on studies from Prof. Narahashi's pioneer work and more recent mutational and computational modeling analyses which collectively elucidated the elusive pyrethroid receptor sites as well as the molecular basis of differential sensitivities of insect and mammalian sodium channels to pyrethroids. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

PubMed Central

Miao, Yinglong; Baudry, Jerome

2011-01-01

Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site. PMID:21943431

Cysteine palmitoylation of the γ subunit has a dominant role in modulating activity of the epithelial sodium channel.

PubMed

Mukherjee, Anindit; Mueller, Gunhild M; Kinlough, Carol L; Sheng, Nan; Wang, Zhijian; Mustafa, S Atif; Kashlan, Ossama B; Kleyman, Thomas R; Hughey, Rebecca P

2014-05-16

The epithelial sodium channel (ENaC) is composed of three homologous subunits (α, β, and γ) with cytoplasmic N and C termini. Our previous work revealed that two cytoplasmic Cys residues in the β subunit, βCys-43 and βCys-557, are Cys-palmitoylated. ENaCs with mutant βC43A/C557A exhibit normal surface expression but enhanced Na(+) self-inhibition and reduced channel open probability. Although the α subunit is not palmitoylated, we now show that the two cytoplasmic Cys residues in the γ subunit are palmitoylated. ENaCs with mutant γC33A, γC41A, or γC33A/C41A exhibit reduced activity compared with wild type channels but normal surface expression and normal levels of α and γ subunit-activating cleavage. These mutant channels have significantly enhanced Na(+) self-inhibition and reduced open probability compared with wild type ENaCs. Channel activity was enhanced by co-expression with the palmitoyltransferase DHHC2 that also co-immunoprecipitates with ENaCs. Secondary structure prediction of the N terminus of the γ subunit places γCys-33 within an α-helix and γCys-44 on a coil before the first transmembrane domain within a short tract that includes a well conserved His-Gly motif, where mutations have been associated with altered channel gating. Our current and previous results suggest that palmitoylation of the β and γ subunits of ENaCs enhances interactions of their respective cytoplasmic domains with the plasma membrane and stabilizes the open state of the channel. Comparison of activities of channels lacking palmitoylation sites in individual or multiple subunits revealed that γ subunit palmitoylation has a dominant role over β subunit palmitoylation in modulating ENaC gating. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Piezo proteins are pore-forming subunits of mechanically activated channels.

PubMed

Coste, Bertrand; Xiao, Bailong; Santos, Jose S; Syeda, Ruhma; Grandl, Jörg; Spencer, Kathryn S; Kim, Sung Eun; Schmidt, Manuela; Mathur, Jayanti; Dubin, Adrienne E; Montal, Mauricio; Patapoutian, Ardem

2012-02-19

Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ∼1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.

STIM1 dimers undergo unimolecular coupling to activate Orai1 channels

NASA Astrophysics Data System (ADS)

Zhou, Yandong; Wang, Xizhuo; Wang, Xianming; Loktionova, Natalia A.; Cai, Xiangyu; Nwokonko, Robert M.; Vrana, Erin; Wang, Youjun; Rothberg, Brad S.; Gill, Donald L.

2015-09-01

The endoplasmic reticulum (ER) Ca2+ sensor, STIM1, becomes activated when ER-stored Ca2+ is depleted and translocates into ER-plasma membrane junctions where it tethers and activates Orai1 Ca2+ entry channels. The dimeric STIM1 protein contains a small STIM-Orai-activating region (SOAR)--the minimal sequence sufficient to activate Orai1 channels. Since SOAR itself is a dimer, we constructed SOAR concatemer-dimers and introduced mutations at F394, which is critical for Orai1 coupling and activation. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai1 binding or activation. This reveals an unexpected unimolecular coupling between STIM1 and Orai1 and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai1 channel subunits. The model predicts that STIM1 dimers may be involved in crosslinking between Orai1 channels with implications for the kinetics and localization of Orai1 channel opening.

A G-protein-activated inwardly rectifying K+ channel (GIRK4) from human hippocampus associates with other GIRK channels.

PubMed

Spauschus, A; Lentes, K U; Wischmeyer, E; Dissmann, E; Karschin, C; Karschin, A

1996-02-01

Transcripts of a gene, GIRK4, that encodes for a 419-amino-acid protein and shows high structural similarity to other subfamily members of G-protein-activated inwardly rectifying K+ channels (GIRK) have been identified in the human hippocampus. When expressed in Xenopus oocytes, GIRK4 yielded functional GIRK channels with activity that was enhanced by the stimulation of coexpressed serotonin 1A receptors. GIRK4 potentiated basal and agonist-induced currents mediated by other GIRK channels, possibly because of channel heteromerization. Despite the structural similarity to a putative rat KATP channel, no ATP sensitivity or KATP-typical pharmacology was observed for GIRK4 alone or GIRK4 transfected in conjunction with other GIRK channels in COS-7 cells. In rat brain, GIRK4 is expressed together with three other subfamily members, GIRK1-3, most likely in identical hippocampal neurons. Thus, heteromerization or an unknown molecular interaction may cause the physiological diversity observed within this class of K+ channels.

Site-specific electronic structure analysis by channeling EELS and first-principles calculations.

PubMed

Tatsumi, Kazuyoshi; Muto, Shunsuke; Yamamoto, Yu; Ikeno, Hirokazu; Yoshioka, Satoru; Tanaka, Isao

2006-01-01

Site-specific electronic structures were investigated by electron energy loss spectroscopy (EELS) under electron channeling conditions. The Al-K and Mn-L(2,3) electron energy loss near-edge structure (ELNES) of, respectively, NiAl2O4 and Mn3O4 were measured. Deconvolution of the raw spectra with the instrumental resolution function restored the blunt and hidden fine features, which allowed us to interpret the experimental spectral features by comparing with theoretical spectra obtained by first-principles calculations. The present method successfully revealed the electronic structures specific to the differently coordinated cationic sites.

Ion Channel Conformation and Oligomerization Assessment by Site-Directed Spin Labeling and Pulsed-EPR.

PubMed

Pliotas, Christos

2017-01-01

Mechanosensitive (MS) ion channels are multimeric integral membrane proteins that respond to increased lipid bilayer tension by opening their nonselective pores to release solutes and relieve increased cytoplasmic pressure. These systems undergo major conformational changes during gating and the elucidation of their mechanism requires a deep understanding of the interplay between lipids and proteins. Lipids are responsible for transmitting lateral tension to MS channels and therefore play a key role in obtaining a molecular-detail model for mechanosensation. Site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful spectroscopic tool in the study of proteins. The main bottleneck for its use relates to challenges associated with successful isolation of the protein of interest, introduction of paramagnetic labels on desired sites, and access to specialized instrumentation and expertise. The design of sophisticated experiments, which combine a variety of existing EPR methodologies to address a diversity of specific questions, require knowledge of the limitations and strengths, characteristic of each particular EPR method. This chapter is using the MS ion channels as paradigms and focuses on the application of different EPR techniques to ion channels, in order to investigate oligomerization, conformation, and the effect of lipids on their regulation. The methodology we followed, from the initial strategic selection of mutants and sample preparation, including protein purification, spin labeling, reconstitution into lipid mimics to the complete set-up of the pulsed-EPR experiments, is described in detail. © 2017 Elsevier Inc. All rights reserved.

The activation of calcium and calcium-activated potassium channels in mammalian colonic smooth muscle by substance P.

PubMed Central

Mayer, E A; Loo, D D; Snape, W J; Sachs, G

1990-01-01

1. The regulation of Ca2(+)-activated K+ channels by the agonist substance P in freshly dissociated smooth muscle cells from the rabbit longitudinal colonic muscle was characterized using the patch clamp technique. 2. In the cell-attached recording mode, when pipette and bath solutions contained equal [K+] (126 mM), the Ca2(+)-activated K+ channels showed a linear current-voltage relationship (between -50 mV and 50 mV) with a slope conductance of 210 +/- 35 pS (n = 12). Reversal potential measurements indicated that the channel was highly selective for K+ over Na+ (PK/PNa = 110). 3. Channels were activated by depolarizing membrane voltages and cytosolic Ca2+, and in inside-out patches channel activation depended sigmoidally on voltage and [Ca2+]. The potential for half-activation at a cytosolic [Ca2+] of 5 x 10(-6) M was 0 mV. A tenfold increase in cytosolic Ca2+ resulted in a 60 mV shift of the sigmoidal voltage activation curve to more negative potentials. 4. Threshold concentrations of substance P (10(-12) M), which did not result in cell contraction, caused a prolonged activation of K+ channels. The K+ channels were observed to open in clusters: simultaneous opening of multiple channels was interrupted by complete, prolonged channel closure. 5. Lowering bath [Ca2+] to submicromolar concentrations abolished the effect of substance P. The activation of K+ channels by substance P (10(-12) M) was also inhibited by the dihydropyridine nifedipine (10(-6) M), a blocker of L-type Ca2+ channels. 6. In the whole-cell recording mode, with the pipette solution containing 126 mM-KCl, 0.77 mM-EGTA and 1 mM-ATP, depolarization from a holding potential of -70 mV elicited outward currents which increased to steady-state values. These were K+ currents as they were blocked by TEA (tetraethylammonium, 30 mM) and Ba2+ (1 mM) and were abolished when pipette K+ was replaced by Cs+. 7. The depolarization-activated outward current was not affected by lowering extracellular [Ca2+] or by

Site-specific distribution of claudin-based paracellular channels with roles in biological fluid flow and metabolism.

PubMed

Tanaka, Hiroo; Tamura, Atsushi; Suzuki, Koya; Tsukita, Sachiko

2017-10-01

The claudins are a family of membrane proteins with at least 27 members in humans and mice. The extracellular regions of claudin proteins play essential roles in cell-cell adhesion and the paracellular barrier functions of tight junctions (TJs) in epithelial cell sheets. Furthermore, the extracellular regions of some claudins function as paracellular channels in the paracellular barrier that allow the selective passage of water, ions, and/or small organic solutes across the TJ in the extracellular space. Structural analyses have revealed a common framework of transmembrane, cytoplasmic, and extracellular regions among the claudin-based paracellular barriers and paracellular channels; however, differences in the claudins' extracellular regions, such as their charges and conformations, determine their properties. Among the biological systems that involve fluid flow and metabolism, it is noted that hepatic bile flow, renal Na + reabsorption, and intestinal nutrient absorption are dynamically regulated via site-specific distributions of paracellular channel-forming claudins in tissue. Here, we focus on how site-specific distributions of claudin-2- and claudin-15-based paracellular channels drive their organ-specific functions in the liver, kidney, and intestine. © 2017 New York Academy of Sciences.

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.

Mapping the lipoylation site of Arabidopsis thaliana plastidial dihydrolipoamide S-acetyltransferase using mass spectrometry and site-directed mutagenesis

USDA-ARS?s Scientific Manuscript database

Background: The catalytic enhancement achieved by the pyruvate dehydrogenase complex (PDC) results from a combination of substrate channeling plus active-site coupling. The mechanism for active-site coupling involves lipoic acid prosthetic groups covalently attached to Lys residues in the primary ...

Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

PubMed Central

Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R

1992-01-01

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

Zinc-dependent multi-conductance channel activity in mitochondria isolated from ischemic brain.

PubMed

Bonanni, Laura; Chachar, Mushtaque; Jover-Mengual, Teresa; Li, Hongmei; Jones, Adrienne; Yokota, Hidenori; Ofengeim, Dimitry; Flannery, Richard J; Miyawaki, Takahiro; Cho, Chang-Hoon; Polster, Brian M; Pypaert, Marc; Hardwick, J Marie; Sensi, Stefano L; Zukin, R Suzanne; Jonas, Elizabeth A

2006-06-21

Transient global ischemia is a neuronal insult that induces delayed cell death. A hallmark event in the early post-ischemic period is enhanced permeability of mitochondrial membranes. The precise mechanisms by which mitochondrial function is disrupted are, as yet, unclear. Here we show that global ischemia promotes alterations in mitochondrial membrane contact points, a rise in intramitochondrial Zn2+, and activation of large, multi-conductance channels in mitochondrial outer membranes by 1 h after insult. Mitochondrial channel activity was associated with enhanced protease activity and proteolytic cleavage of BCL-xL to generate its pro-death counterpart, deltaN-BCL-xL. The findings implicate deltaN-BCL-xL in large, multi-conductance channel activity. Consistent with this, large channel activity was mimicked by introduction of recombinant deltaN-BCL-xL to control mitochondria and blocked by introduction of a functional BCL-xL antibody to post-ischemic mitochondria via the patch pipette. Channel activity was also inhibited by nicotinamide adenine dinucleotide, indicative of a role for the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. In vivo administration of the membrane-impermeant Zn2+ chelator CaEDTA before ischemia or in vitro application of the membrane-permeant Zn2+ chelator tetrakis-(2-pyridylmethyl) ethylenediamine attenuated channel activity, suggesting a requirement for Zn2+. These findings reveal a novel mechanism by which ischemic insults disrupt the functional integrity of the outer mitochondrial membrane and implicate deltaN-BCL-xL and VDAC in the large, Zn2+-dependent mitochondrial channels observed in post-ischemic hippocampal mitochondria.

Zinc-Dependent Multi-Conductance Channel Activity in Mitochondria Isolated from Ischemic Brain

PubMed Central

Bonanni, Laura; Chachar, Mushtaque; Jover-Mengual, Teresa; Li, Hongmei; Jones, Adrienne; Yokota, Hidenori; Ofengeim, Dimitry; Flannery, Richard J.; Miyawaki, Takahiro; Cho, Chang-Hoon; Polster, Brian M.; Pypaert, Marc; Hardwick, J. Marie; Sensi, Stefano L.; Zukin, R. Suzanne; Jonas, Elizabeth A.

2015-01-01

Transient global ischemia is a neuronal insult that induces delayed cell death. A hallmark event in the early post-ischemic period is enhanced permeability of mitochondrial membranes. The precise mechanisms by which mitochondrial function is disrupted are, as yet, unclear.Here we show that global ischemia promotes alterations in mitochondrial membrane contact points, a rise in intramitochondrial Zn2+, and activation of large, multi-conductance channels in mitochondrial outer membranes by 1 h after insult. Mitochondrial channel activity was associated with enhanced protease activity and proteolytic cleavage of BCL-xL to generate its pro-death counterpart, ΔN-BCL-xL. The findings implicate ΔN-BCL-xL in large, multi-conductance channel activity. Consistent with this, large channel activity was mimicked by introduction of recombinant ΔN-BCL-xL to control mitochondria and blocked by introduction of a functional BCL-xL antibody to post-ischemic mitochondria via the patch pipette. Channel activity was also inhibited by nicotinamide adenine dinucleotide, indicative of a role for the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. In vivo administration of the membrane-impermeant Zn2+ chelator CaEDTA before ischemia or in vitro application of the membrane-permeant Zn2+ chelator tetrakis-(2-pyridylmethyl) ethylenediamine attenuated channel activity, suggesting a requirement for Zn2+. These findings reveal a novel mechanism by which ischemic insults disrupt the functional integrity of the outer mitochondrial membrane and implicate ΔNBCL-xL and VDAC in the large, Zn2+-dependent mitochondrial channels observed in post-ischemic hippocampal mitochondria. PMID:16793892

Identification of a putative binding site critical for general anesthetic activation of TRPA1.

PubMed

Ton, Hoai T; Phan, Thieu X; Abramyan, Ara M; Shi, Lei; Ahern, Gerard P

2017-04-04

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABA A receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

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.

An anion channel in Arabidopsis hypocotyls activated by blue light

NASA Technical Reports Server (NTRS)

Cho, M. H.; Spalding, E. P.; Evans, M. L. (Principal Investigator)

1996-01-01

A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition.

Actions and Mechanisms of Polyunsaturated Fatty Acids on Voltage-Gated Ion Channels.

PubMed

Elinder, Fredrik; Liin, Sara I

2017-01-01

Polyunsaturated fatty acids (PUFAs) act on most ion channels, thereby having significant physiological and pharmacological effects. In this review we summarize data from numerous PUFAs on voltage-gated ion channels containing one or several voltage-sensor domains, such as voltage-gated sodium (Na V ), potassium (K V ), calcium (Ca V ), and proton (H V ) channels, as well as calcium-activated potassium (K Ca ), and transient receptor potential (TRP) channels. Some effects of fatty acids appear to be channel specific, whereas others seem to be more general. Common features for the fatty acids to act on the ion channels are at least two double bonds in cis geometry and a charged carboxyl group. In total we identify and label five different sites for the PUFAs. PUFA site 1 : The intracellular cavity. Binding of PUFA reduces the current, sometimes as a time-dependent block, inducing an apparent inactivation. PUFA site 2 : The extracellular entrance to the pore. Binding leads to a block of the channel. PUFA site 3 : The intracellular gate. Binding to this site can bend the gate open and increase the current. PUFA site 4 : The interface between the extracellular leaflet of the lipid bilayer and the voltage-sensor domain. Binding to this site leads to an opening of the channel via an electrostatic attraction between the negatively charged PUFA and the positively charged voltage sensor. PUFA site 5 : The interface between the extracellular leaflet of the lipid bilayer and the pore domain. Binding to this site affects slow inactivation. This mapping of functional PUFA sites can form the basis for physiological and pharmacological modifications of voltage-gated ion channels.

Actions and Mechanisms of Polyunsaturated Fatty Acids on Voltage-Gated Ion Channels

PubMed Central

Elinder, Fredrik; Liin, Sara I.

2017-01-01

Polyunsaturated fatty acids (PUFAs) act on most ion channels, thereby having significant physiological and pharmacological effects. In this review we summarize data from numerous PUFAs on voltage-gated ion channels containing one or several voltage-sensor domains, such as voltage-gated sodium (NaV), potassium (KV), calcium (CaV), and proton (HV) channels, as well as calcium-activated potassium (KCa), and transient receptor potential (TRP) channels. Some effects of fatty acids appear to be channel specific, whereas others seem to be more general. Common features for the fatty acids to act on the ion channels are at least two double bonds in cis geometry and a charged carboxyl group. In total we identify and label five different sites for the PUFAs. PUFA site 1: The intracellular cavity. Binding of PUFA reduces the current, sometimes as a time-dependent block, inducing an apparent inactivation. PUFA site 2: The extracellular entrance to the pore. Binding leads to a block of the channel. PUFA site 3: The intracellular gate. Binding to this site can bend the gate open and increase the current. PUFA site 4: The interface between the extracellular leaflet of the lipid bilayer and the voltage-sensor domain. Binding to this site leads to an opening of the channel via an electrostatic attraction between the negatively charged PUFA and the positively charged voltage sensor. PUFA site 5: The interface between the extracellular leaflet of the lipid bilayer and the pore domain. Binding to this site affects slow inactivation. This mapping of functional PUFA sites can form the basis for physiological and pharmacological modifications of voltage-gated ion channels. PMID:28220076

Intermolecular Interactions in the TMEM16A Dimer Controlling Channel Activity.

PubMed

Scudieri, Paolo; Musante, Ilaria; Gianotti, Ambra; Moran, Oscar; Galietta, Luis J V

2016-12-08

TMEM16A and TMEM16B are plasma membrane proteins with Ca 2+ -dependent Cl - channel function. By replacing the carboxy-terminus of TMEM16A with the equivalent region of TMEM16B, we obtained channels with potentiation of channel activity. Progressive shortening of the chimeric region restricted the "activating domain" to a short sequence close to the last transmembrane domain and led to TMEM16A channels with high activity at very low intracellular Ca 2+ concentrations. To elucidate the molecular mechanism underlying this effect, we carried out experiments based on double chimeras, Forster resonance energy transfer, and intermolecular cross-linking. We also modeled TMEM16A structure using the Nectria haematococca TMEM16 protein as template. Our results indicate that the enhanced activity in chimeric channels is due to altered interaction between the carboxy-terminus and the first intracellular loop in the TMEM16A homo-dimer. Mimicking this perturbation with a small molecule could be the basis for a pharmacological stimulation of TMEM16A-dependent Cl - transport.

