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Sample records for affect channel activity

  1. Ethanol Affects Network Activity in Cultured Rat Hippocampus: Mediation by Potassium Channels

    PubMed Central

    Korkotian, Eduard; Bombela, Tatyana; Odegova, Tatiana; Zubov, Petr; Segal, Menahem

    2013-01-01

    The effects of ethanol on neuronal network activity were studied in dissociated cultures of rat hippocampus. Exposure to low (0.25–0.5%) ethanol concentrations caused an increase in synchronized network spikes, and a decrease in the duration of individual spikes. Ethanol also caused an increase in rate of miniature spontaneous excitatory postsynaptic currents. Higher concentrations of ethanol eliminated network spikes. These effects were reversible upon wash. The effects of the high, but not the low ethanol were blocked by the GABA antagonist bicuculline. The enhancing action of low ethanol was blocked by apamin, an SK potassium channel antagonist, and mimicked by 1-EBIO, an SK channel opener. It is proposed that in cultured hippocampal networks low concentration of ethanol is associated with SK channel activity, rather than the GABAergic receptor. PMID:24260098

  2. Solution structure and alanine scan of a spider toxin that affects the activation of mammalian voltage-gated sodium channels.

    PubMed

    Corzo, Gerardo; Sabo, Jennifer K; Bosmans, Frank; Billen, Bert; Villegas, Elba; Tytgat, Jan; Norton, Raymond S

    2007-02-16

    Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide containing three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltage-gated sodium channels and competes with scorpion beta-toxins, such as Css IV from Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of the mammalian rNav1.2a channel to more hyperpolarized voltages, whereas the insect channel, DmNav1, is not affected. To gain insight into toxin-channel interactions, Magi 5 and 23 analogues were synthesized. The three-dimensional structure of Magi 5 in aqueous solution was determined, and its voltage-gated sodium channel-binding surfaces were mapped onto this structure using data from electrophysiological measurements on a series of Ala-substituted analogues. The structure clearly resembles the inhibitor cystine knot structural motif, although the triple-stranded beta-sheet typically found in that motif is partially distorted in Magi 5. The interactive surface of Magi 5 toward voltage-gated sodium channels resembles in some respects the Janus-faced atracotoxins, with functionally important charged residues on one face of the toxin and hydrophobic residues on the other. Magi 5 also resembles the scorpion beta-toxin Css IV, which has distinct nonpolar and charged surfaces that are critical for channel binding and has a key Glu involved in voltage sensor trapping. These two distinct classes of toxin, with different amino acid sequences and different structures, may utilize similar groups of residues on their surface to achieve the common end of modifying voltage-gated sodium channel function.

  3. 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

  4. Mechanically Activated Ion Channels.

    PubMed

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

    2015-09-23

    Mechanotransduction, the conversion of physical forces into biochemical signals, is essential for various physiological processes such as the conscious sensations of touch and hearing, and the unconscious sensation of blood flow. 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.

  5. Sea Anemone Toxins Affecting Potassium Channels

    NASA Astrophysics Data System (ADS)

    Diochot, Sylvie; Lazdunski, Michel

    The great diversity of K+ channels and their wide distribution in many tissues are associated with important functions in cardiac and neuronal excitability that are now better understood thanks to the discovery of animal toxins. During the past few decades, sea anemones have provided a variety of toxins acting on voltage-sensitive sodium and, more recently, potassium channels. Currently there are three major structural groups of sea anemone K+ channel (SAK) toxins that have been characterized. Radioligand binding and electrophysiological experiments revealed that each group contains peptides displaying selective activities for different subfamilies of K+ channels. Short (35-37 amino acids) peptides in the group I display pore blocking effects on Kv1 channels. Molecular interactions of SAK-I toxins, important for activity and binding on Kv1 channels, implicate a spot of three conserved amino acid residues (Ser, Lys, Tyr) surrounded by other less conserved residues. Long (58-59 amino acids) SAK-II peptides display both enzymatic and K+ channel inhibitory activities. Medium size (42-43 amino acid) SAK-III peptides are gating modifiers which interact either with cardiac HERG or Kv3 channels by altering their voltage-dependent properties. SAK-III toxins bind to the S3C region in the outer vestibule of Kv channels. Sea anemones have proven to be a rich source of pharmacological tools, and some of the SAK toxins are now useful drugs for the diagnosis and treatment of autoimmune diseases.

  6. Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O 2 specificity but not activation by Rubisco activase.

    PubMed

    Esquivel, M Gloria; Genkov, Todor; Nogueira, Ana S; Salvucci, Michael E; Spreitzer, Robert J

    2013-12-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the initial step of carbon metabolism in photosynthesis. The holoenzyme comprises eight large subunits, arranged as a tetramer of dimers around a central solvent channel that defines a fourfold axis of symmetry, and eight small subunits, arranged as two tetramers at the poles of the axis. The phylogenetically divergent small-subunit loops between β-strands A and B form the entrance to the solvent channel. In the green alga Chlamydomonas reinhardtii, Ile-58 from each of the four small-subunit βA-βB loops defines the minimal diameter of the channel opening. To understand the role of the central solvent channel in Rubisco function, directed mutagenesis and transformation of Chlamydomonas were employed to replace Ile-58 with Ala, Lys, Glu, Trp, or three Trp residues (I58W3) to close the entrance to the channel. The I58E, I58K, and I58W substitutions caused only small decreases in photosynthetic growth at 25 and 35 °C, whereas I58W3 had a substantial effect at both temperatures. The mutant enzymes had decreased carboxylation rates, but the I58W3 enzyme had decreases in both carboxylation and CO2/O2 specificity. The I58E, I58W, and I58W3 enzymes were inactivated at lower temperatures than wild-type Rubisco, and were degraded at slower rates under oxidative stress. However, these mutant enzymes were activated by Rubisco activase at normal rates, indicating that the structural transition required for carboxylation is not affected by altering the solvent channel opening. Structural dynamics alone may not be responsible for these distant effects on the Rubisco active site.

  7. A mutation in TRPC6 channels abolishes their activation by hypoosmotic stretch but does not affect activation by diacylglycerol or G protein signaling cascades.

    PubMed

    Wilson, Cory; Dryer, Stuart E

    2014-05-01

    Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathogenesis of kidney disease and in the regulation of vascular smooth muscle tone, podocyte function, and a variety of processes in other cell types. The question of whether their gating is intrinsically mechanosensitive has been controversial. In this study we have examined activation of two alleles of TRPC6 transiently expressed in CHO-K1 cells: the wild-type human TRPC6 channel, and TRPC6-N143S, an allele originally identified in a family with autosomal dominant familial focal and segmental glomerulosclerosis (FSGS). We observed that both channel variants carried robust cationic currents that could be evoked by application of membrane-permeable analogs of diacylglycerol (DAG) or by the P2Y receptor agonist ATP. The amplitudes and characteristics of currents evoked by the DAG analog or ATP were indistinguishable in cells expressing the two TRPC6 alleles. By contrast, hypoosmotic stretch evoked robust currents in wild-type TRPC6 channels but had no discernible effect on currents in cells expressing TRPC6-N143S, indicating that the mutant form lacks mechanosensitivity. Coexpression of TRPC6-N143S with wild-type TRPC6 or TRPC3 channels did not alter stretch-evoked responses compared with when TRPC3 channels were expressed by themselves, indicating that TRPC6-N143S does not function as a dominant-negative. These data indicate that mechanical activation and activation evoked by DAG or ATP occur through fundamentally distinct biophysical mechanisms, and they provide support for the hypothesis that protein complexes containing wild-type TRPC6 subunits can be intrinsically mechanosensitive.

  8. Fe2O3 nanoparticles suppress Kv1.3 channels via affecting the redox activity of Kvβ2 subunit in Jurkat T cells

    NASA Astrophysics Data System (ADS)

    Yan, Li; Liu, Xiao; Liu, Wei-Xia; Tan, Xiao-Qiu; Xiong, Fei; Gu, Ning; Hao, Wei; Gao, Xue; Cao, Ji-Min

    2015-12-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) are promising nanomaterials in medical practice due to their special magnetic characteristics and nanoscale size. However, their potential impacts on immune cells are not well documented. This study aims to investigate the effects of Fe2O3 nanoparticles (Fe2O3-NPs) on the electrophysiology of Kv1.3 channels in Jurkat T cells. Using the whole-cell patch-clamp technique, we demonstrate that incubation of Jurkat cells with Fe2O3-NPs dose- and time-dependently decreased the current density and shifted the steady-state inactivation curve and the recovery curve of Kv1.3 channels to a rightward direction. Fe2O3-NPs increased the NADP level but decreased the NADPH level of Jurkat cells. Direct induction of NADPH into the cytosole of Jurkat cells via the pipette abolished the rightward shift of the inactivation curve. In addition, transmission electron microscopy showed that Fe2O3-NPs could be endocytosed by Jurkat cells with relatively low speed and capacity. Fe2O3-NPs did not significantly affect the viability of Jurkat cells, but suppressed the expressions of certain cytokines (TNFα, IFNγ and IL-2) and interferon responsive genes (IRF-1 and PIM-1), and the time courses of Fe2O3-NPs endocytosis and effects on the expressions of cytokines and interferon responsive genes were compatible. We conclude that Fe2O3-NPs can be endocytosed by Jurkat cells and act intracellularly. Fe2O3-NPs decrease the current density and delay the inactivation and recovery kinetics of Kv1.3 channels in Jurkat cells by oxidizing NADPH and therefore disrupting the redox activity of the Kvβ2 auxiliary subunit, and as a result, lead to changes of the Kv1.3 channel function. These results suggest that iron oxide nanoparticles may affect T cell function by disturbing the activity of Kv1.3 channels. Further, the suppressing effects of Fe2O3-NPs on the expressions of certain inflammatory cytokines and interferon responsive genes suggest that iron

  9. UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling

    PubMed Central

    Moore, Carlene; Cevikbas, Ferda; Pasolli, H. Amalia; Chen, Yong; Kong, Wei; Kempkes, Cordula; Parekh, Puja; Lee, Suk Hee; Kontchou, Nelly-Ange; Yeh, Iwei; Jokerst, Nan Marie; Fuchs, Elaine; Steinhoff, Martin; Liedtke, Wolfgang B.

    2013-01-01

    At our body surface, the epidermis absorbs UV radiation. UV overexposure leads to sunburn with tissue injury and pain. To understand how, we focus on TRPV4, a nonselective cation channel highly expressed in epithelial skin cells and known to function in sensory transduction, a property shared with other transient receptor potential channels. We show that following UVB exposure mice with induced Trpv4 deletions, specifically in keratinocytes, are less sensitive to noxious thermal and mechanical stimuli than control animals. Exploring the mechanism, we find that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage and increased expression of the proalgesic/algogenic mediator endothelin-1. In culture, UVB causes a direct, TRPV4-dependent Ca2+ response in keratinocytes. In mice, topical treatment with a TRPV4-selective inhibitor decreases UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression. In humans, sunburn enhances epidermal expression of TRPV4 and endothelin-1, underscoring the potential of keratinocyte-derived TRPV4 as a therapeutic target for UVB-induced sunburn, in particular pain. PMID:23929777

  10. UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.

    PubMed

    Moore, Carlene; Cevikbas, Ferda; Pasolli, H Amalia; Chen, Yong; Kong, Wei; Kempkes, Cordula; Parekh, Puja; Lee, Suk Hee; Kontchou, Nelly-Ange; Yeh, Iwei; Ye, Iwei; Jokerst, Nan Marie; Fuchs, Elaine; Steinhoff, Martin; Liedtke, Wolfgang B

    2013-08-20

    At our body surface, the epidermis absorbs UV radiation. UV overexposure leads to sunburn with tissue injury and pain. To understand how, we focus on TRPV4, a nonselective cation channel highly expressed in epithelial skin cells and known to function in sensory transduction, a property shared with other transient receptor potential channels. We show that following UVB exposure mice with induced Trpv4 deletions, specifically in keratinocytes, are less sensitive to noxious thermal and mechanical stimuli than control animals. Exploring the mechanism, we find that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage and increased expression of the proalgesic/algogenic mediator endothelin-1. In culture, UVB causes a direct, TRPV4-dependent Ca(2+) response in keratinocytes. In mice, topical treatment with a TRPV4-selective inhibitor decreases UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression. In humans, sunburn enhances epidermal expression of TRPV4 and endothelin-1, underscoring the potential of keratinocyte-derived TRPV4 as a therapeutic target for UVB-induced sunburn, in particular pain.