Intermolecular Interactions in the TMEM16A Dimer Controlling Channel Activity

PubMed Central

Scudieri, Paolo; Musante, Ilaria; Gianotti, Ambra; Moran, Oscar; Galietta, Luis J. V.

2016-01-01

TMEM16A and TMEM16B are plasma membrane proteins with Ca2+-dependent Cl− channel function. By replacing the carboxy-terminus of TMEM16A with the equivalent region of TMEM16B, we obtained channels with potentiation of channel activity. Progressive shortening of the chimeric region restricted the “activating domain” to a short sequence close to the last transmembrane domain and led to TMEM16A channels with high activity at very low intracellular Ca2+ concentrations. To elucidate the molecular mechanism underlying this effect, we carried out experiments based on double chimeras, Forster resonance energy transfer, and intermolecular cross-linking. We also modeled TMEM16A structure using the Nectria haematococca TMEM16 protein as template. Our results indicate that the enhanced activity in chimeric channels is due to altered interaction between the carboxy-terminus and the first intracellular loop in the TMEM16A homo-dimer. Mimicking this perturbation with a small molecule could be the basis for a pharmacological stimulation of TMEM16A-dependent Cl− transport. PMID:27929144

Intermolecular Interactions in the TMEM16A Dimer Controlling Channel Activity

NASA Astrophysics Data System (ADS)

Scudieri, Paolo; Musante, Ilaria; Gianotti, Ambra; Moran, Oscar; Galietta, Luis J. V.

2016-12-01

TMEM16A and TMEM16B are plasma membrane proteins with Ca2+-dependent Cl- channel function. By replacing the carboxy-terminus of TMEM16A with the equivalent region of TMEM16B, we obtained channels with potentiation of channel activity. Progressive shortening of the chimeric region restricted the “activating domain” to a short sequence close to the last transmembrane domain and led to TMEM16A channels with high activity at very low intracellular Ca2+ concentrations. To elucidate the molecular mechanism underlying this effect, we carried out experiments based on double chimeras, Forster resonance energy transfer, and intermolecular cross-linking. We also modeled TMEM16A structure using the Nectria haematococca TMEM16 protein as template. Our results indicate that the enhanced activity in chimeric channels is due to altered interaction between the carboxy-terminus and the first intracellular loop in the TMEM16A homo-dimer. Mimicking this perturbation with a small molecule could be the basis for a pharmacological stimulation of TMEM16A-dependent Cl- transport.

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 Active Site of the Tungsten, Iron-Sulfur Enzyme Acetylene Hydratase▿ †

PubMed Central

tenBrink, Felix; Schink, Bernhard; Kroneck, Peter M. H.

2011-01-01

The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H2O to acetylene (H—C☰C—H) to form acetaldehyde (CH3CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme. PMID:21193613

Substrate channel in nitrogenase revealed by a molecular dynamics approach.

PubMed

Smith, Dayle; Danyal, Karamatullah; Raugei, Simone; Seefeldt, Lance C

2014-04-15

Mo-dependent nitrogenase catalyzes the biological reduction of N2 to two NH3 molecules at FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized submicrosecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel. The viability of this observed channel was tested by examining the free energy of passage of N2 from the surface through the channel to FeMo-cofactor, resulting in the discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment and that approaches a face of FeMo-cofactor earlier implicated in substrate binding.

STIM and Orai proteins and the non-capacitative ARC channels

PubMed Central

Shuttleworth, Trevor J.

2012-01-01

The ARC channel is a small conductance, highly Ca2+-selective ion channel whose activation is specifically dependent on low concentrations of arachidonic acid acting at an intracellular site. They are widely distributed in diverse cell types where they provide an alternative, store-independent pathway for agonist-activated Ca2+ entry. Although biophysically similar to the store-operated CRAC channels, these two conductances function under distinct conditions of agonist stimulation, with the ARC channels providing the predominant route of Ca2+ entry during the oscillatory signals generated at low agonist concentrations. Despite these differences in function, like the CRAC channel, activation of the ARC channels is dependent on STIM1, but it is the pool of STIM1 that constitutively resides in the plasma membrane that is responsible. Similarly, both channels are formed by Orai proteins but, whilst the CRAC channel pore is a tetrameric assembly of Orai1 subunits, the ARC channel pore is formed by a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits. There is increasing evidence that the activity of these channels plays a critical role a variety of different cellular activities. PMID:22201777

P/Q-type calcium channels activate neighboring calcium-dependent potassium channels in mouse motor nerve terminals.

PubMed

Protti, D A; Uchitel, O D

1997-08-01

The identity of the voltage-dependent calcium channels (VDCC), which trigger the Ca2+-gated K+ currents (IK(Ca)) in mammalian motor nerve terminals, was investigated by means of perineurial recordings. The effects of Ca2+ chelators with different binding kinetics on the activation of IK(Ca) were also examined. The calcium channel blockers of the P/Q family, omega-agatoxin IVA (omega-Aga-IVA) and funnel-web spider toxin (FTX), have been shown to exert a strong blocking effect on IK(Ca). In contrast, nitrendipine and omega-conotoxin GVIA (omega-CgTx) did not affect the Ca2+-activated K+ currents. The intracellular action of the fast Ca2+ buffers BAPTA and DM-BAPTA prevented the activation of the IK(Ca), while the slow Ca2+ buffer EGTA was ineffective at blocking it. These data indicate that P/Q-type VDCC mediate the Ca2+ influx which activates IK(Ca). The spatial association between Ca2+ and Ca2+-gated K+ channels is discussed, on the basis of the differential effects of the fast and slow Ca2+ chelators.

Atomic basis for therapeutic activation of neuronal potassium channels

NASA Astrophysics Data System (ADS)

Kim, Robin Y.; Yau, Michael C.; Galpin, Jason D.; Seebohm, Guiscard; Ahern, Christopher A.; Pless, Stephan A.; Kurata, Harley T.

2015-09-01

Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2-5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators.

Oxidative Stress and Maxi Calcium-Activated Potassium (BK) Channels

PubMed Central

Hermann, Anton; Sitdikova, Guzel F.; Weiger, Thomas M.

2015-01-01

All cells contain ion channels in their outer (plasma) and inner (organelle) membranes. Ion channels, similar to other proteins, are targets of oxidative impact, which modulates ion fluxes across membranes. Subsequently, these ion currents affect electrical excitability, such as action potential discharge (in neurons, muscle, and receptor cells), alteration of the membrane resting potential, synaptic transmission, hormone secretion, muscle contraction or coordination of the cell cycle. In this chapter we summarize effects of oxidative stress and redox mechanisms on some ion channels, in particular on maxi calcium-activated potassium (BK) channels which play an outstanding role in a plethora of physiological and pathophysiological functions in almost all cells and tissues. We first elaborate on some general features of ion channel structure and function and then summarize effects of oxidative alterations of ion channels and their functional consequences. PMID:26287261

Role of different types of potassium channels in the antidepressant-like effect of agmatine in the mouse forced swimming test.

PubMed

Budni, Josiane; Gadotti, Vinícius M; Kaster, Manuella P; Santos, Adair R S; Rodrigues, Ana Lúcia S

2007-12-01

The administration of agmatine elicits an antidepressant-like effect in the mouse forced swimming test by a mechanism dependent on the inhibition of the NMDA receptors and the L-arginine-nitric oxide (NO) pathway. Since it has been reported that the NO can activate different types of potassium (K(+)) channels in several tissues, the present study investigates the possibility of synergistic interactions between different types of K(+) channel inhibitors and agmatine in the forced swimming test. Treatment of mice by i.c.v. route with subeffective doses of tetraethylammonium (a non specific inhibitor of K(+) channels, 25 pg/site), glibenclamide (an ATP-sensitive K(+) channels inhibitor, 0.5 pg/site), charybdotoxin (a large- and intermediate-conductance calcium-activated K(+) channel inhibitor, 25 pg/site) or apamin (a small-conductance calcium-activated K(+) channel inhibitor, 10 pg/site), augmented the effect of agmatine (0.001 mg/kg, i.p.) in the forced swimming test. Furthermore, the administration of agmatine and the K(+) channel inhibitors, alone or in combination, did not affect locomotion in the open-field test. Moreover, the reduction in the immobility time elicited by an active dose of agmatine (10 mg/kg, i.p.) in the forced swimming test was prevented by the pre-treatment of mice with the K(+) channel openers cromakalim (10 microg/site, i.c.v.) and minoxidil (10 microg/site, i.c.v.), without affecting locomotion. Together these data raise the possibility that the antidepressant-like effect of agmatine in the forced swimming test is related to its modulatory effects on neuronal excitability, via inhibition of K(+) channels.

Movement of gating machinery during the activation of rod cyclic nucleotide-gated channels.

PubMed Central

Brown, R L; Snow, S D; Haley, T L

1998-01-01

In the visual and olfactory systems, cyclic nucleotide-gated (CNG) ion channels convert stimulus-induced changes in the internal concentrations of cGMP and cAMP into changes in membrane potential. Although it is known that significant activation of these channels requires the binding of three or more molecules of ligand, the detailed molecular mechanism remains obscure. We have probed the structural changes that occur during channel activation by using sulfhydryl-reactive methanethiosulfonate (MTS) reagents and N-ethylmaleimide (NEM). When expressed in Xenopus oocytes, the alpha-subunit of the bovine retinal channel forms homomultimeric channels that are activated by cGMP with a K1/2 of approximately 100 microM. Cyclic AMP, on the other hand, is a very poor activator; a saturating concentration elicits only 1% of the maximum current produced by cGMP. Treatment of excised patches with MTS-ethyltrimethylamine (MTSET) or NEM dramatically potentiated the channel's response to both cyclic nucleotides. After MTSET treatment, the dose-response relation for cGMP was shifted by over two orders of magnitude to lower concentrations. The effect on channel activation by cAMP was even more striking. After modification, the channels were fully activated by cAMP with a K1/2 of approximately 60 microM. This potentiation was abolished by conversion of Cys481 to a nonreactive alanine residue. Potentiation occurred more rapidly in the presence of saturating cGMP, indicating that this region of the channel is more accessible when the channel is open. Cys481 is located in a linker region between the transmembrane and cGMP-binding domains of the channel. These results suggest that this region of the channel undergoes significant movement during the activation process and is critical for coupling ligand binding to pore opening. Potentiation, however, is not mediated by the recently reported interaction between the amino- and carboxy-terminal regions of the alpha-subunit. Deletion of the

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.

On the estimation of cooperativity in ion channel kinetics: activation free energy and kinetic mechanism of Shaker K+ channel.

PubMed

Banerjee, Kinshuk; Das, Biswajit; Gangopadhyay, Gautam

2013-04-28

In this paper, we have explored generic criteria of cooperative behavior in ion channel kinetics treating it on the same footing with multistate receptor-ligand binding in a compact theoretical framework. We have shown that the characterization of cooperativity of ion channels in terms of the Hill coefficient violates the standard Hill criteria defined for allosteric cooperativity of ligand binding. To resolve the issue, an alternative measure of cooperativity is proposed here in terms of the cooperativity index that sets a unified criteria for both the systems. More importantly, for ion channel this index can be very useful to describe the cooperative kinetics as it can be readily determined from the experimentally measured ionic current combined with theoretical modelling. We have analyzed the correlation between the voltage value and slope of the voltage-activation curve at the half-activation point and consequently determined the standard free energy of activation of the ion channel using two well-established mechanisms of cooperativity, namely, Koshland-Nemethy-Filmer (KNF) and Monod-Wyman-Changeux (MWC) models. Comparison of the theoretical results for both the models with appropriate experimental data of mutational perturbation of Shaker K(+) channel supports the experimental fact that the KNF model is more suitable to describe the cooperative behavior of this class of ion channels, whereas the performance of the MWC model is unsatisfactory. We have also estimated the mechanistic performance through standard free energy of channel activation for both the models and proposed a possible functional disadvantage in the MWC scheme.

Interaction of the BKCa channel gating ring with dendrotoxins

PubMed Central

Takacs, Zoltan; Imredy, John P; Bingham, Jon-Paul; Zhorov, Boris S; Moczydlowski, Edward G

2014-01-01

Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca2+-activated K+ channel (BKCa, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BKCa channel gating. In this work we examined the subconductance behavior of 9 natural dendrotoxin homologs and 6 charge neutralization mutants of δ-dendrotoxin in the context of current structural information on the intracellular gating ring domain of the BKCa channel. Calculation of an electrostatic surface map of the BKCa gating ring based on the Poisson-Boltzmann equation reveals a predominantly electronegative surface due to an abundance of solvent-accessible side chains of negatively charged amino acids. Available structure-activity information suggests that cationic DTX/BPTI molecules bind by electrostatic attraction to site(s) on the gating ring located in or near the cytoplasmic side portals where the inactivation ball peptide of the β2 subunit enters to block the channel. Such an interaction may decrease the apparent unitary conductance by altering the dynamic balance of open versus closed states of BKCa channel activation gating. PMID:25483585

Active Brownian particles escaping a channel in single file.

PubMed

Locatelli, Emanuele; Baldovin, Fulvio; Orlandini, Enzo; Pierno, Matteo

2015-02-01

Active particles may happen to be confined in channels so narrow that they cannot overtake each other (single-file conditions). This interesting situation reveals nontrivial physical features as a consequence of the strong interparticle correlations developed in collective rearrangements. We consider a minimal two-dimensional model for active Brownian particles with the aim of studying the modifications introduced by activity with respect to the classical (passive) single-file picture. Depending on whether their motion is dominated by translational or rotational diffusion, we find that active Brownian particles in single file may arrange into clusters that are continuously merging and splitting (active clusters) or merely reproduce passive-motion paradigms, respectively. We show that activity conveys to self-propelled particles a strategic advantage for trespassing narrow channels against external biases (e.g., the gravitational field).

Active Brownian particles escaping a channel in single file

NASA Astrophysics Data System (ADS)

Locatelli, Emanuele; Baldovin, Fulvio; Orlandini, Enzo; Pierno, Matteo

2015-02-01

Active particles may happen to be confined in channels so narrow that they cannot overtake each other (single-file conditions). This interesting situation reveals nontrivial physical features as a consequence of the strong interparticle correlations developed in collective rearrangements. We consider a minimal two-dimensional model for active Brownian particles with the aim of studying the modifications introduced by activity with respect to the classical (passive) single-file picture. Depending on whether their motion is dominated by translational or rotational diffusion, we find that active Brownian particles in single file may arrange into clusters that are continuously merging and splitting (active clusters) or merely reproduce passive-motion paradigms, respectively. We show that activity conveys to self-propelled particles a strategic advantage for trespassing narrow channels against external biases (e.g., the gravitational field).

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.

Ferritin ion channel disorder inhibits Fe(II)/O2 reactivity at distant sites.

PubMed

Tosha, Takehiko; Behera, Rabindra K; Theil, Elizabeth C

2012-11-05

Ferritins, a complex, mineralized, protein nanocage family essential for life, provide iron concentrates and oxidant protection. Protein-based ion channels and Fe(II)/O(2) catalysis initiate conversion of thousands of Fe atoms to caged, ferritin Fe(2)O(3)·H(2)O minerals. The ion channels consist of six helical segments, contributed by 3 of 12 or 24 polypeptide subunits, around the 3-fold cage axes. The channel structure guides entering Fe(II) ions toward multiple, catalytic, diiron sites buried inside ferritin protein helices, ~20 Å away from channel internal exits. The catalytic product, Fe(III)-O(H)-Fe(III), is a mineral precursor; mineral nucleation begins inside the protein cage with mineral growth in the central protein cavity (5-8 nm diameter). Amino acid substitutions that changed ionic or hydrophobic channel interactions R72D, D122R, and L134P increased ion channel structural disorder (protein crystallographic analyses) and increased Fe(II) exit [chelated Fe(II) after ferric mineral reduction/dissolution]. Since substitutions of some channel carboxylate residues diminished ferritin catalysis with no effect on Fe(II) exit, such as E130A and D127A, we investigated catalysis in ferritins with altered Fe(II) exit, R72D, D122R and L134P. The results indicate that simply changing the ionic properties of the channels, as in the R72D variant, need not change the forward catalytic rate. However, both D122R and L134P, which had dramatic effects on ferritin catalysis, also caused larger effects on channel structure and order, contrasting with R72D. All three amino acid substitutions, however, decreased the stability of the catalytic intermediate, diferric peroxo, even though overall ferritin cage structure is very stable, resisting 80 °C and 6 M urea. The localized structural changes in ferritin subdomains that affect ferritin function over long distances illustrate new properties of the protein cage in natural ferritin function and for applied ferritin uses.

Selective disruption of high sensitivity heat activation but not capsaicin activation of TRPV1 channels by pore turret mutations

PubMed Central

Cui, Yuanyuan; Yang, Fan; Cao, Xu; Yarov-Yarovoy, Vladimir

2012-01-01

The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate. PMID:22412190

Mutation of I696 and W697 in the TRP box of vanilloid receptor subtype I modulates allosteric channel activation.

PubMed

Gregorio-Teruel, Lucia; Valente, Pierluigi; González-Ros, José Manuel; Fernández-Ballester, Gregorio; Ferrer-Montiel, Antonio

2014-03-01

The transient receptor potential vanilloid receptor subtype I (TRPV1) channel acts as a polymodal sensory receptor gated by chemical and physical stimuli. Like other TRP channels, TRPV1 contains in its C terminus a short, conserved domain called the TRP box, which is necessary for channel gating. Substitution of two TRP box residues-I696 and W697-with Ala markedly affects TRPV1's response to all activating stimuli, which indicates that these two residues play a crucial role in channel gating. We systematically replaced I696 and W697 with 18 native l-amino acids (excluding cysteine) and evaluated the effect on voltage- and capsaicin-dependent gating. Mutation of I696 decreased channel activation by either voltage or capsaicin; furthermore, gating was only observed with substitution of hydrophobic amino acids. Substitution of W697 with any of the 18 amino acids abolished gating in response to depolarization alone, shifting the threshold to unreachable voltages, but not capsaicin-mediated gating. Moreover, vanilloid-activated responses of W697X mutants showed voltage-dependent gating along with a strong voltage-independent component. Analysis of the data using an allosteric model of activation indicates that mutation of I696 and W697 primarily affects the allosteric coupling constants of the ligand and voltage sensors to the channel pore. Together, our findings substantiate the notion that inter- and/or intrasubunit interactions at the level of the TRP box are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions markedly reduces the efficacy and potency of the activating stimuli. Furthermore, our results identify these interactions as potential sites for pharmacological intervention.

Thermodynamics of Activation Gating in Olfactory-Type Cyclic Nucleotide-Gated (CNGA2) Channels

PubMed Central

Nache, Vasilica; Kusch, Jana; Biskup, Christoph; Schulz, Eckhard; Zimmer, Thomas; Hagen, Volker; Benndorf, Klaus

2008-01-01

Olfactory-type cyclic nucleotide-gated (CNG) ion channels open by the binding of cyclic nucleotides to a binding domain in the C-terminus. Employing the Eyring rate theory, we performed a thermodynamic analysis of the activation gating in homotetrameric CNGA2 channels. Lowering the temperature shifted the concentration-response relationship to lower concentrations, resulting in a decrease of both the enthalpy ΔH and entropy ΔS upon channel opening, suggesting that the order of an open CNGA2 channel plus its environment is higher than that of the closed channel. Activation time courses induced by cGMP concentration jumps were used to study thermodynamics of the transition state. The activation enthalpies ΔH‡ were positive at all cGMP concentrations. In contrast, the activation entropy ΔS‡ was positive at low cGMP concentrations and became then negative at increasing cGMP concentrations. The enthalpic and entropic parts of the activation energies approximately balance each other at all cGMP concentrations, leaving the free enthalpy of activation in the range between 19 and 21 kcal/mol. We conclude that channel activation proceeds through different pathways at different cGMP concentrations. Compared to the unliganded channel, low cGMP concentrations generate a transitional state of lower order whereas high cGMP concentrations generate a transitional state of higher order. PMID:18567637

Comparison of erosion and channel characteristics

USGS Publications Warehouse

Parker, Gene W.

1998-01-01

Erosion was observed at 33 percent of 22,495 bridge sites in nine States. Among sites with erosion, 56 percent were associated with skewed flows, curved channels, or a combination of these two conditions, and at 18 percent of the sites, channels were straight with steep bank angles. The remaining 26 percent are sites with observable erosion at piers or abutments on straight channels. Comparison of the sites with erosion to channel bed-material indicate that 44 percent of the single-span sites had gravel-size or smaller bed material and 70 percent of the multiple-span sites had gravel-size or smaller bed material.

Loperamide: A positive modulator for store-operated calcium channels?

PubMed Central

Harper, Jacquie L.; Shin, Yangmee; Daly, John W.

1997-01-01

The depletion of inositol trisphosphate-sensitive intracellular pools of calcium causes activation of store-operated calcium (SOC) channels. Loperamide at 10–30 μM has no effect on intracellular calcium levels alone, but augments calcium levels in cultured cells when SOC channels have been activated. In HL-60 leukemic cells, the apparent positive modulatory effect of loperamide on SOC channels occurs when these channels have been activated after ATP, thapsigargin, or ionomycin-elicited depletion of calcium from intracellular storage sites. Loperamide has no effect when levels of intracellular calcium are elevated through a mechanism not involving SOC channels by using sphingosine. Loperamide caused augmentation of intracellular calcium levels after activation of SOC channels in NIH 3T3 fibroblasts, astrocytoma 1321N cells, smooth muscle DDT-MF2 cells, RBL-2H3 mast cells, and pituitary GH4C1 cells. Only in astrocytoma cells did loperamide cause an elevation in intracellular calcium in the absence of activation of SOC channels. The augmentation of intracellular calcium elicited by loperamide in cultured cells was dependent on extracellular calcium and was somewhat resistant to agents (SKF 96365, miconazole, clotrimazole, nitrendipine, and trifluoperazine) that in the absence of loperamide effectively blocked SOC channels. It appears that loperamide augments influx of calcium through activated SOC channels. PMID:9405713

α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels.

PubMed

Li, Peiyao; Miao, Yong; Dani, Adish; Vig, Monika

2016-08-15

Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP-deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP-depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1. © 2016 Li, Miao, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Adenosine triphosphate regulates the activity of guinea pig Cav1.2 channel by direct binding to the channel in a dose-dependent manner.

PubMed

Feng, Rui; Xu, Jianjun; Minobe, Etsuko; Kameyama, Asako; Yang, Lei; Yu, Lifeng; Hao, Liying; Kameyama, Masaki

2014-05-01

The present study is to investigate the mechanism by which ATP regulates Cav1.2 channel activity. Ventricular tissue was obtained from adult guinea pig hearts using collagenase. Ca(2+) channel activity was monitored using the patch-clamp technique. Proteins were purified using wheat germ agglutinin-Sepharose, and the concentration was determined using the Coomassie brilliant blue technique. ATP binding to the Cav1.2 channel was examined using the photoaffinity method. EDA-ATP-biotin maintains Ca(2+) channel activity in inside-out membrane patches. ATP directly bound to the Cav1.2 channel in a dose-dependent manner, and at least two molecules of ATP bound to one molecule of the Cav1.2 channel. Low levels of calmodulin (CaM) increased ATP binding to the Cav1.2 channel, but higher levels of CaM decreased ATP binding to the Cav1.2 channel. In addition, Ca(2+) was another regulator for ATP binding to the Cav1.2 channel. Furthermore, ATP bound to GST-fusion peptides of NH2-terminal region (amino acids 6-140) and proximal COOH-terminal region (amino acids 1,509-1,789) of the main subunit (α1C) of the Cav1.2 channel. Our data suggest that ATP might regulate Cav1.2 channel activity by directly binding to the Cav1.2 channel in a dose-dependent manner. In addition, the ATP-binding effect to the Cav1.2 channel was both CaM- and Ca(2+) dependent.

Mean annual runoff and peak flow estimates based on channel geometry of streams in southeastern Montana

USGS Publications Warehouse

Omang, R.J.; Parrett, Charles; Hull, J.A.

1983-01-01

Equations using channel-geometry measurements were developed for estimating mean runoff and peak flows of ungaged streams in southeastern Montana. Two separate sets of esitmating equations were developed for determining mean annual runoff: one for perennial streams and one for ephemeral and intermittent streams. Data from 29 gaged sites on perennial streams and 21 gaged sites on ephemeral and intermittent streams were used in these analyses. Data from 78 gaged sites were used in the peak-flow analyses. Southeastern Montana was divided into three regions and separate multiple-regression equations for each region were developed that relate channel dimensions to peak discharge having recurrence intervals of 2, 5, 10, 25, 50, and 100 years. Channel-geometery relations were developed using measurements of the active-channel width and bankfull width. Active-channel width and bankfull width were the most significant channel features for estimating mean annual runoff for al types of streams. Use of this method requires that onsite measurements be made of channel width. The standard error of estimate for predicting mean annual runoff ranged from about 38 to 79 percent. The standard error of estimate relating active-channel width or bankfull width to peak flow ranged from about 37 to 115 percent. (USGS)

[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.

Allosteric modulation of ATP-gated P2X receptor channels

PubMed Central

Coddou, Claudio; Stojilkovic, Stanko S.; Huidobro-Toro, J. Pablo

2013-01-01

Seven mammalian purinergic receptor subunits, denoted P2X1 to P2X7, and several spliced forms of these subunits have been cloned. When heterologously expressed, these cDNAs encode ATP-gated non-selective cation channels organized as trimers. All activated receptors produce cell depolarization and promote Ca2+ influx through their pores and indirectly by activating voltage-gated calcium channels. However, the biophysical and pharmacological properties of these receptors differ considerably, and the majority of these subunits are also capable of forming heterotrimers with other members of the P2X receptor family, which confers further different properties. These channels have three ATP binding domains, presumably located between neighboring subunits, and occupancy of at least two binding sites is needed for their activation. In addition to the orthosteric binding sites for ATP, these receptors have additional allosteric sites that modulate the agonist action at receptors, including sites for trace metals, protons, neurosteroids, reactive oxygen species and phosphoinositides. The allosteric regulation of P2X receptors is frequently receptor-specific and could be a useful tool to identify P2X members in native tissues and their roles in signaling. The focus of this review is on common and receptor-specific allosteric modulation of P2X receptors and the molecular base accounting for allosteric binding sites. PMID:21639805

Detection of single ion channel activity with carbon nanotubes

NASA Astrophysics Data System (ADS)

Zhou, Weiwei; Wang, Yung Yu; Lim, Tae-Sun; Pham, Ted; Jain, Dheeraj; Burke, Peter J.

2015-03-01

Many processes in life are based on ion currents and membrane voltages controlled by a sophisticated and diverse family of membrane proteins (ion channels), which are comparable in size to the most advanced nanoelectronic components currently under development. Here we demonstrate an electrical assay of individual ion channel activity by measuring the dynamic opening and closing of the ion channel nanopores using single-walled carbon nanotubes (SWNTs). Two canonical dynamic ion channels (gramicidin A (gA) and alamethicin) and one static biological nanopore (α-hemolysin (α-HL)) were successfully incorporated into supported lipid bilayers (SLBs, an artificial cell membrane), which in turn were interfaced to the carbon nanotubes through a variety of polymer-cushion surface functionalization schemes. The ion channel current directly charges the quantum capacitance of a single nanotube in a network of purified semiconducting nanotubes. This work forms the foundation for a scalable, massively parallel architecture of 1d nanoelectronic devices interrogating electrophysiology at the single ion channel level.

Hyperforin activates nonselective cation channels (NSCCs).

PubMed

Treiber, Kristina; Singer, Andrea; Henke, Bettina; Müller, Walter E

2005-05-01

A large body of evidence supports the preclinical antidepressant profile of hyperforin including inhibition of the synaptosomal uptake of several neurotransmitters by hyperforin and studies in behavioural models. In contrast to other antidepressants, hyperforin does not directly inhibit neurotransmitter transporters, but instead uptake inhibition seems to be the consequence of an elevated intracellular sodium concentration ([Na+]i). The mechanism of hyperforin-induced elevation of [Na+]i was investigated using two different cell types: human platelets and rat pheochromocytoma cells (PC12 cells). In both cell systems, hyperforin increased both [Na+]i and free intracellular Ca2+ concentration ([Ca2+]i). One pathway for Na+ and Ca2+ entry is mediated by nonselective cation channels (NSCCs), which can be blocked by SK&F 96365 and LOE 908. LOE 908 is a blocker of both NSCC1 and NSCC2 subclasses, while SK&F 96365 blocks NSCC2 only. Both SK&F 96365 and LOE 908 completely inhibited the hyperforin-induced influx of Na+ and Ca2+ into platelets and PC12 cells. This indicates that hyperforin is mainly active upon NSCC2. The effect of hyperforin is inhibited by La3+ and Gd3+, indicating that there is a potential homology with canonical transient receptor potential protein channels (TRPC channels). Moreover, La3+ and Gd3+ attenuate the effect of hyperforin on serotonin uptake in human platelets. Additionally, hyperforin induces barium influx in PC12 cells and this influx can be inhibited by SK&F 96365, LOE 908, Gd3+ and La3+. In summary, these findings suggest that hyperforin represents a new principle for preclinical antidepressant activity, modulating brain neurotransmission by inhibition of neurotransmitter uptake via activation of NSCCs.British Journal of Pharmacology (2005) 145, 75-83. doi:10.1038/sj.bjp.0706155.

Hyperforin activates nonselective cation channels (NSCCs)

PubMed Central

Treiber, Kristina; Singer, Andrea; Henke, Bettina; Müller, Walter E

2005-01-01

A large body of evidence supports the preclinical antidepressant profile of hyperforin including inhibition of the synaptosomal uptake of several neurotransmitters by hyperforin and studies in behavioural models. In contrast to other antidepressants, hyperforin does not directly inhibit neurotransmitter transporters, but instead uptake inhibition seems to be the consequence of an elevated intracellular sodium concentration ([Na+]i). The mechanism of hyperforin-induced elevation of [Na+]i was investigated using two different cell types: human platelets and rat pheochromocytoma cells (PC12 cells). In both cell systems, hyperforin increased both [Na+]i and free intracellular Ca2+ concentration ([Ca2+]i). One pathway for Na+ and Ca2+ entry is mediated by nonselective cation channels (NSCCs), which can be blocked by SK&F 96365 and LOE 908. LOE 908 is a blocker of both NSCC1 and NSCC2 subclasses, while SK&F 96365 blocks NSCC2 only. Both SK&F 96365 and LOE 908 completely inhibited the hyperforin-induced influx of Na+ and Ca2+ into platelets and PC12 cells. This indicates that hyperforin is mainly active upon NSCC2. The effect of hyperforin is inhibited by La3+ and Gd3+, indicating that there is a potential homology with canonical transient receptor potential protein channels (TRPC channels). Moreover, La3+ and Gd3+ attenuate the effect of hyperforin on serotonin uptake in human platelets. Additionally, hyperforin induces barium influx in PC12 cells and this influx can be inhibited by SK&F 96365, LOE 908, Gd3+ and La3+. In summary, these findings suggest that hyperforin represents a new principle for preclinical antidepressant activity, modulating brain neurotransmission by inhibition of neurotransmitter uptake via activation of NSCCs. PMID:15723093

Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR

NASA Astrophysics Data System (ADS)

Lange, Adam; Giller, Karin; Hornig, Sönke; Martin-Eauclaire, Marie-France; Pongs, Olaf; Becker, Stefan; Baldus, Marc

2006-04-01

The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane-similar to the catalytic function of the active site of an enzyme-and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel-scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton-proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein-inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin-K+ channel interactions.

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

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

The binding site on cochlear stereocilia for antisera raised against renal Na+ channels is blocked by amiloride and dihydrostreptomycin.

PubMed

Furness, D N; Hackney, C M; Benos, D J

1996-04-01

The mechanoelectrical transduction channels on hair cells have been suggested to be operated by tip links that are stretched when the hair bundle is deflected in the direction of the tallest row of stereocilia. Localising these channels is therefore an important test of this hypothesis. The transduction channels are known to be amiloride-sensitive and immunogold labelling with antibodies raised against the amiloride-sensitive epithelial Na+ channel from kidney (alpha NaCh), has suggested that sites with similar characteristics are located in the region where the tips of the shorter stereocilia appear to come into contact with the sides of the adjacent taller stereocilia rather than being associated directly with the tip links. Now, further immunocytochemical experiments have been performed to determine if amiloride and dihydrostreptomycin, both of which can block transduction, can affect this labelling. Immunofluorescent labelling of the stereocilia is obtained when surface preparations of the organ of Corti are fixed and incubated with alpha NaCh followed by an appropriate secondary antibody. This labelling is abolished by trypsinization prior to fixation but retained if the tissue is pretreated with amiloride and then trypsinized in its presence. Because amiloride is known to protect amiloride-binding sites from degradation by trypsin, these results suggest that alpha NaCh is revealing amiloride-binding sites on the stereocilia. Similarly, immunofluorescent labelling of the stereocilia is abolished if cochlear tissue is pretreated with dihydrostreptomycin (DHS) and fixed in its presence prior to incubation with alpha NaCh. Quantitative analysis of colloidal gold labelling using transmission electron microscopy shows that DHS treatment produces a significant reduction in the number of gold particles on stereocilia, especially in the region of contact between them. These results suggest that anti-Na+ recognises a site with characteristics similar to the

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

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

Channel-forming activity in the venom of the cockroach-hunting wasp, Ampulex compressa.

PubMed

Gincel, Dan; Haspel, Gal; Libersat, Frederic

2004-05-01

The parasitoid solitary wasp Ampulex compressa uses the cockroach Periplaneta americana as a food supply for its larvae. To subdue its prey, the wasp injects a venom cocktail into the brain of the cockroach. We investigated channel activity of A. compressa venom by collecting venom and incorporating it into a planar lipid bilayer. The venom, reconstituted into the bilayer, showed ion channel activity, forming a fast-fluctuating channel with a small conductance of 20+/-0.1pS, with no voltage sensitivity. These channels were not observed when the venom was digested with proteases before application to the bilayer, but were not affected by exposure to protease after their incorporation into the bilayer, indicating that the active venom component is a peptide. The channels were found to be cation selective with similar selectivity for the monovalent cations K(+), Li(+) and Na(+), but showed high selectivity against anions (Cl(-)) and divalent cations (Ca(2+) and Mg(2+)). This study is the first demonstration and biophysical characterization of channel activity in the venom of A. compressa. The possible functional significance of this channel activity is discussed in light of the unusual nature of the effects of this wasp venom on the behavior of its prey.

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.

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

Dynamic ion-ion and water-ion interactions in ion channels.

PubMed Central

Wu, J V

1992-01-01

The dynamic interactions among ions and water molecules in ion channels are treated based on an assumption that ions at binding sites can be knocked off by both transient entering ions and local water molecules. The theory, when applied to a single-site model K+ channel, provides solutions for super- and subsaturations, flux-ratio exponent (n') greater than 1, osmotic streaming current, activity-dependent reversal potentials, and anomalous mole-fraction behavior. The analysis predicts that: (a) the saturation may but, in general, does not follow the Michaelis-Menten relation; (b) streaming current results from imbalanced water-ion knock-off interactions; (c) n' greater than 1 even for single-site channels, but it is unlikely to exceed 1.4 unless the pore is occupied by one or more ion(s); (d) in the calculation involving two permeant ion species with similar radii, the heavier ions show higher affinity; the ion-ion knock-off dissociation from the site is more effective when two interacting ions are identical. Therefore, the "multi-ion behaviors" found in most ion channels are the consequences of dynamic ion-ion and water-ion interactions. The presence of these interactions does not require two or more binding sites in channels. PMID:1376158

Coupling of activation and inactivation gate in a K+-channel: potassium and ligand sensitivity

PubMed Central

Ader, Christian; Schneider, Robert; Hornig, Sönke; Velisetty, Phanindra; Vardanyan, Vitya; Giller, Karin; Ohmert, Iris; Becker, Stefan; Pongs, Olaf; Baldus, Marc

2009-01-01

Potassium (K+)-channel gating is choreographed by a complex interplay between external stimuli, K+ concentration and lipidic environment. We combined solid-state NMR and electrophysiological experiments on a chimeric KcsA–Kv1.3 channel to delineate K+, pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH-induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K+ and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K+ ion on the inactivation gate modulate activation-gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K+-channel pore. PMID:19661921

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

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

Structure-function study on a de novo synthetic hydrophobic ion channel.

PubMed Central

Qi, Z; Sokabe, M; Donowaki, K; Ishida, H

1999-01-01

Ion conduction properties of a de novo synthesized channel, formed from cyclic octa-peptides consisting of four alternate L-alanine (Ala) and N'-acylated 3-aminobenzoic acid (Aba) moieties, were studied in bilayer membranes. The single-channel conductance was 9 pS in symmetrical 500 mM KCl. The channel favored permeation of cations over anions with a permeability ratio (PCl-/PK+) of 0.15. The selectivity sequence among monovalent cations based on permeability ratio (PX+/PK+) fell into an order: NH4+(1.4) > Cs+(1. 1) >/= K+(1.0) > Na+(0.4) >> Li+(0). The conductance-activity relationship of the channel in K+ solutions followed simple Michaelis-Menten kinetics with a half-maximal saturating activity of 8 mM and a maximal conductance of 9 pS. The permeability ratio PNa+/PK+ remained constant ( approximately 0.40) under biionic concentrations from 10 to 500 mM. These results suggests that the channel is a one-ion channel. The pore diameter probed by a set of organic cations was approximately 6 A. The single-channel current was blocked by Ca2+ in a dose-dependent manner that followed a single-site titration curve with a voltage-dependent dissociation constant of 0.6 mM at 100 mV. The electric distance of the binding site for Ca2+ was 0.07 from both entrances of the channel, indicating the presence of two symmetrical binding sites in each vicinity of the channel entrance. Correlations between conduction properties and structural aspects of the channel are discussed in terms of a three-barrier and two-binding-site (3B2S) model of Eyring rate theory. All available structural information supported an idea that the channel was formed from a tail-to-tail associated dimer of the molecule, the pore of which was lined with hydrophobic acyl chains. This is the first report to have made a systematic analysis of ion permeation through a hydrophobic pore. PMID:9929469

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.

Multiple-channel detection of cellular activities by ion-sensitive transistors

NASA Astrophysics Data System (ADS)

Machida, Satoru; Shimada, Hideto; Motoyama, Yumi

2018-04-01

An ion-sensitive field-effect transistor to record cellular activities was demonstrated. This field-effect transistor (bio transistor) includes cultured cells on the gate insulator instead of gate electrode. The bio transistor converts a change in potential underneath the cells into variation of the drain current when ion channels open. The bio transistor has high detection sensitivity to even minute variations in potential utilizing a subthreshold swing region. To open ion channels, a reagent solution (acetylcholine) was added to a human-originating cell cultured on the bio transistor. The drain current was successfully decreased with the addition of acetylcholine. Moreover, we attempted to detect the opening of ion channels using a multiple-channel measurement circuit containing several bio transistors. As a consequence, the drain current distinctly decreased only after the addition of acetylcholine. We confirmed that this measurement system including bio transistors enables to observation of cellular activities sensitively and simultaneously.

Equivalence of two approaches for modeling ion permeation through a transmembrane channel with an internal binding site

NASA Astrophysics Data System (ADS)

Zhou, Huan-Xiang

2011-04-01

Ion permeation through transmembrane channels has traditionally been modeled using two different approaches. In one approach, the translocation of the permeant ion through the channel pore is modeled as continuous diffusion and the rate of ion transport is obtained from solving the steady-state diffusion equation. In the other approach, the translocation of the permeant ion through the pore is modeled as hopping along a discrete set of internal binding sites and the rate of ion transport is obtained from solving a set of steady-state rate equations. In a recent work [Zhou, J. Phys. Chem. Lett. 1, 1973 (2010)], the rate constants for binding to an internal site were further calculated by modeling binding as diffusion-influenced reactions. That work provided the foundation for bridging the two approaches. Here we show that, by representing a binding site as an energy well, the two approaches indeed give the same result for the rate of ion transport.