  11. Ischemia-induced cell depolarization: does the hyperpolarization-activated cation channel HCN2 affect the outcome after stroke in mice?

    PubMed Central

    2013-01-01

    Background Brain ischemia is known to include neuronal cell death and persisting neurological deficits. A lack of oxygen and glucose are considered to be key mediators of ischemic neurodegeneration while the exact mechanisms are yet unclear. In former studies the expression of two different two-pore domain potassium (K2P) channels (TASK1, TREK1) were shown to ameliorate neuronal damage due to cerebral ischemia. In neurons, TASK channels carrying hyperpolarizing K+ leak currents, and the pacemaker channel HCN2, carrying depolarizing Ih, stabilize the membrane potential by a mutual functional interaction. It is assumed that this ionic interplay between TASK and HCN2 channels enhances the resistance of neurons to insults accompanied by extracellular pH shifts. Methods In C57Bl/6 (wildtype, WT), hcn2+/+ and hcn2-/- mice we used an in vivo model of cerebral ischemia (transient middle cerebral artery occlusion (tMCAO)) to depict a functional impact of HCN2 in stroke formation. Subsequent analyses comprise behavioural tests and hcn2 gene expression assays. Results After 60 min of tMCAO induction in WT mice, we collected tissue samples at 6, 12, and 24 h after reperfusion. In the infarcted neocortex, hcn2 expression analyses revealed a nominal peak of hcn2 expression 6 h after reperfusion with a tendency towards lower expression levels with longer reperfusion times. Hcn2 gene expression levels in infarcted basal ganglia did not change after 6 h and 12 h. Only at 24 h after reperfusion, hcn2 expression significantly decreases by ~55%. However, 30 min of tMCAO in hcn2-/- as well as hcn2+/+ littermates induced similar infarct volumes. Behavioural tests for global neurological function (Bederson score) and motor function/coordination (grip test) were performed at day 1 after surgery. Again, we found no differences between the groups. Conclusions Here, we hypothesized that the absence of HCN2, an important functional counter player of TASK channels, affects neuronal survival

  12. Hydroxyproline-induced Helical Disruption in Conantokin Rl-B Affects Subunit-selective Antagonistic Activities toward Ion Channels of N-Methyl-d-aspartate Receptors*

    PubMed Central

    Kunda, Shailaja; Yuan, Yue; Balsara, Rashna D.; Zajicek, Jaroslav; Castellino, Francis J.

    2015-01-01

    Conantokins are ∼20-amino acid peptides present in predatory marine snail venoms that function as allosteric antagonists of ion channels of the N-methyl-d-aspartate receptor (NMDAR). These peptides possess a high percentage of post-/co-translationally modified amino acids, particularly γ-carboxyglutamate (Gla). Appropriately spaced Gla residues allow binding of functional divalent cations, which induces end-to-end α-helices in many conantokins. A smaller number of these peptides additionally contain 4-hydroxyproline (Hyp). Hyp should prevent adoption of the metal ion-induced full α-helix, with unknown functional consequences. To address this disparity, as well as the role of Hyp in conantokins, we have solved the high resolution three-dimensional solution structure of a Gla/Hyp-containing 18-residue conantokin, conRl-B, by high field NMR spectroscopy. We show that Hyp10 disrupts only a small region of the α-helix of the Mn2+·peptide complex, which displays cation-induced α-helices on each terminus of the peptide. The function of conRl-B was examined by measuring its inhibition of NMDA/Gly-mediated current through NMDAR ion channels in mouse cortical neurons. The conRl-B displays high inhibitory selectivity for subclasses of NMDARs that contain the functionally important GluN2B subunit. Replacement of Hyp10 with N8Q results in a Mg2+-complexed end-to-end α-helix, accompanied by attenuation of NMDAR inhibitory activity. However, replacement of Hyp10 with Pro10 allowed the resulting peptide to retain its inhibitory property but diminished its GluN2B specificity. Thus, these modified amino acids, in specific peptide backbones, play critical roles in their subunit-selective inhibition of NMDAR ion channels, a finding that can be employed to design NMDAR antagonists that function at ion channels of distinct NMDAR subclasses. PMID:26048991

  13. 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.

  14. Is microrheometry affected by channel deformation?

    PubMed Central

    Greco, Francesco; Netti, Paolo Antonio

    2016-01-01

    Microrheometry is very important for exploring rheological behaviours of several systems when conventional techniques fail. Microrheometrical measurements are usually carried out in microfluidic devices made of Poly(dimethylsiloxane) (PDMS). Although PDMS is a very cheap material, it is also very easy to deform. In particular, a liquid flowing in a PDMS device, in some circumstances, can effectively deform the microchannel, thus altering the flow conditions. The measure of the fluid relaxation time might be performed through viscoelasticity induced particle migration in microfluidics devices. If the channel walls are deformed by the flow, the resulting measured value of the relaxation time could be not reliable. In this work, we study the effect of channel deformation on particle migration in square-shaped microchannel. Experiments are carried out in several PolyEthylene Oxyde solutions flowing in two devices made of PDMS and Poly(methylmethacrylate) (PMMA). The relevance of wall rigidity on particle migration is investigated, and the corresponding importance of wall rigidity on the determination of the relaxation time of the suspending liquid is examined. PMID:27098237

  15. Calcium Activation of Mougeotia Potassium Channels 1

    PubMed Central

    Lew, Roger R.; Serlin, Bruce S.; Schauf, Charles L.; Stockton, Marsha E.

    1990-01-01

    Phytochrome mediates chloroplast movement in the alga Mougeotia, possibly via changes in cytosolic calcium. It is known to regulate a calcium-activated potassium channel in the algal plasma membrane. As part of a characterization of the potassium channel, we examined the properties of calcium activation. The calcium ionophore A23187 activates the channel at external [Ca2+] as low as 20 micromolar. However, external [Ca2+] is not required for activation of the channel by photoactivated phytochrome. Furthermore, when an inhibitor of calcium release from internal stores, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8), is present, red light no longer stimulates channel activity. We conclude that phytochrome activates the plasma membrane potassium channel by releasing calcium from intracellular calcium vesicles; the elevated cytosolic calcium then stimulates channel activity by an unknown mechanism. In the presence of TMB-8, red light does induce chloroplast rotation; thus, potassium channel activation may not be coupled to chloroplast rotation. PMID:16667356

  16. Thermally activated TRPV3 channels.

    PubMed

    Luo, Jialie; Hu, Hongzhen

    2014-01-01

    TRPV3 is a temperature-sensitive transient receptor potential (TRP) ion channel. The TRPV3 protein functions as a Ca(2+)-permeable nonselective cation channel with six transmembrane domains forming a tetrameric complex. TRPV3 is known to be activated by warm temperatures, synthetic small-molecule chemicals, and natural compounds from plants. Its function is regulated by a variety of physiological factors including extracellular divalent cations and acidic pH, intracellular adenosine triphosphate, membrane voltage, and arachidonic acid. TRPV3 shows a broad expression pattern in both neuronal and non-neuronal tissues including epidermal keratinocytes, epithelial cells in the gut, endothelial cells in blood vessels, and neurons in dorsal root ganglia and CNS. TRPV3 null mice exhibit abnormal hair morphogenesis and compromised skin barrier function. Recent advances suggest that TRPV3 may play critical roles in inflammatory skin disorders, itch, and pain sensation. Thus, identification of selective TRPV3 activators and inhibitors could potentially lead to beneficial pharmacological interventions in several diseases. The intent of this review is to summarize our current knowledge of the tissue expression, structure, function, and mechanisms of activation of TRPV3.

  17. Scorpion venom components that affect ion-channels function

    PubMed Central

    Quintero-Hernández, V.; Jiménez-Vargas, J.M.; Gurrola, G.B.; Valdivia, H.H.F.; Possani, L.D.

    2014-01-01

    SUMMARY The number and types of venom components that affect ion-channel function are reviewed. These are the most important venom components responsible for human intoxication, deserving medical attention, often requiring the use of specific anti-venoms. Special emphasis is given to peptides that recognize Na+-, K+- and Ca++-channels of excitable cells. Knowledge generated by direct isolation of peptides from venom and components deduced from cloned genes, whose amino acid sequences are deposited into databanks are now adays in the order of 1.5 thousands, out of an estimate biodiversity closed to 300,000. Here the diversity of components is briefly reviewed with mention to specific references. Structural characteristic are discussed with examples taken from published work. The principal mechanisms of action of the three different types of peptides are also reviewed. Na+-channel specific venom components usually are modifier of the open and closing kinetic mechanisms of the ion-channels, whereas peptides affecting K+-channels are normally pore blocking agents. The Ryanodine Ca++-channel specific peptides are known for causing sub-conducting stages of the channels conductance and some were shown to be able to internalize penetrating inside the muscle cells. PMID:23891887

  18. Target Promiscuity and Heterogeneous Effects of Tarantula Venom Peptides Affecting Na+ and K+ Ion Channels*

    PubMed Central

    Redaelli, Elisa; Cassulini, Rita Restano; Silva, Deyanira Fuentes; Clement, Herlinda; Schiavon, Emanuele; Zamudio, Fernando Z.; Odell, George; Arcangeli, Annarosa; Clare, Jeffrey J.; Alagón, Alejandro; de la Vega, Ricardo C. Rodríguez; Possani, Lourival D.; Wanke, Enzo

    2010-01-01

    Venom-derived peptide modulators of ion channel gating are regarded as essential tools for understanding the molecular motions that occur during the opening and closing of ion channels. In this study, we present the characterization of five spider toxins on 12 human voltage-gated ion channels, following observations about the target promiscuity of some spider toxins and the ongoing revision of their “canonical” gating-modifying mode of action. The peptides were purified de novo from the venom of Grammostola rosea tarantulas, and their sequences were confirmed by Edman degradation and mass spectrometry analysis. Their effects on seven tetrodotoxin-sensitive Na+ channels, the three human ether-à-go-go (hERG)-related K+ channels, and two human Shaker-related K+ channels were extensively characterized by electrophysiological techniques. All the peptides inhibited ion conduction through all the Na+ channels tested, although with distinctive patterns. The peptides also affected the three pharmaceutically relevant hERG isoforms differently. At higher concentrations, all peptides also modified the gating of the Na+ channels by shifting the activation to more positive potentials, whereas more complex effects were recorded on hERG channels. No effects were evident on the two Shaker-related K+ channels at concentrations well above the IC50 value for the affected channels. Given the sequence diversity of the tested peptides, we propose that tarantula toxins should be considered both as multimode and target-promiscuous ion channel modulators; both features should not be ignored when extracting mechanistic interpretations about ion channel gating. Our observations could also aid in future structure-function studies and might help the development of novel ion channel-specific drugs. PMID:19955179

  19. Single Na+ channels activated by veratridine and batrachotoxin

    PubMed Central

    1987-01-01

    Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded. PMID:2435846

  20. Agonist-activated ion channels

    PubMed Central

    Colquhoun, David

    2006-01-01

    This paper looks at ion channels as an example of the pharmacologist's stock in trade, the action of an agonist on a receptor to produce a response. Looked at in this way, ion channels have been helpful because they are still the only system which is simple enough for quantitative investigation of transduction mechanisms. A short history is given of attempts to elucidate what happens between the time when agonist first binds, and the time when the channel opens. PMID:16402101

  1. Cortical activation changes and sub-threshold affective symptoms are associated with social functioning in a non-clinical population: A multi-channel near-infrared spectroscopy study.