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.

The Gárdos channel: a review of the Ca2+-activated K+ channel in human erythrocytes.

PubMed

Maher, Anthony D; Kuchel, Philip W

2003-08-01

Ca(2+)-dependent K(+) efflux from human erythrocytes was first described in the 1950s. Subsequent studies revealed that a K(+)-specific membrane protein (the Gárdos channel) was responsible for this phenomenon (the Gárdos effect). In recent years several types of Ca-activated K(+) channel have been identified and studied in a wide range of cells, with the erythrocyte Gárdos channel serving as both a model for a broader physiological perspective, and an intriguing component of erythrocyte function. The existence of this channel has raised a number of questions. For example, what is its role in the establishment and maintenance of ionic distribution across the red cell membrane? What role might it play in erythrocyte development? To what extent is it active in circulating erythrocytes? What are the cell-physiological implications of its dysfunction?This review summarises current knowledge of this membrane protein with respect to its function and structure, its physiological roles (some putative) and its contribution to various disease states, and it provides an introduction to adaptable NMR methods, which is our own area of technical expertise, for such ion transport analysis.

Updating In Vivo and In Vitro Phosphorylation and Methylation Sites of Voltage-Gated Kv7.2 Potassium Channels.

PubMed

Erdem, Fatma Asli; Salzer, Isabella; Heo, Seok; Chen, Wei-Qiang; Jung, Gangsoo; Lubec, Gert; Boehm, Stefan; Yang, Jae-Won

2017-10-01

Voltage-gated Kv7.2 potassium channels regulate neuronal excitability. The gating of these channels is tightly controlled by various mediators and neurotransmitters acting via G protein-coupled receptors; the underlying signaling cascades involve phosphatidylinositol-4,5-bisphosphate (PIP 2 ), Ca 2+ /calmodulin, and phosphorylation. Recent studies found that the PIP 2 sensitivity of Kv7.2 channels is affected by two posttranslational modifications, phosphorylation and methylation, harboured within putative PIP 2 -binding domains. In this study, we updated phosphorylation and methylation sites in Kv7.2 either heterologously expressed in mammalian cells or as GST-fusion proteins exposed to recombinant protein kinases by using LC-MS/MS. In vitro kinase assays revealed that CDK5, protein kinase C (PKC) alpha, PKA, p38 MAPK, CamKIIα, and GSK3β could mediate phosphorylation. Taken together, we provided a comprehensive map of phosphorylation and methylation in Kv7.2 within protein-protein and protein-lipid interaction domains. This may help to interpret the functional roles of individual PTM sites in Kv7.2 channels. All MS data are available via ProteomeXchange with the identifier PXD005567. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of a pre-active conformation of a pentameric channel receptor

PubMed Central

Menny, Anaïs; Lefebvre, Solène N; Schmidpeter, Philipp AM; Drège, Emmanuelle; Fourati, Zaineb; Delarue, Marc; Edelstein, Stuart J; Nimigean, Crina M; Joseph, Delphine; Corringer, Pierre-Jean

2017-01-01

Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical signaling through global allosteric transitions. Despite the existence of several high-resolution structures of pLGICs, their dynamical properties remain elusive. Using the proton-gated channel GLIC, we engineered multiple fluorescent reporters, each incorporating a bimane and a tryptophan/tyrosine, whose close distance causes fluorescence quenching. We show that proton application causes a global compaction of the extracellular subunit interface, coupled to an outward motion of the M2-M3 loop near the channel gate. These movements are highly similar in lipid vesicles and detergent micelles. These reorganizations are essentially completed within 2 ms and occur without channel opening at low proton concentration, indicating that they report a pre-active intermediate state in the transition pathway toward activation. This provides a template to investigate the gating of eukaryotic neurotransmitter receptors, for which intermediate states also participate in activation. DOI: http://dx.doi.org/10.7554/eLife.23955.001 PMID:28294942

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.

A negative charge in transmembrane segment 1 of domain II of the cockroach sodium channel is critical for channel gating and action of pyrethroid insecticides

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

Du Yuzhe; Song Weizhong; Groome, James R.

2010-08-15

Voltage-gated sodium channels are the primary target of pyrethroids, an important class of synthetic insecticides. Pyrethroids bind to a distinct receptor site on sodium channels and prolong the open state by inhibiting channel deactivation and inactivation. Recent studies have begun to reveal sodium channel residues important for pyrethroid binding. However, how pyrethroid binding leads to inhibition of sodium channel deactivation and inactivation remains elusive. In this study, we show that a negatively charged aspartic acid residue at position 802 (D802) located in the extracellular end of transmembrane segment 1 of domain II (IIS1) is critical for both the action ofmore » pyrethroids and the voltage dependence of channel activation. Charge-reversing or -neutralizing substitutions (K, G, or A) of D802 shifted the voltage dependence of activation in the depolarizing direction and reduced channel sensitivity to deltamethrin, a pyrethroid insecticide. The charge-reversing mutation D802K also accelerated open-state deactivation, which may have counteracted the inhibition of sodium channel deactivation by deltamethrin. In contrast, the D802G substitution slowed open-state deactivation, suggesting an additional mechanism for neutralizing the action of deltamethrin. Importantly, Schild analysis showed that D802 is not involved in pyrethroid binding. Thus, we have identified a sodium channel residue that is critical for regulating the action of pyrethroids on the sodium channel without affecting the receptor site of pyrethroids.« less

Characterization of the slow calcium channel binding sites for ( sup 3 H)SR 33557 in rat heart sarcolemmal membranes

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

Chatelain, P.; Beaufort, P.; Meysmans, L.

1991-01-01

SR 33557 represents a new class of compounds (indolizine sulfone) that inhibit L-type Ca2+ channels. ({sup 3}H)SR 33557 has been shown to bind with high affinity (Kd congruent to 0.36 nM, calculated from saturation isotherms and association/dissociation kinetics) to a single class of sites in a purified preparation of rat cardiac sarcolemmal membranes. The binding was found to be saturable and reversible. The maximal binding capacity was in approximately 1:1 stoichiometry with that of other Ca2+ channel antagonists. Various divalent cations (Mg2+, Mn2+, Ca2+, Ba2+, and Cd2+) were shown to inhibit specific ({sup 3}H)SR 33557 binding, with Cd2+ being themore » most potent. Among several receptor or channel ligands (including omega-conotoxin and Na+ and K+ channel modulators), only the L-type Ca2+ channel antagonists were found to displace ({sup 3}H)SR 33557. However, dihydropyridines, phenylalkylamines, benzothiazepines, and diphenylbutylpiperidines were found to inhibit ({sup 3}H)SR 33557 in a noncompetitive manner as demonstrated by displacement and saturation experiments in addition to dissociation kinetics. From these results, we suggest that SR 33557 binds with high affinity to a unique site on the L-type Ca2+ channel found in rat cardiac sarcolemmal membranes.« less

Optical control of neuronal activity using a light-operated GIRK channel opener (LOGO).

PubMed

Barber, David M; Schönberger, Matthias; Burgstaller, Jessica; Levitz, Joshua; Weaver, C David; Isacoff, Ehud Y; Baier, Herwig; Trauner, Dirk

2016-01-01

G-protein coupled inwardly rectifying potassium channels (GIRKs) are ubiquitously expressed throughout the human body and are an integral part of inhibitory signal transduction pathways. Upon binding of G βγ subunits released from G-protein coupled receptors (GPCRs), GIRK channels open and reduce the activity of excitable cells via hyperpolarization. As such, they play a role in cardiac output, the coordination of movement and cognition. Due to their involvement in a multitude of pathways, the precision control of GIRK channels is an important endeavour. Here, we describe the development of the photoswitchable agonist LOGO (the L ight O perated G IRK-channel O pener), which activates GIRK channels in the dark and is rapidly deactivated upon exposure to long wavelength UV irradiation. LOGO is the first K + channel opener and selectively targets channels that contain the GIRK1 subunit. It can be used to optically silence action potential firing in dissociated hippocampal neurons and LOGO exhibits activity in vivo , controlling the motility of zebrafish larvae in a light dependent fashion. We envisage that LOGO will be a valuable research tool to dissect the function of GIRK channels from other GPCR dependent signalling pathways.

Activation of KV7 channels stimulates vasodilatation of human placental chorionic plate arteries.

PubMed

Mills, T A; Greenwood, S L; Devlin, G; Shweikh, Y; Robinson, M; Cowley, E; Hayward, C E; Cottrell, E C; Tropea, T; Brereton, M F; Dalby-Brown, W; Wareing, M

2015-06-01

Potassium (K(+)) channels are key regulators of vascular smooth muscle cell (VSMC) excitability. In systemic small arteries, Kv7 channel expression/activity has been noted and a role in vascular tone regulation demonstrated. We aimed to demonstrate functional Kv7 channels in human fetoplacental small arteries. Human placental chorionic plate arteries (CPAs) were obtained at term. CPA responses to Kv7 channel modulators was determined by wire myography. Presence of Kv7 channel mRNA (encoded by KCNQ1-5) and protein expression were assessed by RT-PCR and immunohistochemistry/immunofluorescence, respectively. Kv7 channel blockade with linopirdine increased CPA basal tone and AVP-induced contraction. Pre-contracted CPAs (AVP; 80 mM K(+) depolarization solution) exhibited significant relaxation to flupirtine, retigabine, the acrylamide (S)-1, and (S) BMS-204352, differential activators of Kv7.1 - Kv7.5 channels. All CPAs assessed expressed KCNQ1 and KCNQ3-5 mRNA; KCNQ2 was expressed only in a subset of CPAs. Kv7 protein expression was confirmed in intact CPAs and isolated VSMCs. Kv7 channels are present and active in fetoplacental vessels, contributing to vascular tone regulation in normal pregnancy. Targeting these channels may represent a therapeutic intervention in pregnancies complicated by increased vascular resistance. Copyright © 2015 Elsevier Ltd. All rights reserved.

Lipid modulation of thermal transient receptor potential channels.

PubMed

Hernández-García, Enrique; Rosenbaum, Tamara

2014-01-01

There is a subgroup of transient receptor potential (TRP) ion channels that are responsive to temperature (thermo-TRP channels). These are important to a variety of sensory and physiological phenomena such as pain and taste perception. All thermo-TRP channels known to date are subject to modulation by lipidic molecules of many kinds, from the ubiquitous cholesterol to more specialized molecules such as prostaglandins. Although the mechanisms and sites of binding of lipids on thermo-TRPs are largely unknown, the explosion on research of lipids and ion channels has revealed previously unsuspected roles for them. Diacyl glycerol is a lipid produced by phospholipase C (PLC) and it was discovered to modulate TRP channels in the eye of the fly, and many mammal TRP channels have been found to interact with lipids. While most of the lipids acting on thermo-TRP channels have been found to activate them, there are a few capable of inhibition. Phosphatidylinositol 4,5-bisphosphate is even capable of both inhibition and activation on a couple of thermo-TRPs, depending on the cellular context. More data is required to assess the mechanism through which lipids affect thermo-TRP channel activity and the physiological importance of this interaction.

Relative transmembrane segment rearrangements during BK channel activation resolved by structurally assigned fluorophore–quencher pairing

PubMed Central

Pantazis, Antonios

2012-01-01

Voltage-activated proteins can sense, and respond to, changes in the electric field pervading the cell membrane by virtue of a transmembrane helix bundle, the voltage-sensing domain (VSD). Canonical VSDs consist of four transmembrane helices (S1–S4) of which S4 is considered a principal component because it possesses charged residues immersed in the electric field. Membrane depolarization compels the charges, and by extension S4, to rearrange with respect to the field. The VSD of large-conductance voltage- and Ca-activated K+ (BK) channels exhibits two salient inconsistencies from the canonical VSD model: (1) the BK channel VSD possesses an additional nonconserved transmembrane helix (S0); and (2) it exhibits a “decentralized” distribution of voltage-sensing charges, in helices S2 and S3, in addition to S4. Considering these unique features, the voltage-dependent rearrangements of the BK VSD could differ significantly from the standard model of VSD operation. To understand the mode of operation of this unique VSD, we have optically tracked the relative motions of the BK VSD transmembrane helices during activation, by manipulating the quenching environment of site-directed fluorescent labels with native and introduced Trp residues. Having previously reported that S0 and S4 diverge during activation, in this work we demonstrate that S4 also diverges from S1 and S2, whereas S2, compelled by its voltage-sensing charged residues, moves closer to S1. This information contributes spatial constraints for understanding the BK channel voltage-sensing process, revealing the structural rearrangements in a non-canonical VSD. PMID:22802360

Characterization, performance modeling, and design of an active capping remediation project in a heavily polluted urban channel.

PubMed

Yin, Ke; Viana, Priscilla; Zhao, Xiuhong; Rockne, Karl

2010-07-15

Collateral Channel is a heavily polluted former navigation slip to the Chicago Sanitary and Ship Canal (Illinois, USA). Characterization of sediment cores taken in the channel show high levels of heavy metals, polycyclic aromatic hydrocarbons (PAHs) and other contaminants in deposited sediment dating back to the 1800's. Of these, PAHs were the contaminants of greatest concern based upon exceedance of sediment contamination criteria (Sigma(16) PAHs up to 1500mg/kg). Benthic animal counts revealed a lack of biodiversity, with relatively low levels of small tubificid oligochaetes (generally <3000/m(2)) in surficial sediments. Comparison of surficial sediment contaminant levels between 1995 and 2005 showed few decreases in contaminant levels, indicating a lack of "natural recovery" processes occurring in the channel. These results led to an analysis of sediment amendments for an active capping demonstration project in the channel using transport models developed in our previous work (Viana et al., 2008). Based on the sediment characterization and modeling results, the active capping design will be focused on organic contaminant sequestration through the use of organoclay. A site-specific difficulty is the substantial rates of gas ebullition from anaerobic organic matter biodegradation in the sediments, particularly in the summer months. These gases can open advective channels that may result in substantial pollution release and compromise cap effectiveness, and thus the capping scenario must control for such releases. The active capping layer will underlay a sloped sand layer and a high permeability gas venting system to allow biogenically-produced gas migration to shoreline collectors through an innovative support grid. The cap will include an overlaying wetland to remove nutrients from the adjoining Chicago River and provide a public recreational space. Copyright 2010 Elsevier B.V. All rights reserved.

Exterior Site Occupancy Infers Chloride-Induced Proton Gating in a Prokaryotic Homolog of the ClC Chloride Channel

PubMed Central

Bostick, David L.; Berkowitz, Max L.

2004-01-01

The ClC family of anion channels mediates the efficient, selective permeation of Cl− across the biological membranes of living cells under the driving force of an electrochemical gradient. In some eukaryotes, these channels are known to exhibit a unique gating mechanism, which appears to be triggered by the permeant Cl− anion. We infer details of this gating mechanism by studying the free energetics of Cl− occupancy in the pore of a prokaryotic ClC homolog. These free energetics were gleaned from 30 ns of molecular dynamics simulation on an ∼133,000-atom system consisting of a hydrated membrane embedded StClC transporter. The binding sites for Cl− in the transporter were determined for the cases where the putative gating residue, Glu148, was protonated and unprotonated. When the glutamate gate is protonated, Cl− favorably occupies an exterior site, Sext, to form a queue of anions in the pore. However, when the glutamate gate is unprotonated, Cl− cannot occupy this site nor, consequently, pass through the pore. An additional, previously undetected, site was found in the pore near the outer membrane that exists regardless of the protonation state of Glu148. Although this suggests that, for the prokaryotic homolog, protonation of Glu148 may be the first step in transporting Cl− at the expense of H+ transport in the opposite direction, an evolutionary argument might suggest that Cl− opens the ClC gate in eukaryotic channels by inducing the conserved glutamate's protonation. During an additional 20 ns free dynamics simulation, the newly discovered outermost site, Sout, and the innermost site, Sint, were seen to allow spontaneous exchange of Cl− ions with the bulk electrolyte while under depolarization conditions. PMID:15345547

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.

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.

Divalent cations potentiate TRPV1 channel by lowering the heat activation threshold

PubMed Central

Cao, Xu; Ma, Linlin; Yang, Fan

2014-01-01

Transient receptor potential vanilloid type 1 (TRPV1) channel responds to a wide spectrum of physical and chemical stimuli. In doing so, it serves as a polymodal cellular sensor for temperature change and pain. Many chemicals are known to strongly potentiate TRPV1 activation, though how this is achieved remains unclear. In this study we investigated the molecular mechanism underlying the gating effects of divalent cations Mg2+ and Ba2+. Using a combination of fluorescence imaging and patch-clamp analysis, we found that these cations potentiate TRPV1 gating by most likely promoting the heat activation process. Mg2+ substantially lowers the activation threshold temperature; as a result, a significant fraction of channels are heat-activated at room temperature. Although Mg2+ also potentiates capsaicin- and voltage-dependent activation, these processes were found either to be not required (in the case of capsaicin) or insufficient (in the case of voltage) to mediate the activating effect. In support of a selective effect on heat activation, Mg2+ and Ba2+ cause a Ca2+-independent desensitization that specifically prevents heat-induced channel activation but does not prevent capsaicin-induced activation. These results can be satisfactorily explained within an allosteric gating framework in which divalent cations strongly promote the heat-dependent conformational change or its coupling to channel activation, which is further coupled to the voltage- and capsaicin-dependent processes. PMID:24344247

Zinc activates damage-sensing TRPA1 ion channels.

PubMed

Hu, Hongzhen; Bandell, Michael; Petrus, Matt J; Zhu, Michael X; Patapoutian, Ardem

2009-03-01

Zinc is an essential biological trace element. It is required for the structure or function of over 300 proteins, and it is increasingly recognized for its role in cell signaling. However, high concentrations of zinc have cytotoxic effects, and overexposure to zinc can cause pain and inflammation through unknown mechanisms. Here we show that zinc excites nociceptive somatosensory neurons and causes nociception in mice through TRPA1, a cation channel previously shown to mediate the pungency of wasabi and cinnamon through cysteine modification. Zinc activates TRPA1 through a unique mechanism that requires zinc influx through TRPA1 channels and subsequent activation via specific intracellular cysteine and histidine residues. TRPA1 is highly sensitive to intracellular zinc, as low nanomolar concentrations activate TRPA1 and modulate its sensitivity. These findings identify TRPA1 as an important target for the sensory effects of zinc and support an emerging role for zinc as a signaling molecule that can modulate sensory transmission.

On the mechanism of TBA block of the TRPV1 channel.

PubMed

Oseguera, Andrés Jara; Islas, León D; García-Villegas, Refugio; Rosenbaum, Tamara

2007-06-01

The transient receptor potential vanilloid 1 (TRPV1) channel is a nonselective cation channel activated by capsaicin and responsible for thermosensation. To date, little is known about the gating characteristics of these channels. Here we used tetrabutylammonium (TBA) to determine whether this molecule behaves as an ion conduction blocker in TRPV1 channels and to gain insight into the nature of the activation gate of this protein. TBA belongs to a family of classic potassium channel blockers that have been widely used as tools for determining the localization of the activation gate and the properties of the pore of several ion channels. We found TBA to be a voltage-dependent pore blocker and that the properties of block are consistent with an open-state blocker, with the TBA molecule binding to multiple open states, each with different blocker affinities. Kinetics of channel closure and burst-length analysis in the presence of blocker are consistent with a state-dependent blocking mechanism, with TBA interfering with closing of an activation gate. This activation gate may be located cytoplasmically with respect to the binding site of TBA ions, similar to what has been observed in potassium channels. We propose an allosteric model for TRPV1 activation and block by TBA, which explains our experimental data.

Equalization filters for multiple-channel electromyogram arrays

PubMed Central

Clancy, Edward A.; Xia, Hongfang; Christie, Anita; Kamen, Gary

2007-01-01

Multiple channels of electromyogram activity are frequently transduced via electrodes, then combined electronically to form one electrophysiologic recording, e.g. bipolar, linear double difference and Laplacian montages. For high quality recordings, precise gain and frequency response matching of the individual electrode potentials is achieved in hardware (e.g., an instrumentation amplifier for bipolar recordings). This technique works well when the number of derived signals is small and the montages are pre-determined. However, for array electrodes employing a variety of montages, hardware channel matching can be expensive and tedious, and limits the number of derived signals monitored. This report describes a method for channel matching based on the concept of equalization filters. Monopolar potentials are recorded from each site without precise hardware matching. During a calibration phase, a time-varying linear chirp voltage is applied simultaneously to each site and recorded. Based on the calibration recording, each monopolar channel is digitally filtered to “correct” for (equalize) differences in the individual channels, and then any derived montages subsequently created. In a hardware demonstration system, the common mode rejection ratio (at 60 Hz) of bipolar montages improved from 35.2 ± 5.0 dB (prior to channel equalization) to 69.0 ± 5.0 dB (after equalization). PMID:17614134

Entrapment of metal clusters in metal-organic framework channels by extended hooks anchored at open metal sites.

PubMed

Zheng, Shou-Tian; Zhao, Xiang; Lau, Samuel; Fuhr, Addis; Feng, Pingyun; Bu, Xianhui

2013-07-17

Reported here are the new concept of utilizing open metal sites (OMSs) for architectural pore design and its practical implementation. Specifically, it is shown here that OMSs can be used to run extended hooks (isonicotinates in this work) from the framework walls to the channel centers to effect the capture of single metal ions or clusters, with the concurrent partitioning of the large channel spaces into multiple domains, alteration of the host-guest charge relationship and associated guest-exchange properties, and transfer of OMSs from the walls to the channel centers. The concept of the extended hook, demonstrated here in the multicomponent dual-metal and dual-ligand system, should be generally applicable to a range of framework types.

Nerve membrane ion channels as the target site of environmental toxicants

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

Narahashi, T.

1987-04-01

There are many environmentally important chemicals which exhibit potent effects on the nervous system. Since nerve excitation takes place in a fraction of a second, electrophysiological methods provide the authors with the most straightforward approach to the study of the mechanisms of action of environmental toxicants on the nervous system. Aquatic animals such as crayfish, lobster, squid, and marine snails represent extremely useful materials for such electrophysiological studies, because much of the authors knowledge of nerve excitation is derived from those animals. Nerve excitation takes place as a result of opening and closing of ion channels of the membrane. Thesemore » functions are independent of metabolic energy, and can be measured most effectively by voltage clamp techniques as applied to the giant axons of the crayfish and the squid. Patch clamp techniques developed during the past 10 years have added a new dimension to the electrophysiological investigation. These techniques allow them to measure the activity of individual ion channels, thereby making it possible to analyze the interaction of toxic molecules directly with single ion channels. Examples are given summarizing electrophysiological studies of environmental neurotoxicants. The abdominal nerve cords and neuromuscular preparations isolated from the crayfish are convenient materials for bioassay of certain environmental toxicants such as pyrethroids, chlorinated hydrocarbons, and other insecticides. Only a small fraction of the flux through the sodium channel, less than 1%, must be modified by pyrethroids for the animal to develop symptoms of poisoning. Such a toxicological application from channel to animal is important is understanding the potent toxic effect.« less

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

HCN Channels Modulators: The Need for Selectivity

PubMed Central

Romanelli, Maria Novella; Sartiani, Laura; Masi, Alessio; Mannaioni, Guido; Manetti, Dina; Mugelli, Alessandro; Cerbai, Elisabetta

2016-01-01

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, the molecular correlate of the hyperpolarization-activated current (If/Ih), are membrane proteins which play an important role in several physiological processes and various pathological conditions. In the Sino Atrial Node (SAN) HCN4 is the target of ivabradine, a bradycardic agent that is, at the moment, the only drug which specifically blocks If. Nevertheless, several other pharmacological agents have been shown to modulate HCN channels, a property that may contribute to their therapeutic activity and/or to their side effects. HCN channels are considered potential targets for developing drugs to treat several important pathologies, but a major issue in this field is the discovery of isoform-selective compounds, owing to the wide distribution of these proteins into the central and peripheral nervous systems, heart and other peripheral tissues. This survey is focused on the compounds that have been shown, or have been designed, to interact with HCN channels and on their binding sites, with the aim to summarize current knowledge and possibly to unveil useful information to design new potent and selective modulators. PMID:26975509

Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening.

PubMed

Zaydman, Mark A; Silva, Jonathan R; Delaloye, Kelli; Li, Yang; Liang, Hongwu; Larsson, H Peter; Shi, Jingyi; Cui, Jianmin

2013-08-06

Voltage-gated ion channels generate dynamic ionic currents that are vital to the physiological functions of many tissues. These proteins contain separate voltage-sensing domains, which detect changes in transmembrane voltage, and pore domains, which conduct ions. Coupling of voltage sensing and pore opening is critical to the channel function and has been modeled as a protein-protein interaction between the two domains. Here, we show that coupling in Kv7.1 channels requires the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). We found that voltage-sensing domain activation failed to open the pore in the absence of PIP2. This result is due to loss of coupling because PIP2 was also required for pore opening to affect voltage-sensing domain activation. We identified a critical site for PIP2-dependent coupling at the interface between the voltage-sensing domain and the pore domain. This site is actually a conserved lipid-binding site among different K(+) channels, suggesting that lipids play an important role in coupling in many ion channels.

Hydroclimatic signal and LBK cultural activity in the Upper and Lower Rhine, inferred from abandoned channel fill deposits

NASA Astrophysics Data System (ADS)

Berger, J. F.; Salvador, P. G.; Erkens, G.; Toonen, W. H. J.; Purdue, L.; Barra, A.; Houben, P.

2012-04-01

The Linear Band Ceramic (LBK) culture represents a major event in the spread of agriculture in Europe. Occupation particularly occurred in river valleys, with largest densities found along the rivers Danube, Elbe and Rhine. The interaction between the emergence of this culture and the dominant climatic and hydrological conditions is not yet fully established. As part of the ANR OBRESOC project, in which LBK activity is investigated in a transect from France (Marne river) to the catchment of the Danube river (Tisza), we studied palaeo-environmental changes in the Rhine valley between 7600-6600 cal. yrs. BP. Focus is on the Upper Rhine Graben and the Lower Rhine valley near the Rhine Delta apex, which is thought to be a peripheral region of LBK-activity. In these regions, a total of five cores from abandoned channels were analysed to reconstruct palaeo-environmental dynamics in vegetation and fluvial activity during the period of LBK development. Abandoned channel fills are excellent sites to perform detailed studies of palaeo-environmental dynamics, as they (i) form proximal locations to occupation sites of the LBK culture, (ii) act as efficient traps of sediments in which different environmental proxies are well preserved, (iii) contain well-datable material for the construction of detailed age-depth models, and (iv) provide a long proxy record, potentially over more than a millennium at a single site. On all cores, high resolution analysis of channel fill deposits (grain size and geophysical properties) and biotic proxies (micro-charcoal fluxes and pollen assemblages) were preformed to reconstruct palaeo-environmental signals, such as changes in fluvial activity, forest fires, and vegetation evolution, which may be related to agricultural activity, and climatic and hydrogeomorphic changes in the region. In this contribution we compare the results of the high-resolution core analyses (1,5 to 5m sequences for the studied timeframe) derived from the more densely

Desensitization of chemical activation by auxiliary subunits: convergence of molecular determinants critical for augmenting KCNQ1 potassium channels.

PubMed

Gao, Zhaobing; Xiong, Qiaojie; Sun, Haiyan; Li, Min

2008-08-15

Chemical openers for KCNQ potassium channels are useful probes both for understanding channel gating and for developing therapeutics. The five KCNQ isoforms (KCNQ1 to KCNQ5, or Kv7.1 to Kv7.5) are differentially localized. Therefore, the molecular specificity of chemical openers is an important subject of investigation. Native KCNQ1 normally exists in complex with auxiliary subunits known as KCNE. In cardiac myocytes, the KCNQ1-KCNE1 (IsK or minK) channel is thought to underlie the I(Ks) current, a component critical for membrane repolarization during cardiac action potential. Hence, the molecular and pharmacological differences between KCNQ1 and KCNQ1-KCNE1 channels have been important topics. Zinc pyrithione (ZnPy) is a newly identified KCNQ channel opener, which potently activates KCNQ2, KCNQ4, and KCNQ5. However, the ZnPy effects on cardiac KCNQ1 potassium channels remain largely unknown. Here we show that ZnPy effectively augments the KCNQ1 current, exhibiting an increase in current amplitude, reduction of inactivation, and slowing of both activation and deactivation. Some of these are reminiscent of effects by KCNE1. In addition, neither the heteromultimeric KCNQ1-KCNE1 channels nor native I(Ks) current displayed any sensitivity to ZnPy, indicating that the static occupancy by a KCNE subunit desensitizes the reversible effects by a chemical opener. Site-directed mutagenesis of KCNQ1 reveals that residues critical for the potentiation effects by either ZnPy or KCNE are clustered together in the S6 region overlapping with the critical gating determinants. Thus, the convergence of potentiation effects and molecular determinants critical for both an auxiliary subunit and a chemical opener argue for a mechanistic overlap in causing potentiation.

Pungent products from garlic activate the sensory ion channel TRPA1

PubMed Central

Bautista, Diana M.; Movahed, Pouya; Hinman, Andrew; Axelsson, Helena E.; Sterner, Olov; Högestätt, Edward D.; Julius, David; Jordt, Sven-Eric; Zygmunt, Peter M.

2005-01-01

Garlic belongs to the Allium family of plants that produce organosulfur compounds, such as allicin and diallyl disulfide (DADS), which account for their pungency and spicy aroma. Many health benefits have been ascribed to Allium extracts, including hypotensive and vasorelaxant activities. However, the molecular mechanisms underlying these effects remain unknown. Intriguingly, allicin and DADS share structural similarities with allyl isothiocyanate, the pungent ingredient in wasabi and other mustard plants that induces pain and inflammation by activating TRPA1, an excitatory ion channel on primary sensory neurons of the pain pathway. Here we show that allicin and DADS excite an allyl isothiocyanate-sensitive subpopulation of sensory neurons and induce vasodilation by activating capsaicin-sensitive perivascular sensory nerve endings. Moreover, allicin and DADS activate the cloned TRPA1 channel when expressed in heterologous systems. These and other results suggest that garlic excites sensory neurons primarily through activation of TRPA1. Thus different plant genera, including Allium and Brassica, have developed evolutionary convergent strategies that target TRPA1 channels on sensory nerve endings to achieve chemical deterrence. PMID:16103371

Normal Modes Expose Active Sites in Enzymes.

PubMed

Glantz-Gashai, Yitav; Meirson, Tomer; Samson, Abraham O

2016-12-01

Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes.

Normal Modes Expose Active Sites in Enzymes

PubMed Central

Glantz-Gashai, Yitav; Samson, Abraham O.

2016-01-01

Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

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

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

Clinical Trial of the Potassium Channel Activator Diazoxide for Major Depressive Disorder Halted Due to Intolerability.

PubMed

Kadriu, Bashkim; Yuan, Shiwen; Farmer, Cristan; Nugent, Allison C; Lener, Marc S; Niciu, Mark J; Park, Minkyung; Yazdian, Aaron; Ballard, Elizabeth D; Henn, Fritz A; Henter, Ioline D; Park, Lawrence T; Zarate, Carlos A

2018-06-01

Some glutamatergic modulators have demonstrated rapid and relatively sustained antidepressant properties in patients with major depressive disorder. Because the potassium channel activator diazoxide increases glutamate uptake via potassium channel activation, we hypothesized that it might exert antidepressant effects by increasing the removal of glutamate from the synaptic cleft, thereby reducing excessive glutamate transmission. This randomized, double-blind, placebo-controlled, crossover, single-site inpatient clinical study was conducted at the National Institute of Mental Health to assess the efficacy and safety of a 3-week course of diazoxide (200-400 mg daily, twice a day) versus a 3-week course of placebo in 6 participants with treatment-refractory major depressive disorder. The primary clinical outcome measure was change in Montgomery-Asberg Depression Rating Scale score from baseline to posttreatment. Quantitative insulin sensitivity check index, as well as concomitant imaging measures (electroencephalography, proton magnetic resonance spectroscopy, magnetoencephalography), were used as potential surrogate markers of target (KATP channel) engagement. The study was halted due to severe adverse effects. Given the small sample size, statistical evaluation of the effect of diazoxide on Montgomery-Asberg Depression Rating Scale scores or the imaging measures was not pursued. Visual inspection of the quantitative insulin sensitivity check index test revealed no evidence of target engagement. Although the results are negative, they are an important addition to the literature in this rapidly changing field.

Use of Small Fluorescent Molecules to Monitor Channel Activity

NASA Astrophysics Data System (ADS)

Jones, Sharon; Stringer, Sarah; Naik, Rajesh; Stone, Morley

2001-03-01

The Mechanosensitive channel of Large conductance (MscL) allows bacteria to rapidly adapt to changing environmental conditions such as osmolarity. The MscL channel opens in response to increases in membrane tension, which allows for the efflux of cytoplasmic constituents. Here we describe the cloning and expression of Salmonella typhimurium MscL (St-MscL). Using a fluorescence efflux assay, we demonstrate that efflux through the MscL channel during hypoosmotic shock can be monitored using endogenously produced fluorophores. In addition, we observe that thermal stimulation, i.e., heat shock, can also induce efflux through MscL. We present the first evidence of thermal activation of MscL efflux by heat shocking cells expressing the S. typhimurium protein variant. This finding has significant biosensor implications, especially for investigators exploring the use of channel proteins in biosensor applications. Thermal biosensors are relatively unexplored, but would have considerable commercial and military utility.

Calcium-Activated Cl- Channel: Insights on the Molecular Identity in Epithelial Tissues.

PubMed

Rottgen, Trey S; Nickerson, Andrew J; Rajendran, Vazhaikkurichi M

2018-05-10

Calcium-activated chloride secretion in epithelial tissues has been described for many years. However, the molecular identity of the channel responsible for the Ca 2+ -activated Cl − secretion in epithelial tissues has remained a mystery. More recently, TMEM16A has been identified as a new putative Ca 2+ -activated Cl − channel (CaCC). The primary goal of this article will be to review the characterization of TMEM16A, as it relates to the physical structure of the channel, as well as important residues that confer voltage and Ca 2+ -sensitivity of the channel. This review will also discuss the role of TMEM16A in epithelial physiology and potential associated-pathophysiology. This will include discussion of developed knockout models that have provided much needed insight on the functional localization of TMEM16A in several epithelial tissues. Finally, this review will examine the implications of the identification of TMEM16A as it pertains to potential novel therapies in several pathologies.

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

Novel 384-well population patch clamp electrophysiology assays for Ca2+-activated K+ channels.

PubMed

John, Victoria H; Dale, Tim J; Hollands, Emma C; Chen, Mao Xiang; Partington, Leanne; Downie, David L; Meadows, Helen J; Trezise, Derek J

2007-02-01

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.

Activation of the mechanosensitive ion channel MscL by mechanical stimulation of supported Droplet-Hydrogel bilayers

PubMed Central

Rosholm, Kadla R.; Baker, Matthew A. B.; Ridone, Pietro; Nakayama, Yoshitaka; Rohde, Paul R.; Cuello, Luis G.; Lee, Lawrence K.; Martinac, Boris

2017-01-01

The droplet on hydrogel bilayer (DHB) is a novel platform for investigating the function of ion channels. Advantages of this setup include tight control of all bilayer components, which is compelling for the investigation of mechanosensitive (MS) ion channels, since they are highly sensitive to their lipid environment. However, the activation of MS ion channels in planar supported lipid bilayers, such as the DHB, has not yet been established. Here we present the activation of the large conductance MS channel of E. coli, (MscL), in DHBs. By selectively stretching the droplet monolayer with nanolitre injections of buffer, we induced quantifiable DHB tension, which could be related to channel activity. The MscL activity response revealed that the droplet monolayer tension equilibrated over time, likely by insertion of lipid from solution. Our study thus establishes a method to controllably activate MS channels in DHBs and thereby advances studies of MS channels in this novel platform. PMID:28345591

Rational design and validation of a vanilloid-sensitive TRPV2 ion channel.

PubMed

Yang, Fan; Vu, Simon; Yarov-Yarovoy, Vladimir; Zheng, Jie

2016-06-28

Vanilloids activation of TRPV1 represents an excellent model system of ligand-gated ion channels. Recent studies using cryo-electron microcopy (cryo-EM), computational analysis, and functional quantification revealed the location of capsaicin-binding site and critical residues mediating ligand-binding and channel activation. Based on these new findings, here we have successfully introduced high-affinity binding of capsaicin and resiniferatoxin to the vanilloid-insensitive TRPV2 channel, using a rationally designed minimal set of four point mutations (F467S-S498F-L505T-Q525E, termed TRPV2_Quad). We found that binding of resiniferatoxin activates TRPV2_Quad but the ligand-induced open state is relatively unstable, whereas binding of capsaicin to TRPV2_Quad antagonizes resiniferatoxin-induced activation likely through competition for the same binding sites. Using Rosetta-based molecular docking, we observed a common structural mechanism underlying vanilloids activation of TRPV1 and TRPV2_Quad, where the ligand serves as molecular "glue" that bridges the S4-S5 linker to the S1-S4 domain to open these channels. Our analysis revealed that capsaicin failed to activate TRPV2_Quad likely due to structural constraints preventing such bridge formation. These results not only validate our current working model for capsaicin activation of TRPV1 but also should help guide the design of drug candidate compounds for this important pain sensor.

Pathfinder Landing Site: Alternatives to Catastrophic Floods and An Antarctic Ice-Flow Analog for Outflow Channels on Mars

NASA Technical Reports Server (NTRS)

Lucchitta, B. K.

1998-01-01

The Pathfinder spacecraft landed successfully at the mouth of the outflow channels Ares and Tiu Valles, returning a wealth of information about the surrounding landscape. One goal of the mission was to ascertain that catastrophic floods formed the outflow channels, the prevailing hypothesis for their origin. The follow-up reports on the mission proclaim that observations are "consistent" with an origin by catastrophic flood; no alternative mechanisms for channel origin are considered. Thus, the impression is given that the problem of channel origin has been solved. Yet none of the observations are diagnostic of origin by catastrophic floods. Other origins are possible but have been ignored, for instance origin as liquefaction mudflows, debris flows, mass flows, or ice flows. Here I will examine landing site observations that have been used to infer origin by catastrophic flooding and suggest alternative origins. Finally, I will highlight some new observation from Antarctica that make an ice-flow mechanism plausible for the origin of some of the outflow channels.

Tonantzitlolone is a Nanomolar Potency Activator of TRPC1/4/5 Channels.

PubMed

Rubaiy, Hussein N; Ludlow, Melanie J; Siems, Karsten; Norman, Katherine; Foster, Richard; Wolf, Dietmar; Beutler, John A; Beech, David J

2018-06-02

The diterpene ester tonantzitlonone (TZL) is a natural product which displays cytotoxicity towards certain types of cancer cell such as renal cell carcinoma cells. The effect is similar to that of (-)-Englerin A (EA) and so, although it is chemically distinct, we investigated whether TZL also targets transient receptor potential canonical (TRPC) channels of the TRPC1, TRPC4 and TRPC5 type (TRPC1/4/5 channels). Renal cell carcinoma A498 cells natively expressing TRPC1 and TRPC4, modified HEK 293 cells over expressing TRPC4, TRPC5, TRPC4-TRPC1 or TRPC5-TRPC1 concatemer, TRPC3 or TRPM2 or CHO cells over expressing TRPV4 were studied by intracellular Ca 2+ measurement or whole-cell or excised membrane patch-clamp electrophysiology. TZL evoked intracellular Ca 2+ elevation in A498 cells, similar to that evoked by EA. TZL activated overexpressed channels with concentration for 50% activation (EC 50 ) at 123 nM (TRPC4), 83 nM (TRPC5), 140 nM (TRPC4-TRPC1) and 61 nM (TRPC5-TRPC1). Effects of TZL were reversible on wash-out and potently inhibited by the TRPC1/4/5 inhibitor Pico145. TZL activated TRPC5 channels when bath-applied to excised outside-out but not inside-out patches. TZL failed to activate endogenous store-operated Ca 2+ entry in HEK 293 cells or overexpressed TRPC3, TRPV4 or TRPM2 channels. TZL is a novel potent agonist for TRPC1/4/5 channels which should be useful for testing the functionality of this type of ion channel and understanding how TRPC1/4/5 agonists achieve selective cytotoxicity against certain types of cancer cell. This article is protected by copyright. All rights reserved.