    PubMed

    Fujimaki, Koichiro; Takemoto, Hidenori; Morinobu, Shigeru

    2016-02-28

    Few studies have examined the relationship between social function and brain activation in non-clinical populations. The aim of the present study was to assess this relationship and examine the underlying cortical mechanisms in a non-clinical population. Eighty healthy volunteers performed a serial arithmetic task according to the Uchida-Kraepelin performance test while hemoglobin concentration changes were assessed on the surface of the prefrontal cortex (PFC) using 32-channel near-infrared spectroscopy. Participants were also assessed for quality of life (QOL) using the Short-Form 36-item Questionnaire (SF-36), for affective symptoms using the Zung Self-rating Depression Scale (SDS), for apathy using the Apathy Scale, for feelings of stress using the Stress Arousal Checklist (SACL), and for task performance using the number of answers in a serial arithmetic task. Activity in the frontopolar PFC displayed a significant positive correlation with social functioning on the SF-36. SDS and SACL scores correlated negatively with social functioning. Furthermore, in multiple regression analysis, social functioning was predicted by activity of the frontopolar PFC and SDS scores. These results suggest that the association between changes in cortical activation and sub-threshold affective symptoms may objectively identify individuals with QOL on social functioning.

  2. Molecular mechanism of pharmacological activation of BK channels

    PubMed Central

    Gessner, Guido; Cui, Yong-Mei; Otani, Yuko; Ohwada, Tomohiko; Soom, Malle; Hoshi, Toshinori; Heinemann, Stefan H.

    2012-01-01

    Large-conductance voltage- and Ca2+-activated K+ (Slo1 BK) channels serve numerous cellular functions, and their dysregulation is implicated in various diseases. Drugs activating BK channels therefore bear substantial therapeutic potential, but their deployment has been hindered in part because the mode of action remains obscure. Here we provide mechanistic insight into how the dehydroabietic acid derivative Cym04 activates BK channels. As a representative of NS1619-like BK openers, Cym04 reversibly left-shifts the half-activation voltage of Slo1 BK channels. Using an established allosteric BK gating model, the Cym04 effect can be simulated by a shift of the voltage sensor and the ion conduction gate equilibria toward the activated and open state, respectively. BK activation by Cym04 occurs in a splice variant-specific manner; it does not occur in such Slo1 BK channels using an alternative neuronal exon 9, which codes for the linker connecting the transmembrane segment S6 and the cytosolic RCK1 domain—the S6/RCK linker. In addition, Cym04 does not affect Slo1 BK channels with a two-residue deletion within this linker. Mutagenesis and model-based gating analysis revealed that BK openers, such as Cym04 and NS1619 but not mallotoxin, activate BK channels by functionally interacting with the S6/RCK linker, mimicking site-specific shortening of this purported passive spring, which transmits force from the cytosolic gating ring structure to open the channel's gate. PMID:22331907

  3. 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

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

    PubMed

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

    2015-08-17

    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.

  5. BK channel activation: structural and functional insights

    PubMed Central

    Lee, Urvi S.; Cui, Jianmin

    2010-01-01

    The voltage and Ca2+ activated K+ (BK) channels are involved in the regulation of neurotransmitter release and neuronal excitability. Structurally, BK channels are homologous to voltage- and ligand-gated K+ channels, having a voltage sensor and pore as the membrane-spanning domain and a cytosolic domain containing metal binding sites. Recently published electron cryomicroscopy (cryo-EM) and X-ray crystallographic structures of the BK channel provided the first look into the assembly of these domains, corroborating the close interactions among these domains during channel gating that have been suggested by functional studies. This review discusses these latest findings and an emerging new understanding about BK channel gating and implications for diseases such as epilepsy, in which mutations in BK channel genes have been associated. PMID:20663573

  6. BK channel activators and their therapeutic perspectives

    PubMed Central

    Bentzen, Bo H.; Olesen, Søren-Peter; Rønn, Lars C. B.; Grunnet, Morten

    2014-01-01

    The large conductance calcium- and voltage-activated K+ channel (KCa1.1, BK, MaxiK) is ubiquitously expressed in the body, and holds the ability to integrate changes in intracellular calcium and membrane potential. This makes the BK channel an important negative feedback system linking increases in intracellular calcium to outward hyperpolarizing potassium currents. Consequently, the channel has many important physiological roles including regulation of smooth muscle tone, neurotransmitter release and neuronal excitability. Additionally, cardioprotective roles have been revealed in recent years. After a short introduction to the structure, function and regulation of BK channels, we review the small organic molecules activating BK channels and how these tool compounds have helped delineate the roles of BK channels in health and disease. PMID:25346695

  7. BK channels: multiple sensors, one activation gate.

    PubMed

    Yang, Huanghe; Zhang, Guohui; Cui, Jianmin

    2015-01-01

    Ion transport across cell membranes is essential to cell communication and signaling. Passive ion transport is mediated by ion channels, membrane proteins that create ion conducting pores across cell membrane to allow ion flux down electrochemical gradient. Under physiological conditions, majority of ion channel pores are not constitutively open. Instead, structural region(s) within these pores breaks the continuity of the aqueous ion pathway, thereby serves as activation gate(s) to control ions flow in and out. To achieve spatially and temporally regulated ion flux in cells, many ion channels have evolved sensors to detect various environmental stimuli or the metabolic states of the cell and trigger global conformational changes, thereby dynamically operate the opening and closing of their activation gate. The sensors of ion channels can be broadly categorized as chemical sensors and physical sensors to respond to chemical (such as neural transmitters, nucleotides and ions) and physical (such as voltage, mechanical force and temperature) signals, respectively. With the rapidly growing structural and functional information of different types of ion channels, it is now critical to understand how ion channel sensors dynamically control their gates at molecular and atomic level. The voltage and Ca(2+) activated BK channels, a K(+) channel with an electrical sensor and multiple chemical sensors, provide a unique model system for us to understand how physical and chemical energy synergistically operate its activation gate.

  8. Constitutive Activation of the Shaker Kv Channel

    PubMed Central

    Sukhareva, Manana; Hackos, David H.; Swartz, Kenton J.

    2003-01-01

    In different types of K+ channels the primary activation gate is thought to reside near the intracellular entrance to the ion conduction pore. In the Shaker Kv channel the gate is closed at negative membrane voltages, but can be opened with membrane depolarization. In a previous study of the S6 activation gate in Shaker (Hackos, D.H., T.H. Chang, and K.J. Swartz. 2002. J. Gen. Physiol. 119:521–532.), we found that mutation of Pro 475 to Asp results in a channel that displays a large macroscopic conductance at negative membrane voltages, with only small increases in conductance with membrane depolarization. In the present study we explore the mechanism underlying this constitutively conducting phenotype using both macroscopic and single-channel recordings, and probes that interact with the voltage sensors or the intracellular entrance to the ion conduction pore. Our results suggest that constitutive conduction results from a dramatic perturbation of the closed-open equilibrium, enabling opening of the activation gate without voltage-sensor activation. This mechanism is discussed in the context of allosteric models for activation of Kv channels and what is known about the structure of this critical region in K+ channels. PMID:14557403

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

    PubMed

    Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

    2015-02-10

    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 phosphatidylinositol 4-phosphate [PI(4)P] from the plasma membrane through Ca(2+)-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 and 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.

  10. Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2.

    PubMed

    Ponissery Saidu, Samsudeen; Stephan, Aaron B; Talaga, Anna K; Zhao, Haiqing; Reisert, Johannes

    2013-06-01

    Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca(2+)-activated Cl(-) channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5' rapid amplification of cDNA ends analysis was conducted to characterize the 5' end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5' end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca(2+) sensitivity and that the exon 4-encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.

  11. Stretch-activated cation channel from larval bullfrog skin

    PubMed Central

    Hillyard, Stanley D.; Willumsen, Niels J.; Marrero, Mario B.

    2010-01-01

    Cell-attached patches from isolated epithelial cells from larval bullfrog skin revealed a cation channel that was activated by applying suction (−1 kPa to −4.5 kPa) to the pipette. Activation was characterized by an initial large current spike that rapidly attenuated to a stable value and showed a variable pattern of opening and closing with continuing suction. Current–voltage plots demonstrated linear or inward rectification and single channel conductances of 44–56 pS with NaCl or KCl Ringer's solution as the pipette solution, and a reversal potential (−Vp) of 20–40 mV. The conductance was markedly reduced with N-methyl-D-glucamide (NMDG)-Cl Ringer's solution in the pipette. Neither amiloride nor ATP, which are known to stimulate an apical cation channel in Ussing chamber preparations of larval frog skin, produced channel activation nor did these compounds affect the response to suction. Stretch activation was not affected by varying the pipette concentrations of Ca2+ between 0 mmol l−1 and 4 mmol l−1 or by varying pH between 6.8 and 8.0. However, conductance was reduced with 4 mmol l−1 Ca2+. Western blot analysis of membrane homogenates from larval bullfrog and larval toad skin identified proteins that were immunoreactive with mammalian TRPC1 and TRPC5 (TRPC, canonical transient receptor potential channel) antibodies while homogenates of skin from newly metamorphosed bullfrogs were positive for TRPC1 and TRPC3/6/7 antibodies. The electrophysiological response of larval bullfrog skin resembles that of a stretch-activated cation channel characterized in Xenopus oocytes and proposed to be TRPC1. These results indicate this channel persists in all life stages of anurans and that TRP isoforms may be important for sensory functions of their skin. PMID:20435829

  12. Activation and inhibition of TMEM16A calcium-activated chloride channels.

    PubMed

    Ni, Yu-Li; Kuan, Ai-Seon; Chen, Tsung-Yu

    2014-01-01

    Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca(2+)-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca(2+), Sr(2+), and Ba(2+), and discovered that Mg(2+) competes with Ca(2+) in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore-as revealed by the permeability ratios of these anions-appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1.

  13. Activation and Inhibition of TMEM16A Calcium-Activated Chloride Channels

    PubMed Central

    Ni, Yu-Li; Kuan, Ai-Seon; Chen, Tsung-Yu

    2014-01-01

    Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca2+-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca2+, Sr2+, and Ba2+, and discovered that Mg2+ competes with Ca2+ in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore–as revealed by the permeability ratios of these anions–appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1. PMID:24489780

  14. Cilostazol induces vasodilation through the activation of Ca(2+)-activated K(+) channels in aortic smooth muscle.

    PubMed

    Li, Hongliang; Hong, Da Hye; Son, Youn Kyoung; Na, Sung Hun; Jung, Won-Kyo; Bae, Young Min; Seo, Eun Young; Kim, Sung Joon; Choi, Il-Whan; Park, Won Sun

    2015-07-01

    We investigated the vasorelaxant effect of cilostazol and related signaling pathways in phenylephrine (Phe)-induced pre-contracted aortic rings. Cilostazol induced vasorelaxation in a concentration-dependent manner when aortic rings were pre-contracted with Phe. Application of the voltage-dependent K(+) (Kv) channel inhibitor 4-AP, the ATP-sensitive K(+) (K(ATP)) channel inhibitor glibenclamide, and the inwardly rectifying K(+) (Kir) channel inhibitor Ba(2+) did not alter the vasorelaxant effect of cilostazol; however, pre- and post-treatment with the big-conductance Ca(2+)-activated K(+) (BK(Ca)) channel inhibitor paxilline inhibited the vasorelaxant effect of cilostazol. This vasorelaxant effect of cilostazol was reduced in the presence of an adenylyl cyclase or a protein kinase A (PKA) inhibitor, but not a protein kinase G inhibitor. Inside-out single channel recordings revealed that cilostazol induced the activation of BK(Ca) channel activity. The vasorelaxant effect of cilostazol was not affected by removal of the endothelium. In addition, application of a nitric oxide synthase inhibitor and a small-conductance Ca(2+)-activated K(+) (SK(Ca)) channel inhibitor did not affect cilostazol-induced vasorelaxation. We conclude that cilostazol induced vasorelaxation of the aorta through activation of BK(Ca) channel via a PKA-dependent signaling mechanism independent of endothelium.

  15. Active dendrites, potassium channels and synaptic plasticity.

    PubMed Central

    Johnston, Daniel; Christie, Brian R; Frick, Andreas; Gray, Richard; Hoffman, Dax A; Schexnayder, Lalania K; Watanabe, Shigeo; Yuan, Li-Lian

    2003-01-01

    The dendrites of CA1 pyramidal neurons in the hippocampus express numerous types of voltage-gated ion channel, but the distributions or densities of many of these channels are very non-uniform. Sodium channels in the dendrites are responsible for action potential (AP) propagation from the axon into the dendrites (back-propagation); calcium channels are responsible for local changes in dendritic calcium concentrations following back-propagating APs and synaptic potentials; and potassium channels help regulate overall dendritic excitability. Several lines of evidence are presented here to suggest that back-propagating APs, when coincident with excitatory synaptic input, can lead to the induction of either long-term depression (LTD) or long-term potentiation (LTP). The induction of LTD or LTP is correlated with the magnitude of the rise in intracellular calcium. When brief bursts of synaptic potentials are paired with postsynaptic APs in a theta-burst pairing paradigm, the induction of LTP is dependent on the invasion of the AP into the dendritic tree. The amplitude of the AP in the dendrites is dependent, in part, on the activity of a transient, A-type potassium channel that is expressed at high density in the dendrites and correlates with the induction of the LTP. Furthermore, during the expression phase of the LTP, there are local changes in dendritic excitability that may result from modulation of the functioning of this transient potassium channel. The results support the view that the active properties of dendrites play important roles in synaptic integration and synaptic plasticity of these neurons. PMID:12740112

  16. External barium affects the gating of KCNQ1 potassium channels and produces a pore block via two discrete sites.

    PubMed

    Gibor, Gilad; Yakubovich, Daniel; Peretz, Asher; Attali, Bernard

    2004-07-01

    The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ channels are poorly understood. Here we used external barium to investigate the permeation characteristics of homomeric KCNQ1 channels. We assessed the Ba(2+) binding kinetics and the concentration and voltage dependence of Ba(2+) steady-state block. Our results indicate that extracellular Ba(2+) exerts a series of complex effects, including a voltage-dependent pore blockade as well as unique gating alterations. External barium interacts with the permeation pathway of KCNQ1 at two discrete and nonsequential sites. (a) A slow deep Ba(2+) site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba(2+) site that barely contributes to channel block and mostly affects channel gating by shifting rightward the voltage dependence of activation, slowing activation, speeding up deactivation kinetics, and inhibiting channel inactivation. A model of voltage-dependent block cannot predict the complex impact of Ba(2+) on channel gating in low external K(+) solutions. Ba(2+) binding to this superficial site likely modifies the gating transitions states of KCNQ1. Both sites appear to reside in the permeation pathway as high external K(+) attenuates Ba(2+) inhibition of channel conductance and abolishes its impact on channel gating. Our data suggest that despite the high degree of homology of the pore region among the various K(+) channels, KCNQ1 channels display significant structural and functional uniqueness.

  17. H-Channels Affect Frequency, Power and Amplitude Fluctuations of Neuronal Network Oscillations

    PubMed Central

    Avella Gonzalez, Oscar J.; Mansvelder, Huibert D.; van Pelt, Jaap; van Ooyen, Arjen

    2015-01-01

    Oscillations in network activity are ubiquitous in the brain and are involved in diverse cognitive functions. Oscillation characteristics, such as power, frequency, and temporal structure, depend on both network connectivity and intrinsic cellular properties, such as ion channel composition. An important class of channels, with key roles in regulating cell excitability, are h-channels. The h-current (Ih) is a slow, hyperpolarization-activated, depolarizing current that contributes to neuronal resonance and membrane potential. The impact of Ih on network oscillations, however, remains poorly understood. To elucidate the network effects of Ih, we used a computational model of a generic oscillatory neuronal network consisting of inhibitory and excitatory cells that were externally driven by excitatory action potentials and sustained depolarizing currents. We found that Ih increased the oscillation frequency and, in combination with external action potentials, representing input from areas outside the network, strongly decreased the synchrony of firing. As a consequence, the oscillation power and the duration of episodes during which the network exhibited high-amplitude oscillations were greatly reduced in the presence of Ih. Our results suggest that modulation of Ih or impaired expression of h-channels, as observed in epilepsy, could, by affecting oscillation dynamics, markedly alter network-level activity and potentially influence oscillation-dependent cognitive processes such as learning, memory and attention. PMID:26635594

  18. Regulation of TRPM8 channel activity

    PubMed Central

    Yudin, Yevgen; Rohacs, Tibor

    2011-01-01

    Transient Receptor Potential Melastatin 8 (TRPM8) is a Ca2+ permeable non-selective cation channel directly activated by cold temperatures and chemical agonists such as menthol. It is a well established sensor of environmental cold temperatures, found in peripheral sensory neurons, where its activation evokes depolarization and action potentials. The activity of TRPM8 is regulated by a number of cellular signaling pathways, most notably by phosphoinositides and the activation of phospholipase C. This review will summarize current knowledge on the physiological and pathophysiological roles of TRPM8 and its regulation by various intracellular messenger molecules and signaling pathways. PMID:22061619

  19. Pretreating channel catfish with copper sulfate affects susceptibility to columnaris

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Columnaris disease is a costly bacterial disease of commercially grown channel catfish. Three experiments were conducted to evaluate the effects of pre-exposing channel catfish fingerlings to 0, 1/2, 1, or 2 times the recommended copper sulfate rate for 24 hours on their susceptibility to columnaris...

  20. Sodium channel activation mechanisms. Insights from deuterium oxide substitution

    SciTech Connect

    Alicata, D.A.; Rayner, M.D.; Starkus, J.G. )

    1990-04-01

    Schauf and Bullock, using Myxicola giant axons, demonstrated that solvent substitution with deuterium oxide (D2O) significantly affects both sodium channel activation and inactivation kinetics without corresponding changes in gating current or tail current rates. They concluded that (a) no significant component of gating current derives from the final channel opening step, and (b) channels must deactivate (during tail currents) by a different pathway from that used in channel opening. By contrast, Oxford found in squid axons that when a depolarizing pulse is interrupted by a brief (approximately 100 microseconds) return to holding potential, subsequent reactivation (secondary activation) is very rapid and shows almost monoexponential kinetics. Increasing the interpulse interval resulted in secondary activation rate returning towards control, sigmoid (primary activation) kinetics. He concluded that channels open and close (deactivate) via the same pathway. We have repeated both sets of observations in crayfish axons, confirming the results obtained in both previous studies, despite the apparently contradictory conclusions reached by these authors. On the other hand, we find that secondary activation after a brief interpulse interval (50 microseconds) is insensitive to D2O, although reactivation after longer interpulse intervals (approximately 400 microseconds) returns towards a D2O sensitivity similar to that of primary activation. We conclude that D2O-sensitive primary activation and D2O-insensitive tail current deactivation involve separate pathways. However, D2O-insensitive secondary activation involves reversal of the D2O-insensitive deactivation step. These conclusions are consistent with parallel gate models, provided that one gating particle has a substantially reduced effective valence.

  1. Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2

    PubMed Central

    Ponissery Saidu, Samsudeen; Stephan, Aaron B.; Talaga, Anna K.

    2013-01-01

    Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca2+-activated Cl− channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5′ rapid amplification of cDNA ends analysis was conducted to characterize the 5′ end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5′ end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca2+ sensitivity and that the exon 4–encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties. PMID:23669718

  2. Iron status of channel catfish Ictalurus punctatus affected by channel catfish anemia and response to parenteral iron

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Originally reported in 1983, channel catfish anemia (CCA), also ‘white lip’ or ‘no blood,’ is a major idiopathic disease affecting commercial production in the Mississippi Delta region of the USA. Affected individuals are characterized by lethargy, anorexia, extreme pallor, and packed cell volumes o...

  3. Ca2+-activated K channels in parotid acinar cells

    PubMed Central

    Romanenko, Victor G; Thompson, Jill

    2010-01-01

    Fluid secretion relies on a close interplay between Ca2+-activated Cl and K channels. Salivary acinar cells contain both large conductance, BK, and intermediate conductance, IK1, K channels. Physiological fluid secretion occurs with only modest (<500 nM) increases in intracellular Ca2+ levels but BK channels in many cell types and in heterologous expression systems require very high concentrations for significant activation. We report here our efforts to understand this apparent contradiction. We determined the Ca2+ dependence of IK1 and BK channels in mouse parotid acinar cells. IK1 channels activated with an apparent Ca2+ affinity of about 350 nM and a hill coefficient near 3. Native parotid BK channels activated at similar Ca2+ levels unlike the BK channels in other cell types. Since the parotid BK channel is encoded by an uncommon splice variant, we examined this clone in a heterologous expression system. In contrast to the native parotid channel, activation of this expressed “parslo” channel required very high levels of Ca2+. In order to understand the functional basis for the special properties of the native channels, we analyzed the parotid BK channel in the context of the horrigan-Aldrich model of BK channel gating. We found that the shifted activation of parotid BK channels resulted from a hyperpolarizing shift of the voltage dependence of voltage sensor activation and channel opening and included a large change in the coupling of these two processes. PMID:20519930

  4. Golgi Anti-apoptotic Proteins Are Highly Conserved Ion Channels That Affect Apoptosis and Cell Migration*

    PubMed Central

    Carrara, Guia; Saraiva, Nuno; Parsons, Maddy; Byrne, Bernadette; Prole, David L.; Taylor, Colin W.; Smith, Geoffrey L.

    2015-01-01

    Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca2+ content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently. PMID:25713081

  5. Impact of Volcanic Activity on AMC Channel Operations

    DTIC Science & Technology

    2014-06-13

    IMPACT OF VOLCANIC ACTIVITY ON AMC CHANNEL OPERATIONS GRADUATE RESEARCH PROJECT Matthew D... VOLCANIC ACTIVITY ON AMC CHANNEL OPERATIONS GRADUATE RESEARCH PROJECT Presented to the Faculty Department of Operational Sciences...AFIT-ENS-GRP-14-J-11 IMPACT OF VOLCANIC ACTIVITY ON AMC CHANNEL OPERATIONS Matthew D. Meshanko, BS, MA Major, USAF

  6. Pretreating channel catfish with copper sulfate affects susceptibility to columnaris

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Columnaris disease is one of the most important bacterial diseases of channel catfish, Ictalurus punctatus, commercially grown in the US. This disease can greatly diminish the profitability of aquaculture operations by large-scale mortality events, particularly in the fingerling production phase. ...

  7. Two recombinant depressant scorpion neurotoxins differentially affecting mammalian sodium channels.

    PubMed

    Yuan, Yuzhe; Luo, Lan; Peigneur, Steve; Tytgat, Jan; Zhu, Shunyi

    2010-07-01

    The scorpion depressant toxins are a group of evolutionarily conserved polypeptides targeting sodium channels, which show preferential ability to induce flaccid paralysis in insects, making them attractive candidates for the construction of transgenic plants or viral vectors to control pests. In this study, two new depressant toxin-like peptides (BmKITc and BmKITc2) differing only at position 52 (Lys for Thr) were produced in Escherichia coli. Circular dichroism analysis indicated that these two recombinant peptides display a typical structural feature similar to native scorpion toxins. They both cause a maintained current component at the last phase of inactivation of the insect sodium channel DmNav1/tipE expressed in Xenopus oocytes and interestingly, they do not produce a beta effect despite of their primary structure as beta-toxins. Furthermore, an inhibitory effect with BmKITc but not with BmKITc2 was observed on TTX-R sodium currents in rat DRG neurons. We hypothesize that such differential potency highlights a crucial role of lysine 52 in channel selectivity. Our results therefore indicate that, in spite of the general idea, not all scorpion depressant toxins interact with mammalian and/or insect sodium channels in the same manner.

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

    PubMed

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

    2009-09-16

    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.

  9. The Antibacterial Activity of Human Neutrophils and Eosinophils Requires Proton Channels but Not BK Channels

    PubMed Central

    Femling, Jon K.; Cherny, Vladimir V.; Morgan, Deri; Rada, Balázs; Davis, A. Paige; Czirják, Gabor; Enyedi, Peter; England, Sarah K.; Moreland, Jessica G.; Ligeti, Erzsébet; Nauseef, William M.; DeCoursey, Thomas E.