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

Quantitative autoradiography of the binding sites for ( sup 125 I) iodoglyburide, a novel high-affinity ligand for ATP-sensitive potassium channels in rat brain

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

Gehlert, D.R.; Gackenheimer, S.L.; Mais, D.E.

1991-05-01

We have developed a high specific activity ligand for localization of ATP-sensitive potassium channels in the brain. When brain sections were incubated with ({sup 125}I)iodoglyburide (N-(2-((((cyclohexylamino)carbonyl)amino)sulfonyl)ethyl)-5-{sup 125}I-2- methoxybenzamide), the ligand bound to a single site with a KD of 495 pM and a maximum binding site density of 176 fmol/mg of tissue. Glyburide was the most potent inhibitor of specific ({sup 125}I)iodoglyburide binding to rat forebrain sections whereas iodoglyburide and glipizide were slightly less potent. The binding was also sensitive to ATP which completely inhibited binding at concentrations of 10 mM. Autoradiographic localization of ({sup 125}I)iodoglyburide binding indicated a broadmore » distribution of the ATP-sensitive potassium channel in the brain. The highest levels of binding were seen in the globus pallidus and ventral pallidum followed by the septohippocampal nucleus, anterior pituitary, the CA2 and CA3 region of the hippocampus, ventral pallidum, the molecular layer of the cerebellum and substantia nigra zona reticulata. The hilus and dorsal subiculum of the hippocampus, molecular layer of the dentate gyrus, cerebral cortex, lateral olfactory tract nucleus, olfactory tubercle and the zona incerta contained relatively high levels of binding. A lower level of binding (approximately 3- to 4-fold) was found throughout the remainder of the brain. These results indicate that the ATP-sensitive potassium channel has a broad presence in the rat brain and that a few select brain regions are enriched in this subtype of neuronal potassium channels.« less

Mechanism of activation at the selectivity filter of the KcsA K+ channel

PubMed Central

Heer, Florian T; Posson, David J; Wojtas-Niziurski, Wojciech

2017-01-01

Potassium channels are opened by ligands and/or membrane potential. In voltage-gated K+ channels and the prokaryotic KcsA channel, conduction is believed to result from opening of an intracellular constriction that prevents ion entry into the pore. On the other hand, numerous ligand-gated K+ channels lack such gate, suggesting that they may be activated by a change within the selectivity filter, a narrow region at the extracellular side of the pore. Using molecular dynamics simulations and electrophysiology measurements, we show that ligand-induced conformational changes in the KcsA channel removes steric restraints at the selectivity filter, thus resulting in structural fluctuations, reduced K+ affinity, and increased ion permeation. Such activation of the selectivity filter may be a universal gating mechanism within K+ channels. The occlusion of the pore at the level of the intracellular gate appears to be secondary. PMID:28994652

Characteristics of camel-gate structures with active doping channel profiles

NASA Astrophysics Data System (ADS)

Tsai, Jung-Hui; Lour, Wen-Shiung; Laih, Lih-Wen; Liu, Rong-Chau; Liu, Wen-Chau

1996-03-01

In this paper, we demonstrate the influence of channel doping profile on the performances of camel-gate field effect transistors (CAMFETs). For comparison, single and tri-step doping channel structures with identical doping thickness products are employed, while other parameters are kept unchanged. The results of a theoretical analysis show that the single doping channel FET with lightly doping active layer has higher barrier height and drain-source saturation current. However, the transconductance is decreased. For a tri-step doping channel structure, it is found that the output drain-source saturation current and the barrier height are enhanced. Furthermore, the relatively voltage independent performances are improved. Two CAMFETs with single and tri-step doping channel structures have been fabricated and discussed. The devices exhibit nearly voltage independent transconductances of 144 mS mm -1 and 222 mS mm -1 for single and tri-step doping channel CAMFETs, respectively. The operation gate voltage may extend to ± 1.5 V for a tri-step doping channel CAMFET. In addition, the drain current densities of > 750 and 405 mA mm -1 are obtained for the tri-step and single doping CAMFETs. These experimental results are inconsistent with theoretical analysis.

Differential Activation of Vascular Smooth Muscle Kv7.4, Kv7.5, and Kv7.4/7.5 Channels by ML213 and ICA-069673

PubMed Central

Brueggemann, Lyubov I.; Haick, Jennifer M.; Cribbs, Leanne L.

2014-01-01

Recent research suggests that smooth muscle cells express Kv7.4 and Kv7.5 voltage-activated potassium channels, which contribute to maintenance of their resting membrane voltage. New pharmacologic activators of Kv7 channels, ML213 (N-mesitybicyclo[2.2.1]heptane-2-carboxamide) and ICA-069673 N-(6-chloropyridin-3-yl)-3,4-difluorobenzamide), have been reported to discriminate among channels formed from different Kv7 subtypes. We compared the effects of ML213 and ICA-069673 on homomeric human Kv7.4, Kv7.5, and heteromeric Kv7.4/7.5 channels exogenously expressed in A7r5 vascular smooth muscle cells. We found that, despite its previous description as a selective activator of Kv7.2 and Kv7.4, ML213 significantly increased the maximum conductance of homomeric Kv7.4 and Kv7.5, as well as heteromeric Kv7.4/7.5 channels, and induced a negative shift of their activation curves. Current deactivation rates decreased in the presence of the ML213 (10 μM) for all three channel combinations. Mutants of Kv7.4 (W242L) and Kv7.5 (W235L), previously found to be insensitive to another Kv7 channel activator, retigabine, were also insensitive to ML213 (10 μM). In contrast to ML213, ICA-069673 robustly activated Kv7.4 channels but was significantly less effective on homomeric Kv7.5 channels. Heteromeric Kv7.4/7.5 channels displayed intermediate responses to ICA-069673. In each case, ICA-069673 induced a negative shift of the activation curves without significantly increasing maximal conductance. Current deactivation rates decreased in the presence of ICA-069673 in a subunit-specific manner. Kv7.4 W242L responded to ICA-069673-like wild-type Kv7.4, but a Kv7.4 F143A mutant was much less sensitive to ICA-069673. Based on these results, ML213 and ICA-069673 likely bind to different sites and are differentially selective among Kv7.4, Kv7.5, and Kv7.4/7.5 channel subtypes. PMID:24944189

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.

Recent (circa 1998 to 2011) channel-migration rates of selected streams in Indiana

USGS Publications Warehouse

Robinson, Bret A.

2013-01-01

An investigation was completed to document recent (circa 1998 to 2011) channel-migration rates at 970 meander bends along 38 of the largest streams in Indiana. Data collection was completed by using the Google Earth™ platform and, for each selected site, identifying two images with capture dates separated by multiple years. Within each image, the position of the meander-bend cutbank was measured relative to a fixed local landscape feature visible in both images, and an average channel-migration rate was calculated at the point of maximum cutbank displacement. From these data it was determined that 65 percent of the measured sites have recently been migrating at a rate less than 1 ft/yr, 75 percent of the sites have been migrating at a rate less than 10 ft/yr, and while some sites are migrating in excess of 20 ft/yr, these occurrences are rare. In addition, it is shown that recent channel-migration activity is not evenly distributed across Indiana. For the stream reaches studied, far northern and much of far southern Indiana are drained by streams that recently have been relatively stationary. At the same time, this study shows that most of the largest streams in west-central Indiana and many of the largest streams in east-central Indiana have shown significant channel-migration activity during the recent past. It is anticipated that these results will support several fluvial-erosion-hazard mitigation activities currently being undertaken in Indiana.

Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel

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

Bera, Asim K.; Aukema, Kelly G.; Elias, Mikael

Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas themore » two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel.« less

Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics*

PubMed Central

Lukacs, Peter; Gawali, Vaibhavkumar S.; Cervenka, Rene; Ke, Song; Koenig, Xaver; Rubi, Lena; Zarrabi, Touran; Hilber, Karlheinz; Stary-Weinzinger, Anna; Todt, Hannes

2014-01-01

Despite the availability of several crystal structures of bacterial voltage-gated Na+ channels, the structure of eukaryotic Na+ channels is still undefined. We used predictions from available homology models and crystal structures to modulate an external access pathway for the membrane-impermeant local anesthetic derivative QX-222 into the internal vestibule of the mammalian rNaV1.4 channel. Potassium channel-based homology models predict amino acid Ile-1575 in domain IV segment 6 to be in close proximity to Lys-1237 of the domain III pore-loop selectivity filter. The mutation K1237E has been shown previously to increase the diameter of the selectivity filter. We found that an access pathway for external QX-222 created by mutations of Ile-1575 was abolished by the additional mutation K1237E, supporting the notion of a close spatial relationship between sites 1237 and 1575. Crystal structures of bacterial voltage-gated Na+ channels predict that the side chain of rNaV1.4 Trp-1531 of the domain IV pore-loop projects into the space between domain IV segment 6 and domain III pore-loop and, therefore, should obstruct the putative external access pathway. Indeed, mutations W1531A and W1531G allowed for exceptionally rapid access of QX-222. In addition, W1531G created a second non-selective ion-conducting pore, bypassing the outer vestibule but probably merging into the internal vestibule, allowing for control by the activation gate. These data suggest a strong structural similarity between bacterial and eukaryotic voltage-gated Na+ channels. PMID:24947510

Regulation of Chloride Channels by Protein Kinase C in Normal and Cystic Fibrosis Airway Epithelia

NASA Astrophysics Data System (ADS)

Li, Ming; McCann, John D.; Anderson, Matthew P.; Clancy, John P.; Liedtke, Carole M.; Nairn, Angus C.; Greengard, Paul; Welsh, Michael J.

1989-06-01

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.

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 New Splice Variant of Large Conductance Ca2+-activated K+ (BK) Channel α Subunit Alters Human Chondrocyte Function.

PubMed

Suzuki, Yoshiaki; Ohya, Susumu; Yamamura, Hisao; Giles, Wayne R; Imaizumi, Yuji

2016-11-11

Large conductance Ca 2+ -activated K + (BK) channels play essential roles in both excitable and non-excitable cells. For example, in chondrocytes, agonist-induced Ca 2+ release from intracellular store activates BK channels, and this hyperpolarizes these cells, augments Ca 2+ entry, and forms a positive feed-back mechanism for Ca 2+ signaling and stimulation-secretion coupling. In the present study, functional roles of a newly identified splice variant in the BK channel α subunit (BKαΔe2) were examined in a human chondrocyte cell line, OUMS-27, and in a HEK293 expression system. Although BKαΔe2 lacks exon2, which codes the intracellular S0-S1 linker (Glu-127-Leu-180), significant expression was detected in several tissues from humans and mice. Molecular image analyses revealed that BKαΔe2 channels are not expressed on plasma membrane but can traffic to the plasma membrane after forming hetero-tetramer units with wild-type BKα (BKαWT). Single-channel current analyses demonstrated that BKα hetero-tetramers containing one, two, or three BKαΔe2 subunits are functional. These hetero-tetramers have a smaller single channel conductance and exhibit lower trafficking efficiency than BKαWT homo-tetramers in a stoichiometry-dependent manner. Site-directed mutagenesis of residues in exon2 identified Helix2 and the linker to S1 (Trp-158-Leu-180, particularly Arg-178) as an essential segment for channel function including voltage dependence and trafficking. BKαΔe2 knockdown in OUMS-27 chondrocytes increased BK current density and augmented the responsiveness to histamine assayed as cyclooxygenase-2 gene expression. These findings provide significant new evidence that BKαΔe2 can modulate cellular responses to physiological stimuli in human chondrocyte and contribute under pathophysiological conditions, such as osteoarthritis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Dissection of the components for PIP2 activation and thermosensation in TRP channels

PubMed Central

Brauchi, Sebastian; Orta, Gerardo; Mascayano, Carolina; Salazar, Marcelo; Raddatz, Natalia; Urbina, Hector; Rosenmann, Eduardo; Gonzalez-Nilo, Fernando; Latorre, Ramon

2007-01-01

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in the activation of several transient receptor potential (TRP) channels. The role of PIP2 on temperature gating of thermoTRP channels has not been explored in detail, and the process of temperature activation is largely unexplained. In this work, we have exchanged different segments of the C-terminal region between cold-sensitive (TRPM8) and heat-sensitive (TRPV1) channels, trying to understand the role of the segment in PIP2 and temperature activation. A chimera in which the proximal part of the C-terminal of TRPV1 replaces an equivalent section of TRPM8 C-terminal is activated by PIP2 and confers the phenotype of heat activation. PIP2, but not temperature sensitivity, disappears when positively charged residues contained in the exchanged region are neutralized. Shortening the exchanged segment to a length of 11 aa produces voltage-dependent and temperature-insensitive channels. Our findings suggest the existence of different activation domains for temperature, PIP2, and voltage. We provide an interpretation for channel–PIP2 interaction using a full-atom molecular model of TRPV1 and PIP2 docking analysis. PMID:17548815

Design, synthesis and structure-activity relationship of indoxacarb analogs as voltage-gated sodium channel blocker.

PubMed

Hao, Wenbo; Fu, Chunling; Yu, Huijuan; Chen, Jian; Xu, Hanhong; Shao, Guang; Jiang, Dingxin

2015-10-15

Indoxacarb, the first commercialized pyrazoline-type sodium-channel blocker, is a commonly used insecticide because of high selectivity. To discover sodium-channel blocker with high insecticidal activity, a series of novel indoxacarb analogs were designed and synthesized by judicious structural modifications of the substituent group of C5, C6 in indenone and C'4 in benzene ring. Some analogs exhibited significant insecticidal activities against Spodoptera litura F. and excellent BgNav1-1a channel inhibitory activity. The structure-activity analysis indicated that the presence of strong electron-withdrawing group and decreased steric hindrance of indenone ring (R(1), R(2)) in 5- and 6-position could enhance larvicidal activity and BgNav1-1a channel inhibitory activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

Structure of the skeletal muscle calcium release channel activated with Ca2+ and AMP-PCP.

PubMed Central

Serysheva, I I; Schatz, M; van Heel, M; Chiu, W; Hamilton, S L

1999-01-01

The functional state of the skeletal muscle Ca2+ release channel is modulated by a number of endogenous molecules during excitation-contraction. Using electron cryomicroscopy and angular reconstitution techniques, we determined the three-dimensional (3D) structure of the skeletal muscle Ca2+ release channel activated by a nonhydrolyzable analog of ATP in the presence of Ca2+. These ligands together produce almost maximum activation of the channel and drive the channel population toward a predominately open state. The resulting 30-A 3D reconstruction reveals long-range conformational changes in the cytoplasmic region that might affect the interaction of the Ca2+ release channel with the t-tubule voltage sensor. In addition, a central opening and mass movements, detected in the transmembrane domain of both the Ca(2+)- and the Ca2+/nucleotide-activated channels, suggest a mechanism for channel opening similar to opening-closing of the iris in a camera diaphragm. PMID:10512814

Statistical mechanics of an ideal active fluid confined in a channel

NASA Astrophysics Data System (ADS)

Wagner, Caleb; Baskaran, Aparna; Hagan, Michael

The statistical mechanics of ideal active Brownian particles (ABPs) confined in a channel is studied by obtaining the exact solution of the steady-state Smoluchowski equation for the 1-particle distribution function. The solution is derived using results from the theory of two-way diffusion equations, combined with an iterative procedure that is justified by numerical results. Using this solution, we quantify the effects of confinement on the spatial and orientational order of the ensemble. Moreover, we rigorously show that both the bulk density and the fraction of particles on the channel walls obey simple scaling relations as a function of channel width. By considering a constant-flux steady state, an effective diffusivity for ABPs is derived which shows signatures of the persistent motion that characterizes ABP trajectories. Finally, we discuss how our techniques generalize to other active models, including systems whose activity is modeled in terms of an Ornstein-Uhlenbeck process.

A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

DOE PAGES

Schmandt, Nicolaus; Velisetty, Phanindra; Chalamalasetti, Sreevatsa V.; ...

2015-09-28

Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primarymore » amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators.« less

A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

PubMed Central

Schmandt, Nicolaus; Velisetty, Phanindra; Chalamalasetti, Sreevatsa V.; Stein, Richard A.; Bonner, Ross; Talley, Lauren; Parker, Mark D.; Mchaourab, Hassane S.; Yee, Vivien C.; Lodowski, David T.

2015-01-01

Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators. PMID:26415570

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.

Selective activation of heteromeric SK channels contributes to action potential repolarization in mouse atrial myocytes.

PubMed

Hancock, Jane M; Weatherall, Kate L; Choisy, Stéphanie C; James, Andrew F; Hancox, Jules C; Marrion, Neil V

2015-05-01

Activation of small conductance calcium-activated potassium (SK) channels is proposed to contribute to repolarization of the action potential in atrial myocytes. This role is controversial, as these cardiac SK channels appear to exhibit an uncharacteristic pharmacology. The objectives of this study were to resolve whether activation of SK channels contributes to atrial action potential repolarization and to determine the likely subunit composition of the channel. The effect of 2 SK channel inhibitors was assessed on outward current evoked in voltage clamp and on action potential duration in perforated patch and whole-cell current clamp recording from acutely isolated mouse atrial myocytes. The presence of SK channel subunits was assessed using immunocytochemistry. A significant component of outward current was reduced by the SK channel blockers apamin and UCL1684. Block by apamin displayed a sensitivity indicating that this current was carried by homomeric SK2 channels. Action potential duration was significantly prolonged by UCL1684, but not by apamin. This effect was accompanied by an increase in beat-to-beat variability and action potential triangulation. This pharmacology was matched by that of expressed heteromeric SK2-SK3 channels in HEK293 cells. Immunocytochemistry showed that atrial myocytes express both SK2 and SK3 channels with an overlapping expression pattern. Only proposed heteromeric SK2-SK3 channels are physiologically activated to contribute to action potential repolarization, which is indicated by the difference in pharmacology of evoked outward current and prolongation of atrial action potential duration. The effect of blocking this channel on the action potential suggests that SK channel inhibition during cardiac function has the potential to be proarrhythmic. Copyright © 2015 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Contribution of S4 segments and S4-S5 linkers to the low-voltage activation properties of T-type CaV3.3 channels.