    2006-01-01

    Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K+ (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853–858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA2 or the production of superoxide anion (\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2^{.}}^{-}\\end{equation*}\\end{document}). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn2+ inhibition of NADPH oxidase activity assessed by H2O2 production, thus validating previous studies showing that Zn2+ inhibited \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2^{.}}^{-}\\end{equation*}\\end{document} production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and

  10. Metal interactions with voltage- and receptor-activated ion channels.

    PubMed Central

    Vijverberg, H P; Oortgiesen, M; Leinders, T; van Kleef, R G

    1994-01-01

    Effects of Pb and several other metal ions on various distinct types of voltage-, receptor- and Ca-activated ion channels have been investigated in cultured N1E-115 mouse neuroblastoma cells. Experiments were performed using the whole-cell voltage clamp and single-channel patch clamp techniques. External superfusion of nanomolar to submillimolar concentrations of Pb causes multiple effects on ion channels. Barium current through voltage-activated Ca channels is blocked by micromolar concentrations of Pb, whereas voltage-activated Na current appears insensitive. Neuronal type nicotinic acetylcholine receptor-activated ion current is blocked by nanomolar concentrations of Pb and this block is reversed at micromolar concentrations. Serotonin 5-HT3 receptor-activated ion current is much less sensitive to Pb. In addition, external superfusion with micromolar concentrations of Pb as well as of Cd and aluminum induces inward current, associated with the direct activation of nonselective cation channels by these metal ions. In excised inside-out membrane patches of neuroblastoma cells, micromolar concentrations of Ca activate small (SK) and big (BK) Ca-activated K channels. Internally applied Pb activates SK and BK channels more potently than Ca, whereas Cd is approximately equipotent to Pb with respect to SK channel activation, but fails to activate BK channels. The results show that metal ions cause distinct, selective effects on the various types of ion channels and that metal ion interaction sites of ion channels may be highly selective for particular metal ions. PMID:7531139

  11. Epilepsy-Related Slack Channel Mutants Lead to Channel Over-Activity by Two Different Mechanisms.

    PubMed

    Tang, Qiong-Yao; Zhang, Fei-Fei; Xu, Jie; Wang, Ran; Chen, Jian; Logothetis, Diomedes E; Zhang, Zhe

    2016-01-05

    Twelve sodium-activated potassium channel (KCNT1, Slack) genetic mutants have been identified from severe early-onset epilepsy patients. The changes in biophysical properties of these mutants and the underlying mechanisms causing disease remain elusive. Here, we report that seven of the 12 mutations increase, whereas one mutation decreases, the channel's sodium sensitivity. Two of the mutants exhibit channel over-activity only when the intracellular Na(+) ([Na(+)]i) concentration is ∼80 mM. In contrast, single-channel data reveal that all 12 mutants increase the maximal open probability (Po). We conclude that these mutant channels lead to channel over-activity predominantly by increasing the ability of sodium binding to activate the channel, which is indicated by its maximal Po. The sodium sensitivity of these epilepsy causing mutants probably determines the [Na(+)]i concentration at which these mutants exert their pathological effects.

  12. Single-channel properties of BK-type calcium-activated potassium channels at a cholinergic presynaptic nerve terminal

    PubMed Central

    Sun, Xiao-Ping; Schlichter, Lyanne C; Stanley, Elis F

    1999-01-01

    A high-conductance calcium-activated potassium channel (BK KCa) was characterized at a cholinergic presynaptic nerve terminal using the calyx synapse isolated from the chick ciliary ganglion.The channel had a conductance of 210 pS in a 150 mM:150 mM K+ gradient, was highly selective for K+ over Na+, and was sensitive to block by external charybdotoxin or tetraethylammonium (TEA) and by internal Ba2+. At +60 mV it was activated by cytoplasmic calcium [Ca2+]i with a Kd of ≈0.5 μM and a Hill coefficient of ≈2.0. At 10 μM [Ca2+]i the channel was 50 % activated (V½) at -8.0 mV with a voltage dependence (Boltzmann slope-factor) of 32.7 mV. The V½ values hyperpolarized with an increase in [Ca2+]i while the slope factors decreased. There were no overt differences in conductance or [Ca2+]i sensitivity between BK channels from the transmitter release face and the non-release face.Open and closed times were fitted by two and three exponentials, respectively. The slow time constants were strongly affected by both [Ca2+]i and membrane potential changes.In cell-attached patch recordings BK channel opening was enhanced by a prepulse permissive for calcium influx through the patch, suggesting that the channel can be activated by calcium ion influx through neighbouring calcium channels.The properties of the presynaptic BK channel are well suited for rapid activation during the presynaptic depolarization and Ca2+ influx that are associated with transmitter release. This channel may play an important role in terminating release by rapid repolarization of the action potential. PMID:10420003

  13. Bisandrographolide from Andrographis paniculata activates TRPV4 channels.

    PubMed

    Smith, Paula L; Maloney, Katherine N; Pothen, Randy G; Clardy, Jon; Clapham, David E

    2006-10-06

    Many transient receptor potential (TRP) channels are activated or blocked by various compounds found in plants; two prominent examples include the activation of TRPV1 channels by capsaicin and the activation of TRPM8 channels by menthol. We sought to identify additional plant compounds that are active on other types of TRP channels. We screened a library of extracts from 50 Chinese herbal plants using a calcium-imaging assay to find compounds active on TRPV3 and TRPV4 channels. An extract from the plant Andrographis paniculata potently activated TRPV4 channels. The extract was fractionated further, and the active compound was identified as bisandrographolide A (BAA). We used purified compound to characterize the activity of BAA on certain TRPV channel subtypes. Although BAA activated TRPV4 channels with an EC(50) of 790-950 nm, it did not activate or block activation of TRPV1, TRPV2, or TRPV3 channels. BAA activated a large TRPV4-like current in immortalized mouse keratinocytes (308 cells) that have been shown to express TRPV4 protein endogenously. This compound also activated TRPV4 currents in cell-free outside-out patches from HEK293T cells overexpressing TRPV4 cDNA, suggesting that BAA can activate the channel in a membrane-delimited manner. Another related compound, andrographolide, found in abundance in the plant Andrographis was unable to activate or block activation of TRPV4 channels. These experiments show that BAA activates TRPV4 channels, and we discuss the possibility that activation of TRPV4 by BAA could play a role in some of the effects of Andrographis extract described in traditional medicine.

  14. A quantized mechanism for activation of pannexin channels

    PubMed Central

    Chiu, Yu-Hsin; Jin, Xueyao; Medina, Christopher B.; Leonhardt, Susan A.; Kiessling, Volker; Bennett, Brad C.; Shu, Shaofang; Tamm, Lukas K.; Yeager, Mark; Ravichandran, Kodi S.; Bayliss, Douglas A.

    2017-01-01

    Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels. PMID:28134257

  15. 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(+).

  16. Design of a specific activator for skeletal muscle sodium channels uncovers channel architecture.

    PubMed

    Cohen, Lior; Ilan, Nitza; Gur, Maya; Stühmer, Walter; Gordon, Dalia; Gurevitz, Michael

    2007-10-05

    Gating modifiers of voltage-gated sodium channels (Na(v)s) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion beta-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNa(v)1.2a) and skeletal muscle (rNa(v)1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4(F14A/E15A/E28R), of rNa(v)1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4(F14A/E15A/E28R) was tested using an rNa(v)1.4 mutant carrying the same mutation present in the genetic disorder hypokalemic periodic paralysis. The activator restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin residues involved in the construction of Css4(F14A/E15A/E28R) and residues whose equivalents in the rat brain channel rNa(v)1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and E592A at Domain-2/voltage sensor segments 1-2 (D2/S1-S2), R27Q and E1251N at D3/SS2-S6, and E28R with both E650A at D2/S3-S4 and E1251N at D3/SS2-S6. These results show that despite the differences in interactions with the rat brain and skeletal muscle Na(v)s, Css4 recognizes a similar region on both channel subtypes. Moreover, our data indicate that the S3-S4 loop of the voltage sensor module in Domain-2 is in very close proximity to the SS2-S6 segment of the pore module of Domain-3 in rNa(v)1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Na(v)s resembles that of voltage-gated potassium channels.

  17. Pseudohypoaldosteronism type 1 and Liddle’s syndrome mutations that affect the single-channel properties of the epithelial Na+ channel

    PubMed Central

    Boiko, Nina; Kucher, Volodymyr; Stockand, James D

    2015-01-01

    These studies test whether three disease-causing mutations in genes (SCNN1A and SCNN1G) encoding subunits of the epithelial Na+ channel, ENaC, affect the biophysical and gating properties of this important renal ion channel. The S562P missense mutation in αENaC and the K106_S108delinsN mutation in γENaC are associated with pseudohypoaldosteronism type 1 (PHA1). The N530S missense mutation in γENaC causes Liddle’s syndrome. Incorporation of S562P into αENaC and K106_S108N into γENaC resulted in significant decreases in macroscopic ENaC currents. Conversely, incorporation of N530S into γENaC increased macroscopic ENaC current. The S562P substitution resulted in a nonfunctional channel. The K106_S108N mutation produced a functional channel having a normal macroscopic current–voltage relation, there was a slight but significant decrease in unitary conductance and a marked decrease in single-channel open probability. The N530S substitution increased single-channel open probability having no effect on the macroscopic current–voltage relation or unitary conductance of the channel. These findings are consistent with mutation of residues at 562 in αENaC and 530 in γENaC, and a 3′ splice site in SCNN1G (318-1 G→A; K106_108SdelinsN) resulting in aberrant ENaC activity due to changes in the biophysical and gating properties of the channel. Such changes likely contribute to the cellular mechanism underpinning the PHA1 and Liddle’s syndrome caused by these mutations in ENaC subunits. PMID:26537344

  18. Gating and flickery block differentially affected by rubidium in homomeric KCNQ1 and heteromeric KCNQ1/KCNE1 potassium channels.

    PubMed Central

    Pusch, M; Bertorello, L; Conti, F

    2000-01-01

    The voltage-gated potassium channel KCNQ1 associates with the small KCNE1 subunit to form the cardiac IKs delayed rectifier potassium current and mutations in both genes can lead to the long QT syndrome. KCNQ1 can form functional homotetrameric channels, however with drastically different biophysical properties compared to heteromeric KCNQ1/KCNE1 channels. We analyzed gating and conductance of these channels expressed in Xenopus oocytes using the two-electrode voltage-clamp and the patch-clamp technique and high extracellular potassium (K) and rubidium (Rb) solutions. Inward tail currents of homomeric KCNQ1 channels are increased about threefold upon substitution of 100 mM potassium with 100 mM rubidium despite a smaller rubidium permeability, suggesting an effect of rubidium on gating. However, the kinetics of tail currents and the steady-state activation curve are only slightly changed in rubidium. Single-channel amplitude at negative voltages was estimated by nonstationary noise analysis, and it was found that rubidium has only a small effect on homomeric channels (1.2-fold increase) when measured at a 5-kHz bandwidth. The apparent single-channel conductance was decreased after filtering the data at lower cutoff frequencies indicative of a relatively fast "flickery/block" process. The relative conductance in rubidium compared to potassium increased at lower cutoff frequencies (about twofold at 10 Hz), suggesting that the main effect of rubidium is to decrease the probability of channel blockage leading to an increase of inward currents without large changes in gating properties. Macroscopic inward tail currents of heteromeric KCNQ1/KCNE1 channels in rubidium are reduced by about twofold and show a pronounced sigmoidal time course that develops with a delay similar to the inactivation process of homomeric KCNQ1, and is indicative of the presence of several open states. The single channel amplitude of heteromers is about twofold smaller in rubidium than in

  19. Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels

    PubMed Central

    Proks, Peter; Puljung, Michael C.; Vedovato, Natascia; Sachse, Gregor; Mulvaney, Rachel; Ashcroft, Frances M.

    2016-01-01

    KATP channels act as key regulators of electrical excitability by coupling metabolic cues—mainly intracellular adenine nucleotide concentrations—to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P2 and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing KATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377720

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

    PubMed

    Cui, Yuanyuan; Yang, Fan; Cao, Xu; Yarov-Yarovoy, Vladimir; Wang, KeWei; Zheng, Jie

    2012-04-01

    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.

  1. Regulation of Sodium Channel Activity by Capping of Actin Filaments

    PubMed Central

    Shumilina, Ekaterina V.; Negulyaev, Yuri A.; Morachevskaya, Elena A.; Hinssen, Horst; Khaitlina, Sofia Yu

    2003-01-01

    Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of actin filaments and inactivated by G-actin under ionic conditions promoting rapid actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-actin, CapZ prevented actin-induced channel inactivation, and this effect occurred at CapZ/actin molar ratios from 1:5 to 1:100. When actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of actin filaments at the plasma membrane due to the modification of actin dynamics by CapZ. No effect on the channel activity was observed in response to F-actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that actin-capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells. PMID:12686620

  2. Long distance effect on ligand-gated ion channels extracellular domain may affect interactions with the intracellular machinery.

    PubMed

    Garret, Maurice; Boué-Grabot, Eric; Taly, Antoine

    2014-01-01

    Modulation of receptor trafficking is critical for controlling neurotransmission. A γ2(R43Q) point mutation on GABAA receptor subunit is linked to epilepsy in human. We recently analyzed the effect of this amino-acid substitution on GABAA receptor trafficking and showed that this mutation as well as agonist application, both affecting GABAA receptor extracellular domain, have an effect on receptor endocytosis. By comparing homology models based on ligand gated ion channels in their active and resting states, we reveal that the γ2R43 domain is located in a loop that is affected by motion resulting from receptor activation. Taken together, these results suggest that endocytosis of GABAA receptors is linked to agonist induced conformational changes. We propose that ligand or modulator binding is followed by a whole chain of interconnections, including the intracellular domain, that may influence ligand-gated channel trafficking.

  3. Long distance effect on ligand-gated ion channels extracellular domain may affect interactions with the intracellular machinery

    PubMed Central

    Garret, Maurice; Boué-Grabot, Eric; Taly, Antoine

    2014-01-01

    Modulation of receptor trafficking is critical for controlling neurotransmission. A γ2(R43Q) point mutation on GABAA receptor subunit is linked to epilepsy in human. We recently analyzed the effect of this amino-acid substitution on GABAA receptor trafficking and showed that this mutation as well as agonist application, both affecting GABAA receptor extracellular domain, have an effect on receptor endocytosis. By comparing homology models based on ligand gated ion channels in their active and resting states, we reveal that the γ2R43 domain is located in a loop that is affected by motion resulting from receptor activation. Taken together, these results suggest that endocytosis of GABAA receptors is linked to agonist induced conformational changes. We propose that ligand or modulator binding is followed by a whole chain of interconnections, including the intracellular domain, that may influence ligand-gated channel trafficking. PMID:25254078

  4. Mechanisms of Activation of Voltage-Gated Potassium Channels

    PubMed Central

    Grizel, A. V.; Glukhov, G. S.; Sokolova, O. S.

    2014-01-01

    Voltage-gated potassium ion channels (Kv) play an important role in a variety of cellular processes, including the functioning of excitable cells, regulation of apoptosis, cell growth and differentiation, the release of neurotransmitters and hormones, maintenance of cardiac activity, etc. Failure in the functioning of Kv channels leads to severe genetic disorders and the development of tumors, including malignant ones. Understanding the mechanisms underlying Kv channels functioning is a key factor in determining the cause of the diseases associated with mutations in the channels, and in the search for new drugs. The mechanism of activation of the channels is a topic of ongoing debate, and a consensus on the issue has not yet been reached. This review discusses the key stages in studying the mechanisms of functioning of Kv channels and describes the basic models of their activation known to date. PMID:25558391

  5. 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

  6. Purinergic regulation of CFTR and Ca(2+)-activated Cl(-) channels and K(+) channels in human pancreatic duct epithelium.

    PubMed

    Wang, Jing; Haanes, Kristian A; Novak, Ivana

    2013-04-01

    Purinergic agonists have been considered for the treatment of respiratory epithelia in cystic fibrosis (CF) patients. The pancreas, one of the most seriously affected organs in CF, expresses various purinergic receptors. Studies on the rodent pancreas show that purinergic signaling regulates pancreatic secretion. In the present study we aim to identify Cl(-) and K(+) channels in human pancreatic ducts and their regulation by purinergic receptors. Human pancreatic duct epithelia formed by Capan-1 or CFPAC-1 cells were studied in open-circuit Ussing chambers. In Capan-1 cells, ATP/UTP effects were dependent on intracellular Ca(2+). Apically applied ATP/UTP stimulated CF transmembrane conductance regulator (CFTR) and Ca(2+)-activated Cl(-) (CaCC) channels, which were inhibited by CFTRinh-172 and niflumic acid, respectively. The basolaterally applied ATP stimulated CFTR. In CFPAC-1 cells, which have mutated CFTR, basolateral ATP and UTP had negligible effects. In addition to Cl(-) transport in Capan-1 cells, the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DC-EBIO) and clotrimazole indicated functional expression of the intermediate conductance K(+) channels (IK, KCa3.1). The apical effects of ATP/UTP were greatly potentiated by the IK channel opener DC-EBIO. Determination of RNA and protein levels revealed that Capan-1 cells have high expression of TMEM16A (ANO1), a likely CaCC candidate. We conclude that in human pancreatic duct cells ATP/UTP regulates via purinergic receptors both Cl(-) channels (TMEM16A/ANO1 and CFTR) and K(+) channels (IK). The K(+) channels provide the driving force for Cl(-)-channel-dependent secretion, and luminal ATP provided locally or secreted from acini may potentiate secretory processes. Future strategies in augmenting pancreatic duct function should consider sidedness of purinergic signaling and the essential role of K(+) channels.

  7. Allosterism and Structure in Thermally Activated Transient Receptor Potential Channels.

    PubMed

    Diaz-Franulic, Ignacio; Poblete, Horacio; Miño-Galaz, Germán; González, Carlos; Latorre, Ramón

    2016-07-05

    The molecular sensors that mediate temperature changes in living organisms are a large family of proteins known as thermosensitive transient receptor potential (TRP) ion channels. These membrane proteins are polymodal receptors that can be activated by cold or hot temperatures, depending on the channel subtype, voltage, and ligands. The stimuli sensors are allosterically coupled to a pore domain, increasing the probability of finding the channel in its ion conductive conformation. In this review we first discuss the allosteric coupling between the temperature and voltage sensor modules and the pore domain, and then discuss the thermodynamic foundations of thermo-TRP channel activation. We provide a structural overview of the molecular determinants of temperature sensing. We also posit an anisotropic thermal diffusion model that may explain the large temperature sensitivity of TRP channels. Additionally, we examine the effect of several ligands on TRP channel function and the evidence regarding their mechanisms of action.

  8. Hypoglycemia-activated K+ channels in hippocampal neurons.

    PubMed

    Tromba, C; Salvaggio, A; Racagni, G; Volterra, A

    1992-08-31

    Channels linking the electrical and metabolic activities of cells (KATP channels) have been described in various tissues, including some brain areas (hypothalamus, cerebral cortex and substantia nigra). Here we report the existence in hippocampal neurons of K+ permeant channels whose activity is regulated by extracellular glucose. They are open at the cell resting potential and respond to transient hypoglycemia with a reversible increase in activity. The one type so far characterized has a conductance of approximately 100 pS in isotonic K+, is inhibited by the sulphonylurea glibenclamide (1 microM), and is activated by the potassium channel opener lemakalim (0.1-1 microM). These data provide a direct demonstration of the presence, in hippocampal neurons, of glucose-sensitive channels that could belong to the KATP family.

  9. CNTF-Treated Astrocyte Conditioned Medium Enhances Large-Conductance Calcium-Activated Potassium Channel Activity in Rat Cortical Neurons.

    PubMed

    Sun, Meiqun; Liu, Hongli; Xu, Huanbai; Wang, Hongtao; Wang, Xiaojing

    2016-08-01

    Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM's effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM's effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM's effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKβ3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p < 0.05). CNTF-ACM produced a significant increase in BKα1 and BKβ3 expression (p < 0.05) but had no significant effect upon SK2 or SK3 expression (p > 0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p > 0.05) as well as BKα1 and BKβ3 expression in CNTF-ACM-treated neurons (p > 0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons.

  10. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  11. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  12. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  13. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  14. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  15. Niflumic acid affects store-operated Ca(2+)-permeable (SOC) and Ca (2+)-dependent K (+) and Cl (-) ion channels and induces apoptosis in K562 cells.

    PubMed

    Kucherenko, Yuliya V; Lang, Florian

    2014-07-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells. However, the precise mechanisms by which NSAIDs facilitate apoptosis in tumor cells are not clear. In the present study, we show that niflumic acid (NA), a member of the fenamates group of NSAIDs and Cl(-) and Ca(2+)-activated Cl(-) (CAC) channels blocker, induced apoptosis (by ~8 %, 24 h treatment) and potentiated (by 8-10 %) apoptotic effect of endoplasmic reticulum Ca(2+) mobilizer thapsigargin (Tg) in human erythroleukemic K562 cell line. The whole-cell patch clamp and Fluo-3 flow cytometric experiments confirmed an inhibitory effect of NA (100 and 300 µM) on store-operated (SOC) channels. We also found that NA-blocked CAC channels were activated by acute application of Tg (2 µM) in K562 cells. NA blockage of CAC channels was accompanied by activation of Ca(2+)-activated K(+) (SK4) channels. The observed effects of NA were not connected with COX-2 inhibition since 100-nM NA (IC50 for COX-2 inhibition) did not induce either apoptosis or affect the channels activity. We conclude that inhibition of SOC channels plays a major role in NA-induced apoptosis. Increased apoptotic levels in Tg-treated K562 cells in the presence of NA may be due to the blockage of CAC and stimulation of SK4 channels in addition to SOC channels inhibition.

  16. Zinc pyrithione-mediated activation of voltage-gated KCNQ potassium channels rescues epileptogenic mutants.

    PubMed

    Xiong, Qiaojie; Sun, Haiyan; Li, Min

    2007-05-01

    KCNQ potassium channels are activated by changes in transmembrane voltage and play an important role in controlling electrical excitability. Human mutations of KCNQ2 and KCNQ3 potassium channel genes result in reduction or loss of channel activity and cause benign familial neonatal convulsions (BFNCs). Thus, small molecules capable of augmenting KCNQ currents are essential both for understanding the mechanism of channel activity and for developing therapeutics. We performed a high-throughput screen in search for agonistic compounds potentiating KCNQ potassium channels. Here we report identification of a new opener, zinc pyrithione (1), which activates both recombinant and native KCNQ M currents. Interactions with the channel protein cause an increase of single-channel open probability that could fully account for the overall conductance increase. Separate point mutations have been identified that either shift the concentration dependence or affect potentiation efficacy, thereby providing evidence for residues influencing ligand binding and downstream events. Furthermore, zinc pyrithione is capable of rescuing the mutant channels causal to BFNCs.