PubMed

Sanchez-Sandoval, Ana Laura; Herrera Carrillo, Zazil; Díaz Velásquez, Clara Estela; Delgadillo, Dulce María; Rivera, Heriberto Manuel; Gomora, Juan Carlos

2018-01-01

Voltage-gated calcium channels contain four highly conserved transmembrane helices known as S4 segments that exhibit a positively charged residue every third position, and play the role of voltage sensing. Nonetheless, the activation range between high-voltage (HVA) and low-voltage (LVA) activated calcium channels is around 30-40 mV apart, despite the high level of amino acid similarity within their S4 segments. To investigate the contribution of S4 voltage sensors for the low-voltage activation characteristics of CaV3.3 channels we constructed chimeras by swapping S4 segments between this LVA channel and the HVA CaV1.2 channel. The substitution of S4 segment of Domain II in CaV3.3 by that of CaV1.2 (chimera IIS4C) induced a ~35 mV shift in the voltage-dependence of activation towards positive potentials, showing an I-V curve that almost overlaps with that of CaV1.2 channel. This HVA behavior induced by IIS4C chimera was accompanied by a 2-fold decrease in the voltage-dependence of channel gating. The IVS4 segment had also a strong effect in the voltage sensing of activation, while substitution of segments IS4 and IIIS4 moved the activation curve of CaV3.3 to more negative potentials. Swapping of IIS4 voltage sensor influenced additional properties of this channel such as steady-state inactivation, current decay, and deactivation. Notably, Domain I voltage sensor played a major role in preventing CaV3.3 channels to inactivate from closed states at extreme hyperpolarized potentials. Finally, site-directed mutagenesis in the CaV3.3 channel revealed a partial contribution of the S4-S5 linker of Domain II to LVA behavior, with synergic effects observed in double and triple mutations. These findings indicate that IIS4 and, to a lesser degree IVS4, voltage sensors are crucial in determining the LVA properties of CaV3.3 channels, although the accomplishment of this function involves the participation of other structural elements like S4-S5 linkers.

Contribution of S4 segments and S4-S5 linkers to the low-voltage activation properties of T-type CaV3.3 channels

PubMed Central

Sanchez-Sandoval, Ana Laura; Herrera Carrillo, Zazil; Díaz Velásquez, Clara Estela; Delgadillo, Dulce María; Rivera, Heriberto Manuel

2018-01-01

Voltage-gated calcium channels contain four highly conserved transmembrane helices known as S4 segments that exhibit a positively charged residue every third position, and play the role of voltage sensing. Nonetheless, the activation range between high-voltage (HVA) and low-voltage (LVA) activated calcium channels is around 30–40 mV apart, despite the high level of amino acid similarity within their S4 segments. To investigate the contribution of S4 voltage sensors for the low-voltage activation characteristics of CaV3.3 channels we constructed chimeras by swapping S4 segments between this LVA channel and the HVA CaV1.2 channel. The substitution of S4 segment of Domain II in CaV3.3 by that of CaV1.2 (chimera IIS4C) induced a ~35 mV shift in the voltage-dependence of activation towards positive potentials, showing an I-V curve that almost overlaps with that of CaV1.2 channel. This HVA behavior induced by IIS4C chimera was accompanied by a 2-fold decrease in the voltage-dependence of channel gating. The IVS4 segment had also a strong effect in the voltage sensing of activation, while substitution of segments IS4 and IIIS4 moved the activation curve of CaV3.3 to more negative potentials. Swapping of IIS4 voltage sensor influenced additional properties of this channel such as steady-state inactivation, current decay, and deactivation. Notably, Domain I voltage sensor played a major role in preventing CaV3.3 channels to inactivate from closed states at extreme hyperpolarized potentials. Finally, site-directed mutagenesis in the CaV3.3 channel revealed a partial contribution of the S4-S5 linker of Domain II to LVA behavior, with synergic effects observed in double and triple mutations. These findings indicate that IIS4 and, to a lesser degree IVS4, voltage sensors are crucial in determining the LVA properties of CaV3.3 channels, although the accomplishment of this function involves the participation of other structural elements like S4-S5 linkers. PMID:29474447

Voltage activation and hysteresis of the non-selective voltage-dependent channel in the intact human red cell.

PubMed

Bennekou, Poul; Barksmann, Trine L; Jensen, Lars R; Kristensen, Berit I; Christophersen, Palle

2004-05-01

Suspension of intact human red cells in media with low chloride and sodium concentrations (isotonic sucrose substitution) results in strongly inside positive membrane potentials, which activate the voltage-dependent non-selective cation (NSVDC) channel. By systematic variation of the initial Nernst potentials for chloride (degree of ion substitution) as well as the chloride conductance (block by NS1652), and by exploiting the interplay between the Ca(2+)-permeable NSVDC channel, the Ca(2+)-activated K+ channel (the Gárdos channel) and the Ca(2+)-pump, a graded activation of the NSVDC channel was achieved. Under these conditions, it was shown that the NSVDC channels exist in two states of activation depending on the initial conditions for the activation. The hysteretic behaviour, which in patch clamp experiments has been found for the individual channel unit, is thus retained at the cellular level and can be demonstrated with red cells in suspension.

Activation gating kinetics of GIRK channels are mediated by cytoplasmic residues adjacent to transmembrane domains.

PubMed

Sadja, Rona; Reuveny, Eitan

2009-01-01

G-protein-coupled inwardly rectifying potassium channels (GIRK/Kir3.x) are involved in neurotransmission-mediated reduction of excitability. The gating mechanism following G protein activation of these channels likely proceeds from movement of inner transmembrane helices to allow K(+) ions movement through the pore of the channel. There is limited understanding of how the binding of G-protein betagamma subunits to cytoplasmic regions of the channel transduces the signal to the transmembrane regions. In this study, we examined the molecular basis that governs the activation kinetics of these channels, using a chimeric approach. We identified two regions as being important in determining the kinetics of activation. One region is the bottom of the outer transmembrane helix (TM1) and the cytoplasmic domain immediately adjacent (the slide helix); and the second region is the bottom of the inner transmembrane helix (TM2) and the cytoplasmic domain immediately adjacent. Interestingly, both of these regions are sufficient in mediating the kinetics of fast activation gating. This result suggests that there is a cooperative movement of either one of these domains to allow fast and efficient activation gating of GIRK channels.

Synthesis, QSAR and calcium channel modulator activity of new hexahydroquinoline derivatives containing nitroimidazole.

PubMed

Miri, Ramin; Javidnia, Katayoun; Mirkhani, Hossein; Hemmateenejad, Bahram; Sepeher, Zahra; Zalpour, Masomeh; Behzad, Taherh; Khoshneviszadeh, Mehdi; Edraki, Najmeh; Mehdipour, Ahmad R

2007-10-01

The discovery that 1,4-dihydropyridine class of calcium channel antagonists inhibit Ca2+ influx represented a major therapeutic advance in the treatment of cardiovascular disease. In contrast to the effects of known calcium channel blockers of the Nifedipine-type, the so-called calcium channel agonists, such as Bay K8644 and CGP 28392, increase calcium influx by binding at the same receptor regions. Our goal was to discover a dual cardioselective Ca2+-channel agonist/vascular selective smooth muscle Ca2+ channel antagonist third-generation 1,4-dihydropyridine drug which would have a suitable therapeutic profile for treating congestive heart failure (CHF) patients. A series of unsymmetrical alkyl, cycloalkyl and aryl ester analogues of 2-methyl-4-(1-methyl)-5-nitro-2-imidazolyl-5-oxo-1,4,5,6,7, 8-hexahydroquinolin-3-arboxylate were synthesized using modified Hantzsch reaction. All compounds show calcium antagonist activity on guinea-pig ileum longitudinal smooth muscle and some of them show agonist effect activity on guinea-pig auricle. Effect of structural parameters on the Ca2+ channel agonist/antagonist was evaluated by quantitative structure-activity relationship analysis. These compounds could be considered as a synthon for developing a suitable drug for treating CHF patients.

Cd(2+) sensitivity and permeability of a low voltage-activated Ca(2+) channel with CatSper-like selectivity filter.

PubMed

Garza-López, Edgar; Chávez, Julio César; Santana-Calvo, Carmen; López-González, Ignacio; Nishigaki, Takuya

2016-07-01

CatSper is a sperm-specific Ca(2+) channel that plays an essential role in the male fertility. However, its biophysical properties have been poorly characterized mainly due to its deficient heterologous expression. As other voltage-gated Ca(2+) channels (CaVs), CatSper possesses a conserved Ca(2+)-selective filter motif ([T/S]x[D/E]xW) in the pore region. Interestingly, CatSper conserves four aspartic acids (DDDD) as the negatively charged residues in this motif while high voltage-activated CaVs have four glutamic acids (EEEE) and low voltage-activated CaVs possess two glutamic acids and two aspartic acids (EEDD). Previous studies based on site-directed mutagenesis of L- and T-type channels showed that the number of D seems to have a negative correlation with their cadmium (Cd(2+)) sensitivity. These results suggest that CatSper (DDDD) would have low sensitivity to Cd(2+). To explore Cd(2+)-sensitivity and -permeability of CatSper, we performed two types of experiments: 1) Electrophysiological analysis of heterologously expressed human CaV3.1 channel and three pore mutants (DEDD, EDDD and DDDD), 2) Cd(2+) imaging of human spermatozoa with FluoZin-1. Electrophysiological studies showed a significant increase in Cd(2+) and manganese (Mn(2+)) currents through the CaV3.1 mutants as well as a reduction in the inhibitory effect of Cd(2+) on the Ca(2+) current. In fluorescence imaging with human sperm, we observed an increase in Cd(2+) influx potentiated by progesterone, a potent activator of CatSper. These results support our hypothesis, namely that Cd(2+)-sensitivity and -permeability are related to the absolute number of D in the Ca(2+)-selective filter independently to the type of the Cav channels. Copyright © 2016 Elsevier Ltd. All rights reserved.

The bile acid-sensitive ion channel (BASIC) is activated by alterations of its membrane environment.

PubMed

Schmidt, Axel; Lenzig, Pia; Oslender-Bujotzek, Adrienne; Kusch, Jana; Lucas, Susana Dias; Gründer, Stefan; Wiemuth, Dominik

2014-01-01

The bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC family of ion channels. Channels of this family are characterized by a common structure, their physiological functions and modes of activation, however, are diverse. Rat BASIC is expressed in brain, liver and intestinal tract and activated by bile acids. The physiological function of BASIC and its mechanism of bile acid activation remain a puzzle. Here we addressed the question whether amphiphilic bile acids activate BASIC by directly binding to the channel or indirectly by altering the properties of the surrounding membrane. We show that membrane-active substances other than bile acids also affect the activity of BASIC and that activation by bile acids and other membrane-active substances is non-additive, suggesting that BASIC is sensitive for changes in its membrane environment. Furthermore based on results from chimeras between BASIC and ASIC1a, we show that the extracellular and the transmembrane domains are important for membrane sensitivity.

The calcium-activated potassium channel KCa3.1 plays a central role in the chemotactic response of mammalian neutrophils.

PubMed

Henríquez, C; Riquelme, T T; Vera, D; Julio-Kalajzić, F; Ehrenfeld, P; Melvin, J E; Figueroa, C D; Sarmiento, J; Flores, C A

2016-01-01

Neutrophils are the first cells to arrive at sites of injury. Nevertheless, many inflammatory diseases are characterized by an uncontrolled infiltration and action of these cells. Cell migration depends on volume changes that are governed by ion channel activity, but potassium channels in neutrophil have not been clearly identified. We aim to test whether KCa3.1 participates in neutrophil migration and other relevant functions of the cell. Cytometer and confocal measurements to determine changes in cell volume were used. Cells isolated from human, mouse and horse were tested for KCa3.1-dependent chemotaxis. Chemokinetics, calcium handling and release of reactive oxygen species were measured to determine the role of KCa3.1 in those processes. A mouse model was used to test for neutrophil recruitment after acute lung injury in vivo. We show for the first time that KCa3.1 is expressed in mammalian neutrophils. When the channel is inhibited by a pharmacological blocker or by genetic silencing, it profoundly affects cell volume regulation, and chemotactic and chemokinetic properties of the cells. We also demonstrated that pharmacological inhibition of KCa3.1 did not affect calcium entry or reactive oxygen species production in neutrophils. Using a mouse model of acute lung injury, we observed that Kca3.1(-/-) mice are significantly less effective at recruiting neutrophils into the site of inflammation. These results demonstrate that KCa3.1 channels are key actors in the migration capacity of neutrophils, and its inhibition did not affect other relevant cellular functions. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

The calcium-activated potassium channel KCa3.1 plays a central role in the chemotactic response of mammalian neutrophils

PubMed Central

Henríquez, C.; Riquelme, T. T.; Vera, D.; Julio-Kalajzić, F.; Ehrenfeld, P.; Melvin, J. E.; Figueroa, C. D.; Sarmiento, J.; Flores, C. A.

2017-01-01

Aim Neutrophils are the first cells to arrive at sites of injury. Nevertheless, many inflammatory diseases are characterized by an uncontrolled infiltration and action of these cells. Cell migration depends on volume changes that are governed by ion channel activity, but potassium channels in neutrophil have not been clearly identified. We aim to test whether KCa3.1 participates in neutrophil migration and other relevant functions of the cell. Methods Cytometer and confocal measurements to determine changes in cell volume were used. Cells isolated from human, mouse and horse were tested for KCa3.1-dependent chemotaxis. Chemokinetics, calcium handling and release of reactive oxygen species were measured to determine the role of KCa3.1 in those processes. A mouse model was used to test for neutrophil recruitment after acute lung injury in vivo. Results We show for the first time that KCa3.1 is expressed in mammalian neutrophils. When the channel is inhibited by a pharmacological blocker or by genetic silencing, it profoundly affects cell volume regulation, and chemotactic and chemokinetic properties of the cells. We also demonstrated that pharmacological inhibition of KCa3.1 did not affect calcium entry or reactive oxygen species production in neutrophils. Using a mouse model of acute lung injury, we observed that Kca3.1−/− mice are significantly less effective at recruiting neutrophils into the site of inflammation. Conclusions These results demonstrate that KCa3.1 channels are key actors in the migration capacity of neutrophils, and its inhibition did not affect other relevant cellular functions. PMID:26138196

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

PAD-MAC: Primary User Activity-Aware Distributed MAC for Multi-Channel Cognitive Radio Networks

PubMed Central

Ali, Amjad; Piran, Md. Jalil; Kim, Hansoo; Yun, Jihyeok; Suh, Doug Young

2015-01-01

Cognitive radio (CR) has emerged as a promising technology to solve problems related to spectrum scarcity and provides a ubiquitous wireless access environment. CR-enabled secondary users (SUs) exploit spectrum white spaces opportunistically and immediately vacate the acquired licensed channels as primary users (PUs) arrive. Accessing the licensed channels without the prior knowledge of PU traffic patterns causes severe throughput degradation due to excessive channel switching and PU-to-SU collisions. Therefore, it is significantly important to design a PU activity-aware medium access control (MAC) protocol for cognitive radio networks (CRNs). In this paper, we first propose a licensed channel usage pattern identification scheme, based on a two-state Markov model, and then estimate the future idle slots using previous observations of the channels. Furthermore, based on these past observations, we compute the rank of each available licensed channel that gives SU transmission success assessment during the estimated idle slot. Secondly, we propose a PU activity-aware distributed MAC (PAD-MAC) protocol for heterogeneous multi-channel CRNs that selects the best channel for each SU to enhance its throughput. PAD-MAC controls SU activities by allowing them to exploit the licensed channels only for the duration of estimated idle slots and enables predictive and fast channel switching. To evaluate the performance of the proposed PAD-MAC, we compare it with the distributed QoS-aware MAC (QC-MAC) and listen-before-talk MAC schemes. Extensive numerical results show the significant improvements of the PAD-MAC in terms of the SU throughput, SU channel switching rate and PU-to-SU collision rate. PMID:25831084

Rational design and validation of a vanilloid-sensitive TRPV2 ion channel

PubMed Central

Yang, Fan; Vu, Simon; Yarov-Yarovoy, Vladimir; Zheng, Jie

2016-01-01

Vanilloids activation of TRPV1 represents an excellent model system of ligand-gated ion channels. Recent studies using cryo-electron microcopy (cryo-EM), computational analysis, and functional quantification revealed the location of capsaicin-binding site and critical residues mediating ligand-binding and channel activation. Based on these new findings, here we have successfully introduced high-affinity binding of capsaicin and resiniferatoxin to the vanilloid-insensitive TRPV2 channel, using a rationally designed minimal set of four point mutations (F467S–S498F–L505T–Q525E, termed TRPV2_Quad). We found that binding of resiniferatoxin activates TRPV2_Quad but the ligand-induced open state is relatively unstable, whereas binding of capsaicin to TRPV2_Quad antagonizes resiniferatoxin-induced activation likely through competition for the same binding sites. Using Rosetta-based molecular docking, we observed a common structural mechanism underlying vanilloids activation of TRPV1 and TRPV2_Quad, where the ligand serves as molecular “glue” that bridges the S4–S5 linker to the S1–S4 domain to open these channels. Our analysis revealed that capsaicin failed to activate TRPV2_Quad likely due to structural constraints preventing such bridge formation. These results not only validate our current working model for capsaicin activation of TRPV1 but also should help guide the design of drug candidate compounds for this important pain sensor. PMID:27298359

Hyperpolarization-activated cation channels in fast-spiking interneurons of rat hippocampus

PubMed Central

Aponte, Yexica; Lien, Cheng-Chang; Reisinger, Ellen; Jonas, Peter

2006-01-01

Hyperpolarization-activated channels (Ih or HCN channels) are widely expressed in principal neurons in the central nervous system. However, Ih in inhibitory GABAergic interneurons is less well characterized. We examined the functional properties of Ih in fast-spiking basket cells (BCs) of the dentate gyrus, using hippocampal slices from 17- to 21-day-old rats. Bath application of the Ih channel blocker ZD 7288 at a concentration of 30 μm induced a hyperpolarization of 5.7 ± 1.5 mV, an increase in input resistance and a correlated increase in apparent membrane time constant. ZD 7288 blocked a hyperpolarization-activated current in a concentration-dependent manner (IC50, 1.4 μm). The effects of ZD 7288 were mimicked by external Cs+. The reversal potential of Ih was −27.4 mV, corresponding to a Na+ to K+ permeability ratio (PNa/PK) of 0.36. The midpoint potential of the activation curve of Ih was −83.9 mV, and the activation time constant at −120 mV was 190 ms. Single-cell expression analysis using reverse transcription followed by quantitative polymerase chain reaction revealed that BCs coexpress HCN1 and HCN2 subunit mRNA, suggesting the formation of heteromeric HCN1/2 channels. ZD 7288 increased the current threshold for evoking antidromic action potentials by extracellular stimulation, consistent with the expression of Ih in BC axons. Finally, ZD 7288 decreased the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in hippocampal granule cells, the main target cells of BCs, to 70 ± 4% of the control value. In contrast, the amplitude of mIPSCs was unchanged, consistent with the presence of Ih in inhibitory terminals. In conclusion, our results suggest that Ih channels are expressed in the somatodendritic region, axon and presynaptic elements of fast-spiking BCs in the hippocampus. PMID:16690716

Vascular activation of K+ channels and Na+-K+ ATPase activity of estrogen-deficient female rats.

PubMed

Ribeiro Junior, Rogério Faustino; Fiorim, Jonaina; Marques, Vinicius Bermond; de Sousa Ronconi, Karoline; Botelho, Tatiani; Grando, Marcella D; Bendhack, Lusiane M; Vassallo, Dalton Valentim; Stefanon, Ivanita

2017-12-01

The goal of the present study was to evaluate vascular potassium channels and Na + -K + -ATPase activity in estrogen deficient female rats. Female rats that underwent ovariectomy were assigned to receive daily treatment with placebo (OVX) or estrogen replacement (OVX+E2, 1mg/kg, once a week, i.m.). Aortic rings were used to examine the involvement of K + channels and Na + -K + -ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100μM) and K + channels blockers: tetraethylammonium (TEA, 5mM), 4-aminopyridine (4-AP, 5mM), iberiotoxin (IbTX, 30nM), apamin (0.5mM), charybdotoxin (ChTX, 0.1mM) and iberiotoxin plus apamin. When aortic rings were pre-contracted with KCl (60mM) or pre-incubated with TEA (5mM), 4-aminopyridine (4-AP, 5mM) and iberiotoxin (IbTX, 30nM) plus apamin (0.5μM), the ACh-induced relaxation was less effective in the ovariectomized group. Additionally, 4-AP and IbTX decreased the relaxation by sodium nitroprusside in all groups but this reduction was greater in the ovariectomized group. Estrogen deficiency also increased aortic functional Na + -K + ATPase activity evaluated by K + -induced relaxation. L-NAME or endothelium removal were not able to block the increase in aortic functional Na + -K + ATPase activity, however, TEA (5mM) restored this increase to the control level. We also found that estrogen deficiency increased superoxide anion production and reduced nitric oxide release in aortic ring from ovariectomized animals. In summary, our results emphasize that the process underlying ACh-induced relaxation is preserved in ovariectomized animals due to the activation of K + channels and increased Na + -K + ATPase activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Channel erosion surveys along the TAPS route, Alaska, 1977

USGS Publications Warehouse

Loeffler, Robert M.; Childers, Joseph M.