  17. Nitric Oxide Regulates Neuronal Activity via Calcium-Activated Potassium Channels

    PubMed Central

    Zhong, Lei Ray; Estes, Stephen; Artinian, Liana; Rehder, Vincent

    2013-01-01

    Nitric oxide (NO) is an unconventional membrane-permeable messenger molecule that has been shown to play various roles in the nervous system. How NO modulates ion channels to affect neuronal functions is not well understood. In gastropods, NO has been implicated in regulating the feeding motor program. The buccal motoneuron, B19, of the freshwater pond snail Helisoma trivolvis is active during the hyper-retraction phase of the feeding motor program and is located in the vicinity of NO-producing neurons in the buccal ganglion. Here, we asked whether B19 neurons might serve as direct targets of NO signaling. Previous work established NO as a key regulator of growth cone motility and neuronal excitability in another buccal neuron involved in feeding, the B5 neuron. This raised the question whether NO might modulate the electrical activity and neuronal excitability of B19 neurons as well, and if so whether NO acted on the same or a different set of ion channels in both neurons. To study specific responses of NO on B19 neurons and to eliminate indirect effects contributed by other cells, the majority of experiments were performed on single cultured B19 neurons. Addition of NO donors caused a prolonged depolarization of the membrane potential and an increase in neuronal excitability. The effects of NO could mainly be attributed to the inhibition of two types of calcium-activated potassium channels, apamin-sensitive and iberiotoxin-sensitive potassium channels. NO was found to also cause a depolarization in B19 neurons in situ, but only after NO synthase activity in buccal ganglia had been blocked. The results suggest that NO acts as a critical modulator of neuronal excitability in B19 neurons, and that calcium-activated potassium channels may serve as a common target of NO in neurons. PMID:24236040

  18. Flow-activated ion channels in vascular endothelium.

    PubMed

    Gautam, Mamta; Gojova, Andrea; Barakat, Abdul I

    2006-01-01

    The ability of vascular endothelial cells (ECs) to respond to fluid mechanical forces associated with blood flow is essential for flow-mediated vasoregulation and arterial wall remodeling. Abnormalities in endothelial responses to flow also play a role in the development of atherosclerosis. Although our understanding of the endothelial signaling pathways stimulated by flow has greatly increased over the past two decades, the mechanisms by which ECs sense flow remain largely unknown. Activation of flow-sensitive ion channels is among the fastest known endothelial responses to flow; therefore, these ion channels have been proposed as candidate flow sensors. This review focuses on: 1) describing the various types of flow-sensitive ion channels that have been reported in ECs, 2) discussing the implications of activation of these ion channels for endothelial function, and 3) proposing candidate mechanisms for activation of flow-sensitive ion channels.

  19. 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

  20. The regulation of BK channel activity by pre- and post-translational modifications.

    PubMed

    Kyle, Barry D; Braun, Andrew P

    2014-01-01

    Large conductance, Ca(2+)-activated K(+) (BK) channels represent an important pathway for the outward flux of K(+) ions from the intracellular compartment in response to membrane depolarization, and/or an elevation in cytosolic free [Ca(2+)]. They are functionally expressed in a range of mammalian tissues (e.g., nerve and smooth muscles), where they can either enhance or dampen membrane excitability. The diversity of BK channel activity results from the considerable alternative mRNA splicing and post-translational modification (e.g., phosphorylation) of key domains within the pore-forming α subunit of the channel complex. Most of these modifications are regulated by distinct upstream cell signaling pathways that influence the structure and/or gating properties of the holo-channel and ultimately, cellular function. The channel complex may also contain auxiliary subunits that further affect channel gating and behavior, often in a tissue-specific manner. Recent studies in human and animal models have provided strong evidence that abnormal BK channel expression/function contributes to a range of pathologies in nerve and smooth muscle. By targeting the upstream regulatory events modulating BK channel behavior, it may be possible to therapeutically intervene and alter BK channel expression/function in a beneficial manner.

  1. The regulation of BK channel activity by pre- and post-translational modifications

    PubMed Central

    Kyle, Barry D.; Braun, Andrew P.

    2014-01-01

    Large conductance, Ca2+-activated K+ (BK) channels represent an important pathway for the outward flux of K+ ions from the intracellular compartment in response to membrane depolarization, and/or an elevation in cytosolic free [Ca2+]. They are functionally expressed in a range of mammalian tissues (e.g., nerve and smooth muscles), where they can either enhance or dampen membrane excitability. The diversity of BK channel activity results from the considerable alternative mRNA splicing and post-translational modification (e.g., phosphorylation) of key domains within the pore-forming α subunit of the channel complex. Most of these modifications are regulated by distinct upstream cell signaling pathways that influence the structure and/or gating properties of the holo-channel and ultimately, cellular function. The channel complex may also contain auxiliary subunits that further affect channel gating and behavior, often in a tissue-specific manner. Recent studies in human and animal models have provided strong evidence that abnormal BK channel expression/function contributes to a range of pathologies in nerve and smooth muscle. By targeting the upstream regulatory events modulating BK channel behavior, it may be possible to therapeutically intervene and alter BK channel expression/function in a beneficial manner. PMID:25202279

  2. Small Conductance Ca2+-Activated K+ Channels and Cardiac Arrhythmias

    PubMed Central

    Zhang, Xiao-Dong; Lieu, Deborah K.; Chiamvimonvat, Nipavan

    2015-01-01

    Small conductance Ca2+-activated K+ (SK, KCa2) channels are unique in that they are gated solely by changes in intracellular Ca2+ and hence, function to integrate intracellular Ca2+ and membrane potentials on a beat-to-beat basis. Recent studies have provided evidence for the existence and functional significance of SK channels in the heart. Indeed, our knowledge of cardiac SK channels has been greatly expanded over the past decade. Interests in cardiac SK channels are further driven by recent studies suggesting the critical roles of SK channels in human atrial fibrillation, SK channel as a possible novel therapeutic target in atrial arrhythmias and up-regulation of SK channels in heart failure (HF) in animal models and human HF. However, there remain critical gaps in our knowledge. Specifically, blockade of SK channels in cardiac arrhythmias has been shown to be both anti-arrhythmic and proarrhythmic. This contemporary review will provide an overview of the literature on the role of cardiac SK channels in cardiac arrhythmias and to serve as a discussion platform for the current clinical perspectives. At the translational level, development of SK channel blockers as a new therapeutic target in the treatment of atrial fibrillation and the possible pro-arrhythmic effects merit further considerations and investigations. PMID:25956967

  3. SLO2 Channels Are Inhibited by All Divalent Cations That Activate SLO1 K+ Channels.

    PubMed

    Budelli, Gonzalo; Sun, Qi; Ferreira, Juan; Butler, Alice; Santi, Celia M; Salkoff, Lawrence

    2016-04-01

    Two members of the family of high conductance K(+)channels SLO1 and SLO2 are both activated by intracellular cations. However, SLO1 is activated by Ca(2+)and other divalent cations, while SLO2 (Slack or SLO2.2 from rat) is activated by Na(+) Curiously though, we found that SLO2.2 is inhibited by all divalent cations that activate SLO1, with Zn(2+)being the most effective inhibitor with an IC50of ∼8 μmin contrast to Mg(2+), the least effective, with an IC50of ∼ 1.5 mm Our results suggest that divalent cations are not SLO2 pore blockers, but rather inhibit channel activity by an allosteric modification of channel gating. By site-directed mutagenesis we show that a histidine residue (His-347) downstream of S6 reduces inhibition by divalent cations. An analogous His residue present in some CNG channels is an inhibitory cation binding site. To investigate whether inhibition by divalent cations is conserved in an invertebrate SLO2 channel we cloned the SLO2 channel fromDrosophila(dSLO2) and compared its properties to those of rat SLO2.2. We found that, like rat SLO2.2, dSLO2 was also activated by Na(+)and inhibited by divalent cations. Inhibition of SLO2 channels in mammals andDrosophilaby divalent cations that have second messenger functions may reflect the physiological regulation of these channels by one or more of these ions.

  4. Do recreational activities affect coastal biodiversity?

    NASA Astrophysics Data System (ADS)

    Riera, Rodrigo; Menci, Cristiano; Sanabria-Fernández, José Antonio; Becerro, Mikel A.

    2016-09-01

    Human activities are largely affecting coastal communities worldwide. Recreational perturbations have been overlooked in comparison to other perturbations, yet they are potential threats to marine biodiversity. They affect coastal communities in different ways, underpinning consistent shifts in fish and invertebrates assemblages. Several sites were sampled subjected to varying effects by recreational fishermen (low and high pressure) and scuba divers (low and high) in an overpopulated Atlantic island. Non-consistent differences in ecological, trophic and functional diversity were found in coastal communities, considering both factors ("diving" and "fishing"). Multivariate analyses only showed significant differences in benthic invertebrates between intensively-dived and non-dived sites. The lack of clear trends may be explained by the depletion of coastal resources in the study area, an extensively-affected island by overfishing.

  5. Shikonin Inhibits Intestinal Calcium-Activated Chloride Channels and Prevents Rotaviral Diarrhea

    PubMed Central

    Jiang, Yu; Yu, Bo; Yang, Hong; Ma, Tonghui

    2016-01-01

    Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl- current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl- currents in mouse colonic epithelia but did not affect cytoplasmic Ca2+ concentration as well as the other major enterocyte chloride channel conductance regulator. Characterization study found that shikonin inhibited basolateral K+ channel activity without affecting Na+/K+-ATPase activities. In vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in vivo. Taken together, the results suggested that shikonin inhibited enterocyte calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K+ channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea. PMID:27601995

  6. Shikonin Inhibits Intestinal Calcium-Activated Chloride Channels and Prevents Rotaviral Diarrhea.

    PubMed

    Jiang, Yu; Yu, Bo; Yang, Hong; Ma, Tonghui

    2016-01-01

    Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl(-) current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl(-) currents in mouse colonic epithelia but did not affect cytoplasmic Ca(2+) concentration as well as the other major enterocyte chloride channel conductance regulator. Characterization study found that shikonin inhibited basolateral K(+) channel activity without affecting Na(+)/K(+)-ATPase activities. In vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in vivo. Taken together, the results suggested that shikonin inhibited enterocyte calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K(+) channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea.

  7. Regulation of Voltage-Activated K(+) Channel Gating by Transmembrane β Subunits.

    PubMed

    Sun, Xiaohui; Zaydman, Mark A; Cui, Jianmin

    2012-01-01

    Voltage-activated K(+) (K(V)) channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. K(V) channels contain a central pore-gate domain (PGD) surrounded by four voltage-sensing domains (VSDs). The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many K(V) channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the K(V) β subunits that contain transmembrane (TM) segments including the KCNE family and the β subunits of large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels. These TM β subunits affect the voltage-dependent activation of K(V) α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening, and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into K(V) channel modulation by TM β subunits.

  8. 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.

  9. Chlorpromazine confers neuroprotection against brain ischemia by activating BKCa channel.

    PubMed

    Li, Hua-Juan; Zhang, Yu-Jiao; Zhou, Li; Han, Feng; Wang, Ming-Yan; Xue, Mao-Qiang; Qi, Zhi

    2014-07-15

    Chlorpromazine (CPZ) is a well-known antipsychotic drug, still widely being used to treat symptoms of schizophrenia, psychotic depression and organic psychoses. We have previously reported that CPZ activates the BKCa (KCa1.1) channel at whole cell level. In the present study, we demonstrated that CPZ increased the single channel open probability of the BKCa channels without changing its single channel amplitude. As BKCa channel is one of the molecular targets of brain ischemia, we explored a possible new use of this old drug on ischemic brain injury. In middle cerebral artery occlusion (MCAO) focal cerebral ischemia, a single intraperitoneal injection of CPZ at several dosages (5mg/kg, 10mg/kg and 20mg/kg) could exert a significant neuroprotective effect on the brain damage in a dose- and time-dependent manner. Furthermore, blockade of BKCa channels abolished the neuroprotective effect of CPZ on MCAO, suggesting that the effect of CPZ is mediated by activation of the BKCa channel. These results demonstrate that CPZ could reduce focal cerebral ischemic damage through activating BKCa channels and merits exploration as a potential therapeutic agent for treating ischemic stroke.

  10. Flow-induced activation of TRPV5 and TRPV6 channels stimulates Ca(2+)-activated K(+) channel causing membrane hyperpolarization.

    PubMed

    Cha, Seung-Kuy; Kim, Ji-Hee; Huang, Chou-Long

    2013-12-01

    TRPV5 and TRPV6 channels are expressed in distal renal tubules and play important roles in the transcellular Ca(2+) reabsorption in kidney. They are regulated by multiple intracellular factors including protein kinases A and C, membrane phospholipid PIP2, protons, and divalent ions Ca(2+) and Mg(2+). Here, we report that fluid flow that generates shear force within the physiological range of distal tubular fluid flow activated TRPV5 and TRPV6 channels expressed in HEK cells. Flow-induced activation of channel activity was reversible and did not desensitize over 2min. Fluid flow stimulated TRPV5 and 6-mediated Ca(2+) entry and increased intracellular Ca(2+) concentration. N-glycosylation-deficient TRPV5 channel was relatively insensitive to fluid flow. In cells coexpressing TRPV5 (or TRPV6) and Slo1-encoded maxi-K channels, fluid flow induced membrane hyperpolarization, which could be prevented by the maxi-K blocker iberiotoxin or TRPV5 and 6 blocker La(3+). In contrast, fluid flow did not cause membrane hyperpolarization in cells coexpressing ROMK1 and TRPV5 or 6 channel. These results reveal a new mechanism for the regulation of TRPV5 and TRPV6 channels. Activation of TRPV5 and TRPV6 by fluid flow may play a role in the regulation of flow-stimulated K(+) secretion via maxi-K channels in distal renal tubules and in the mechanism of pathogenesis of thiazide-induced hypocalciuria.