1977-01-01

Channel surveys were made along the trans-Alaska pipeline system (TAPS) route during 1977 at the same 28 sites that were studied in 1976. In addition, a new site at pipeline mile 22 near Deadhorse (alignment No 134) along the Sagavanirktok River was put under surveillance. Except for changes wrought by the completion of construction, most of the sites showed very little change. Significant events include virtual completion of all construction activities along the pipeline, the pipeline startup , and the breakup flood along the Sagavanirktok River which breached many river-training structures. In general, 1977 saw heavy flooding on streams draining the north and south slopes of the Brooks Range and only moderate flooding on streams further south. Aerial photogrammetric surveys were used again in 1977 on the same seven sites as in 1976. Results document the applicability of the method for channel erosion studies. (Woodard-USGS)

Melatonin mediates vasodilation through both direct and indirect activation of BKCa channels.

PubMed

Zhao, T; Zhang, H; Jin, C; Qiu, F; Wu, Y; Shi, L

2017-10-01

Melatonin, synthesized primarily by the pineal gland, is a neuroendocrine hormone with high membrane permeability. The vascular effects of melatonin, including vasoconstriction and vasodilation, have been demonstrated in numerous studies. However, the mechanisms underlying these effects are not fully understood. Large-conductance Ca 2+ -activated K + (BK Ca ) channels are expressed broadly on smooth muscle cells and play an important role in vascular tone regulation. This study explored the mechanisms of myocyte BK Ca channels and endothelial factors underlying the action of melatonin on the mesenteric arteries (MAs). Vascular contractility and patch-clamp studies were performed on myocytes of MAs from Wistar rats. Melatonin induced significant vasodilation on MAs. In the presence of N ω -nitro-l-arginine methyl ester (l-NAME), a potent endothelial oxide synthase (eNOS) inhibitor, melatonin elicited concentration-dependent relaxation, with lowered pIC 50 The effect of melatonin was significantly attenuated in the presence of BK Ca channel blocker iberiotoxin or MT1/MT2 receptor antagonist luzindole in both (+) l-NAME and (-) l-NAME groups. In the (+) l-NAME group, iberiotoxin caused a parallel rightward shift of the melatonin concentration-relaxation curve, with pIC 50 lower than that of luzindole. Both inside-out and cell-attached patch-clamp recordings showed that melatonin significantly increased the open probability, mean open time and voltage sensitivity of BK Ca channels. In a cell-attached patch-clamp configuration, the melatonin-induced enhancement of BK Ca channel activity was significantly suppressed by luzindole. These findings indicate that in addition to the activation of eNOS, melatonin-induced vasorelaxation of MAs is partially attributable to its direct (passing through the cell membrane) and indirect (via MT1/MT2 receptors) activation of the BK Ca channels on mesenteric arterial myocytes. © 2017 Society for Endocrinology.

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.

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.

Channel stability of Turkey Creek, Nebraska

USGS Publications Warehouse

Rus, David L.; Soenksen, Philip J.

1998-01-01

Channelization on Turkey Creek and its receiving stream, the South Fork Big Nemaha River, has disturbed the equilibrium of Turkey Creek and has led to channel-stability problems, such as degradation and channel widening, which pose a threat to bridges and land adjacent to the stream. As part of a multiagency study, the U.S. Geological Survey assessed channel stability at two bridge sites on upper and middle portions of Turkey Creek by analyzing streambed-elevation data for gradation changes, comparing recent cross-section surveys and historic accounts, identifying bank-failure blocks, and analyzing tree-ring samples. These results were compared to gradation data and trend results for a U.S. Geological Survey streamflow-gaging station near the mouth of Turkey Creek from a previous study. Examination of data on streambed elevations reveals that degradation has occurred. The streambed elevation declined 0.5 m at the upper site from 1967-97. The streambed elevation declined by 3.2 m at the middle site from 1948-97 and exposed 2 m of the pilings of the Nebraska Highway 8 bridge. Channel widening could not be verified at the two sites from 1967-97, but a historic account indicates widening at the middle site to be two to three times that of the 1949 channel width. Small bank failures were evident at the upper site and a 4-m-wide bank failure occurred at the middle site in 1987 according to tree ring analyses. Examination of streambed-elevation data from a previous study at the lower site reveals a statistically significant aggrading trend from 1958-93. Further examination of these data suggests minor degradation occurred until 1975, followed by aggradation.

Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel.

PubMed

Jensen, B S; Strobaek, D; Christophersen, P; Jorgensen, T D; Hansen, C; Silahtaroglu, A; Olesen, S P; Ahring, P K

1998-09-01

The human intermediate-conductance, Ca2+-activated K+ channel (hIK) was identified by searching the expressed sequence tag database. hIK was found to be identical to two recently cloned K+ channels, hSK4 and hIK1. RNA dot blot analysis showed a widespread tissue expression, with the highest levels in salivary gland, placenta, trachea, and lung. With use of fluorescent in situ hybridization and radiation hybrid mapping, hIK mapped to chromosome 19q13.2 in the same region as the disease Diamond-Blackfan anemia. Stable expression of hIK in HEK-293 cells revealed single Ca2+-activated K+ channels exhibiting weak inward rectification (30 and 11 pS at -100 and +100 mV, respectively). Whole cell recordings showed a noninactivating, inwardly rectifying K+ conductance. Ionic selectivity estimated from bi-ionic reversal potentials gave the permeability (PK/PX) sequence K+ = Rb+ (1.0) > Cs+ (10.4) > Na+, Li+, N-methyl-D-glucamine (>51). NH+4 blocked the channel completely. hIK was blocked by the classical inhibitors of the Gardos channel charybdotoxin (IC50 28 nM) and clotrimazole (IC50 153 nM) as well as by nitrendipine (IC50 27 nM), Stichodactyla toxin (IC50 291 nM), margatoxin (IC50 459 nM), miconazole (IC50 785 nM), econazole (IC50 2.4 microM), and cetiedil (IC50 79 microM). Finally, 1-ethyl-2-benzimidazolinone, an opener of the T84 cell IK channel, activated hIK with an EC50 of 74 microM.

Molecular mechanism underlying ethanol activation of G-protein–gated inwardly rectifying potassium channels

PubMed Central

Bodhinathan, Karthik; Slesinger, Paul A.

2013-01-01

Alcohol (ethanol) produces a wide range of pharmacological effects on the nervous system through its actions on ion channels. The molecular mechanism underlying ethanol modulation of ion channels is poorly understood. Here we used a unique method of alcohol-tagging to demonstrate that alcohol activation of a G-protein–gated inwardly rectifying potassium (GIRK or Kir3) channel is mediated by a defined alcohol pocket through changes in affinity for the membrane phospholipid signaling molecule phosphatidylinositol 4,5-bisphosphate. Surprisingly, hydrophobicity and size, but not the canonical hydroxyl, were important determinants of alcohol-dependent activation. Altering levels of G protein Gβγ subunits, conversely, did not affect alcohol-dependent activation, suggesting a fundamental distinction between receptor and alcohol gating of GIRK channels. The chemical properties of the alcohol pocket revealed here might extend to other alcohol-sensitive proteins, revealing a unique protein microdomain for targeting alcohol-selective therapeutics in the treatment of alcoholism and addiction. PMID:24145411

Two MscS homologs provide mechanosensitive channel activities in the Arabidopsis root.

PubMed

Haswell, Elizabeth S; Peyronnet, Rémi; Barbier-Brygoo, Hélène; Meyerowitz, Elliot M; Frachisse, Jean-Marie

2008-05-20

In bacterial and animal systems, mechanosensitive (MS) ion channels are thought to mediate the perception of pressure, touch, and sound [1-3]. Although plants respond to a wide variety of mechanical stimuli, and although many mechanosensitive channel activities have been characterized in plant membranes by the patch-clamp method, the molecular nature of mechanoperception in plant systems has remained elusive [4]. Likely candidates are relatives of MscS (Mechanosensitive channel of small conductance), a well-characterized MS channel that serves to protect E. coli from osmotic shock [5]. Ten MscS-Like (MSL) proteins are found in the genome of the model flowering plant Arabidopsis thaliana[4, 6, 7]. MSL2 and MSL3, along with MSC1, a MscS family member from green algae, are implicated in the control of organelle morphology [8, 9]. Here, we characterize MSL9 and MSL10, two MSL proteins found in the plasma membrane of root cells. We use a combined genetic and electrophysiological approach to show that MSL9 and MSL10, along with three other members of the MSL family, are required for MS channel activities detected in protoplasts derived from root cells. This is the first molecular identification and characterization of MS channels in plant membranes.

Pharmacology of the human red cell voltage-dependent cation channel; Part I. Activation by clotrimazole and analogues.

PubMed

Barksmann, Trine L; Kristensen, Berit I; Christophersen, Palle; Bennekou, Poul

2004-01-01

The activation and pharmacological modulation of the nonselective voltage-dependent cation (NSVDC) channel from human erythrocytes were studied. Basic channel activation was achieved by suspending red cells in a low Cl(-) Ringer (2 mM), where a positive membrane potential (V(m) = E(Cl)) immediately developed. Voltage- and time-dependent activation of the NSVDC channel occurred, reaching a cation conductance (g+) of 1.5-2.0 microS cm(-2). In the presence of the classical Gárdos channel blocker clotrimazole (0-50 microM), activation occurred faster, and g+ saturated dose-dependently (EC50 = 14 microM) at a value of about 4 microS cm(-2). The clotrimazole analogues TRAM-34, econazole, and miconazole also stimulated the channel, whereas the chemically more distant Gárdos channel inhibitors nitrendipine and cetiedil had no effects. Although the potency for modulation of the NSVDC channel is much lower than the IC50 value for Gárdos channel inhibition, clotrimazole (and its analogues) constitutes the first chemical class of positive modulators of the NSVDC channel. This may be an important pharmacological "fingerprint" in the identification of the cloned equivalent of the erythrocyte channel.

Sorting Out Effects of Active Stream Restoration: Channel Morphology, Channel Change Processes and Potential Controls

NASA Astrophysics Data System (ADS)

McDowell, P. F.

2017-12-01

In many active restoration projects, instream structures or modifications are designed to produce specific change in channel form, such as reduced W:D or increased pool depth, yet there is little monitoring to evaluate effectiveness. Active restoration often takes place within a context of other land management changes that can have an effect on channel form. Thus, the effects of active restoration are difficult to separate from the effects of other management actions. We measured morphologic response to restoration designs on sections of the Middle Fork John Day River, a gravel-cobble bed river under a cattle grazing regime in the Blue Mountain of Oregon. Since 2000, restoration actions have included elimination of cattle grazing in the riparian zone (passive restoration), riparian planting of woody vegetation, instream log structures for fish habitat and pool maintenance, and elimination of a major flow diversion. We listed the hypothetical effects of each of these management changes, showing overlap among effects of active and passive restoration. Repeat cross-section and longitudinal profile surveys over eight years, and repeat aerial imagery, documented changes in channel width, depth and bed morphology, and processes of change (bank erosion or aggradation, point bar erosion or aggradation, bed incision or aggradation), in two restored reaches and two adjacent control (unrestored) reaches. Morphologic changes were modest. Bankfull cross-section area, width, and W:D all decreased slightly in both restored reaches. Control reaches were unchanged or increased slightly. Processes of change were markedly different among the four reaches, with different reaches dominated by different processes. One restored reach was dominated by slight bed aggradation, increased pool depth and deep pools/km, while the other restored reach was dominated by bank erosion, bar aggradation and slight bed incision, along with increased deep pools/km. The longitudinal profile showed

Characterization of the Body-to-Body Propagation Channel for Subjects during Sports Activities.

PubMed

Mohamed, Marshed; Cheffena, Michael; Moldsvor, Arild

2018-02-18

Body-to-body wireless networks (BBWNs) have great potential to find applications in team sports activities among others. However, successful design of such systems requires great understanding of the communication channel as the movement of the body components causes time-varying shadowing and fading effects. In this study, we present results of the measurement campaign of BBWN during running and cycling activities. Among others, the results indicated the presence of good and bad states with each state following a specific distribution for the considered propagation scenarios. This motivated the development of two-state semi-Markov model, for simulation of the communication channels. The simulation model was validated using the available measurement data in terms of first and second order statistics and have shown good agreement. The first order statistics obtained from the simulation model as well as the measured results were then used to analyze the performance of the BBWNs channels under running and cycling activities in terms of capacity and outage probability. Cycling channels showed better performance than running, having higher channel capacity and lower outage probability, regardless of the speed of the subjects involved in the measurement campaign.

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity*

PubMed Central

Tétreault, Marie-Philippe; Bourdin, Benoîte; Briot, Julie; Segura, Emilie; Lesage, Sylvie; Fiset, Céline; Parent, Lucie

2016-01-01

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca2+ channel complexes. CaVα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type CaV1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant CaVα2δ1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the CaVα2δ1-mediated increase in the peak current density and voltage-dependent gating of CaV1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored CaVα2δ1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of CaVα2δ1 as well as the CaVα2δ1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of CaVα2δ1, and furthermore that N-glycosylation of CaVα2δ1 is essential to produce functional L-type Ca2+ channels. PMID:26742847

Sodium Binding Sites and Permeation Mechanism in the NaChBac Channel: A Molecular Dynamics Study.

PubMed

Guardiani, Carlo; Rodger, P Mark; Fedorenko, Olena A; Roberts, Stephen K; Khovanov, Igor A

2017-03-14

NaChBac was the first discovered bacterial sodium voltage-dependent channel, yet computational studies are still limited due to the lack of a crystal structure. In this work, a pore-only construct built using the NavMs template was investigated using unbiased molecular dynamics and metadynamics. The potential of mean force (PMF) from the unbiased run features four minima, three of which correspond to sites IN, CEN, and HFS discovered in NavAb. During the run, the selectivity filter (SF) is spontaneously occupied by two ions, and frequent access of a third one is often observed. In the innermost sites IN and CEN, Na + is fully hydrated by six water molecules and occupies an on-axis position. In site HFS sodium interacts with a glutamate and a serine from the same subunit and is forced to adopt an off-axis placement. Metadynamics simulations biasing one and two ions show an energy barrier in the SF that prevents single-ion permeation. An analysis of the permeation mechanism was performed both computing minimum energy paths in the axial-axial PMF and through a combination of Markov state modeling and transition path theory. Both approaches reveal a knock-on mechanism involving at least two but possibly three ions. The currents predicted from the unbiased simulation using linear response theory are in excellent agreement with single-channel patch-clamp recordings.

Liposomal quercetin potentiates maxi-K channel openings in smooth muscles and restores its activity after oxidative stress.

PubMed

Melnyk, Mariia I; Dryn, Dariia O; Al Kury, Lina T; Zholos, Alexander V; Soloviev, Anatoly I

2018-04-19

The effects of quercetin-loaded liposomes (PCL-Q) and their constituents, that is, free quercetin (Q) and 'empty' phosphatidylcholine vesicles (PCL), on maxi-K channel activity were studied in single mouse ileal myocytes before and after H 2 O 2 -induced oxidative stress. Macroscopic Maxi-K channel currents were recorded using whole-cell patch clamp techniques, while single BK Ca channel currents were recorded in the cell-attached configuration. Bath application of PCL-Q (100 μg/ml of lipid and 3 μg/ml of quercetin) increased single Maxi-K channel activity more than threefold, from 0.010 ± 0.003 to 0.034 ± 0.004 (n = 5; p < 0.05), whereas single-channel conductance increased non-significantly from 138 to 146 pS. In the presence of PCL-Q multiple simultaneous channel openings were observed, with up to eight active channels in the membrane patch. Surprisingly, 'empty' PCL (100 μg/ml) also produced some channel activation, although it was less potent compared to PCL-Q, that is, these increased NPo from 0.010 ± 0.003 to 0.019 ± 0.003 (n = 5; p < 0.05) and did not affect single-channel conductance (139 pS). Application of PCL-Q restored macroscopic Maxi-K currents suppressed by H 2 O 2 -induced oxidative stress in ileal smooth muscle cells. We conclude that PCL-Q can activate Maxi-K channels in ileal myocytes mainly by increasing channel open probability, as well as maintain Maxi-K-mediated whole-cell current under the conditions of oxidative stress. While fusion of the 'pure' liposomes with the plasma membrane may indirectly activate Maxi-K channels by altering channel's phospholipids environment, the additional potentiating action of quercetin may be due to its better bioavailability.

Cl- channels of the gastric parietal cell that are active at low pH.

PubMed

Cuppoletti, J; Baker, A M; Malinowska, D H

1993-06-01

HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 m

Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor

PubMed Central

Liedtke, Wolfgang; Choe, Yong; Martí-Renom, Marc A.; Bell, Andrea M.; Denis, Charlotte S.; Šali, Andrej; Hudspeth, A. J.; Friedman, Jeffrey M.; Heller, Stefan

2008-01-01

SUMMARY The detection of osmotic stimuli is essential for all organisms, yet few osmoreceptive proteins are known, none of them in vertebrates. By employing a candidate-gene approach based on genes encoding members of the TRP superfamily of ion channels, we cloned cDNAs encoding the vanilloid receptor-related osmotically activated channel (VR-OAC) from the rat, mouse, human, and chicken. This novel cation-selective channel is gated by exposure to hypotonicity within the physiological range. In the central nevous system, the channel is expressed neurons of the circumventricular organs, neurosensory cells responsive to systemic osmotic pressure. The channel also occurs in other neurosensory cells, including inner-ear hair cells, sensory neurons, and Merkel cells. PMID:11081638

Magnesium Excretion in C. elegans Requires the Activity of the GTL-2 TRPM Channel

PubMed Central

Teramoto, Takayuki; Sternick, Laura A.; Kage-Nakadai, Eriko; Sajjadi, Shirine; Siembida, Jakub; Mitani, Shohei; Iwasaki, Kouichi; Lambie, Eric J.

2010-01-01

Systemic magnesium homeostasis in mammals is primarily governed by the activities of the TRPM6 and TRPM7 cation channels, which mediate both uptake by the intestinal epithelial cells and reabsorption by the distal convoluted tubule cells in the kidney. In the nematode, C. elegans, intestinal magnesium uptake is dependent on the activities of the TRPM channel proteins, GON-2 and GTL-1. In this paper we provide evidence that another member of the TRPM protein family, GTL-2, acts within the C. elegans excretory cell to mediate the excretion of excess magnesium. Thus, the activity of GTL-2 balances the activities of the paralogous TRPM channel proteins, GON-2 and GTL-1. PMID:20221407

Single-Pixel Optical Fluctuation Analysis of Calcium Channel Function in Active Zones of Motor Nerve Terminals

PubMed Central

Luo, Fujun; Dittrich, Markus; Stiles, Joel R.; Meriney, Stephen D.

2011-01-01

We used high-resolution fluorescence imaging and single-pixel optical fluctuation analysis to estimate the opening probability of individual voltage-gated calcium (Ca2+) channels during an action potential and the number of such Ca2+ channels within active zones of frog neuromuscular junctions. Analysis revealed ~36 Ca2+ channels within each active zone, similar to the number of docked synaptic vesicles but far less than the total number of transmembrane particles reported based on freeze-fracture analysis (~200–250). The probability that each channel opened during an action potential was only ~0.2. These results suggest why each active zone averages only one quantal release event during every other action potential, despite a substantial number of docked vesicles. With sparse Ca2+ channels and low opening probability, triggering of fusion for each vesicle is primarily controlled by Ca2+ influx through individual Ca2+ channels. In contrast, the entire synapse is highly reliable because it contains hundreds of active zones. PMID:21813687