  11. Flow-Induced Activation of TRPV5 and TRPV6 Channel Stimulates Ca2+-Activated K+ Channel Causing Membrane Hyperpolarization

    PubMed Central

    Cha, Seung-Kuy; Kim, Ji-Hee; Huang, Chou-Long

    2014-01-01

    TRPV5 and TRPV6 channels are expressed in distal renal tubules and play important roles in the transcellular Ca2+ reabsorption in kidney. They are regulated by multiple intracellular factors including protein kinase A and C, membrane phospholipid PIP2, protons, and divalent ions Ca2+ and Mg2+. Here, we report that fluid flow that generates shear force within the physiological range of distal tubular fluid flow activated TRPV5 and TRPV6 channels expressed in HEK cells. Flow-induced activation of channel activity was reversible and did not desensitize over 2 minutes. Fluid flow stimulated TRPV5 and 6-mediated Ca2+ entry and increased intracellular Ca2+ concentration. N-glycosylation-deficient TRPV5 channel was relatively insensitive to fluid flow. In cells coexpressing TRPV5 (or TRPV6) and Slo1-encoded maxi-K channels, fluid flow induced membrane hyperpolarization, which could be prevented by the maxi-K blocker iberiotoxin or TRPV5 and 6 blocker La3+. In contrast, fluid flow did not cause membrane hyperpolarization in cells coexpressing ROMK1 and TRPV5 or 6 channels. These results reveal a new mechanism for regulation of TRPV5 and TRPV6 channels. Activation of TRPV5 and TRPV6 by fluid flow may play a role in the regulation of flow-stimulated K+ secretion via maxi-K channels in distal renal tubules and in the mechanism of pathogenesis of thiazide-induced hypocalciuria. PMID:24001793

  12. Transmission probability and active pumping ability of the channel with moving partition plane

    NASA Astrophysics Data System (ADS)

    Luo, X.; Day, Chr.

    2016-11-01

    In this paper we present Test Particle Monte Carlo (TPMC) simulations of the transmission probability and the active pumping ability of a rectangular channel of different physical parameters. The novel feature of this problem is that the channel has a partition plane in the middle which is moving up and down, which makes it to a dynamical system. The system was simulated by our TPMC code ProVac3D developed with a new subroutine considering the moving partition plane. In order to obtain precise simulation results, at least 1011 test particles were simulated in parallel on the supercomputer Helios in Japan. It is found that the amplitude and the frequency of the movement will affect both transmission probability and the active pumping ability of the channel. Moreover, it is also found that transmission probability and active pumping ability of the channel with the moving partition plane are no longer independent of the mass of the gas species.

  13. Guanosine 5'-monophosphate modulates gating of high-conductance Ca2+-activated K+ channels in vascular smooth muscle cells.

    PubMed Central

    Williams, D L; Katz, G M; Roy-Contancin, L; Reuben, J P

    1988-01-01

    Ca2+-activated K+ channels (PKCa channels) account for the predominant K+ permeability of many types of smooth muscle cells. When activated, they oppose depolarization due to Na+ and Ca2+ channel activity. Several vasodilatory agents that increase intracellular cGMP levels (e.g., nitroprusside, adenosine, and atrial natriuretic factor) enhance the activity of these high-conductance PKCa channels in on-cell patches of bovine aortic smooth muscle cells. In addition, dibutyryl-cGMP (1.0 mM) causes a similar increase in channel activity. To pursue the mechanism of channel modulation by these agents, a series of guanine and adenine nucleotides were evaluated by using inside-out excised patches. Whereas cAMP, AMP, ADP, and ATP were ineffective, all of the corresponding guanine nucleotides potentiated PKCa channel activity when tested at a high concentration (500 microM). However, only GMP consistently enhanced channel activity in the 1-100 microM range by increasing the percent open time and frequency of opening of these channels over a wide range of potentials and Ca2+ levels without affecting single-channel conductance. Thus, GMP is a potent modulator of PKCa channels and it, rather than cGMP, may mediate the action of the vasodilators examined in this study. PMID:2848262

  14. Chloride dependence of hyperpolarization-activated chloride channel gates

    PubMed Central

    Pusch, Michael; Jordt, Sven-Eric; Stein, Valentin; Jentsch, Thomas J

    1999-01-01

    ClC proteins are a class of voltage-dependent Cl− channels with several members mutated in human diseases. The prototype ClC-0 Torpedo channel is a dimeric protein; each subunit forms a pore that can gate independently from the other one. A common slower gating mechanism acts on both pores simultaneously; slow gating activates ClC-0 at hyperpolarized voltages. The ClC-2 Cl− channel is also activated by hyperpolarization, as are some ClC-1 mutants (e.g. D136G) and wild-type (WT) ClC-1 at certain pH values.We studied the dependence on internal Cl− ([Cl−]i) of the hyperpolarization-activated gates of several ClC channels (WT ClC-0, ClC-0 mutant P522G, ClC-1 mutant D136G and an N-terminal deletion mutant of ClC-2), by patch clamping channels expressed in Xenopus oocytes.With all these channels, reducing [Cl−]i shifted activation to more negative voltages and reduced the maximal activation at most negative voltages.We also investigated the external halide dependence of WT ClC-2 using two-electrode voltage-clamp recording. Reducing external Cl− ([Cl−]o) activated ClC-2 currents. Replacing [Cl−]o by the less permeant Br− reduced channel activity and accelerated deactivation.Gating of the ClC-2 mutant K566Q in normal [Cl−]o resembled that of WT ClC-2 in low [Cl−]o, i.e. channels had a considerable open probability (Po) at resting membrane potential. Substituting external Cl− by Br− or I− led to a decrease in Po.The [Cl−]i dependence of the hyperpolarization-activated gates of various ClC channels suggests a similar gating mechanism, and raises the possibility that the gating charge for the hyperpolarization-activated gate is provided by Cl−.The external halide dependence of hyperpolarization-activated gating of ClC-2 suggests that it is mediated or modulated by anions as in other ClC channels. In contrast to the depolarization-activated fast gates of ClC-0 and ClC-1, the absence of Cl− favours channel opening. Lysine 556 may be important

  15. Oxidative Regulation of Large Conductance Calcium-Activated Potassium Channels

    PubMed Central

    Tang, Xiang D.; Daggett, Heather; Hanner, Markus; Garcia, Maria L.; McManus, Owen B.; Brot, Nathan; Weissbach, Herbert; Heinemann, Stefan H.; Hoshi, Toshinori

    2001-01-01

    Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca2+-activated K+ channels (BKCa or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca2+, the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K+ channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism. PMID:11222629

  16. Putative chanzyme activity of TRPM2 cation channel is unrelated to pore gating

    PubMed Central

    Tóth, Balázs; Iordanov, Iordan; Csanády, László

    2014-01-01

    Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel expressed in immune cells of phagocytic lineage, pancreatic β cells, and brain neurons and is activated under oxidative stress. TRPM2 activity is required for immune cell activation and insulin secretion and is responsible for postischemic neuronal cell death. TRPM2 is opened by binding of ADP ribose (ADPR) to its C-terminal cytosolic nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain, which, when expressed in isolation, cleaves ADPR into AMP and ribose-5-phosphate. A suggested coupling of this enzymatic activity to channel gating implied a potentially irreversible gating cycle, which is a unique feature of a small group of channel enzymes known to date. The significance of such a coupling lies in the conceptually distinct pharmacologic strategies for modulating the open probability of channels obeying equilibrium versus nonequilibrium gating mechanisms. Here we examine the potential coupling of TRPM2 enzymatic activity to pore gating. Mutation of several residues proposed to enhance or eliminate NUDT9-H catalytic activity all failed to affect channel gating kinetics. An ADPR analog, α-β-methylene-ADPR (AMPCPR), was shown to be entirely resistant to hydrolysis by NUDT9, but nevertheless supported TRPM2 channel gating, albeit with reduced apparent affinity. The rate of channel deactivation was not slowed but, rather, accelerated in AMPCPR. These findings, as well as detailed analyses of steady-state gating kinetics of single channels recorded in the presence of a range of concentrations of ADPR or AMPCPR, identify TRPM2 as a simple ligand-gated channel that obeys an equilibrium gating mechanism uncoupled from its enzymatic activity. PMID:25385633

  17. Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channels does not affect their voltage sensor

    PubMed Central

    1993-01-01

    Inactivation of currents carried by Ba2+ and Ca2+, as well as intramembrane charge movement from L-type Ca2+ channels were studied in guinea pig ventricular myocytes using the whole-cell patch clamp technique. Prolonged (2 s) conditioning depolarization caused substantial reduction of charge movement between -70 and 10 mV (charge 1, or charge from noninactivated channels). In parallel, the charge mobile between -70 and -150 mV (charge 2, or charge from inactivated channels) was increased. The availability of charge 2 depended on the conditioning pulse voltage as the sum of two Boltzmann components. One component had a central voltage of -75 mV and a magnitude of 1.7 nC/microF. It presumably is the charge movement (charge 2) from Na+ channels. The other component, with a central voltage of approximately - 30 mV and a magnitude of 3.5 nC/microF, is the charge 2 of L-type Ca2+ channels. The sum of charge 1 and charge 2 was conserved after different conditioning pulses. The difference between the voltage dependence of the activation of L-type Ca2+ channels (half-activation voltage, V, of approximately -20 mV) and that of charge 2 (V of -100 mV) made it possible to record the ionic currents through Ca2+ channels and charge 2 in the same solution. In an external solution with Ba2+ as sole metal the maximum available charge 2 of L-type Ca2+ channels was 10-15% greater than that in a Ca(2+)-containing solution. External Cd2+ caused 20-30% reduction of charge 2 both from Na+ and L-type Ca2+ channels. Voltage- and Ca(2+)-dependent inactivation phenomena were compared with a double pulse protocol in cells perfused with an internal solution of low calcium buffering capacity. As the conditioning pulse voltage increased, inactivation monitored with the second pulse went through a minimum at about 0 mV, the voltage at which conditioning current had its maximum. Charge 2, recorded in parallel, did not show any increase associated with calcium entry. Two alternative interpretations

  18. Toward a unifying model of malaria-induced channel activity

    PubMed Central

    Bouyer, Guillaume; Egée, Stéphane; Thomas, Serge L. Y.

    2007-01-01

    Infection of RBC by the malaria parasite Plasmodium falciparum activates, at the trophozoite stage, a membrane current 100- to 150-fold larger than in uninfected RBC. This current is carried by small anion channels initially described in supraphysiological ion concentrations (1.115 M Cl−) and named plasmodial surface anion channels (PSAC), suggesting their plasmodial origin. Our results obtained with physiological ion concentrations (0.145 M Cl−) support the notion that the parasite-induced channels represent enhanced activity versions of anion channels already present in uninfected RBCs. Among them, an 18-pS inwardly rectifying anion channel (IRC) and a 4- to 5-pS small conductance anion channel (SCC) were present in most single-channel recordings of infected membranes. The aim of this study was to clarify disparities in the reported electrophysiological data and to investigate possible technical reasons why these discrepancies have arisen. We demonstrate that PSAC is the supraphysiological correlate of the SCC and is inhibited by Zn2+, suggesting that it is a ClC-2 channel. We show that in physiological solutions 80% of the membrane conductance in infected cells can be accounted for by IRC and 20% can be accounted for by SCC whereas in supraphysiological conditions the membrane conductance is almost exclusively carried by SCC (PSAC) because the IRC is functionally turned off. PMID:17576926

  19. Fear conditioning suppresses large-conductance calcium-activated potassium channels in lateral amygdala neurons.

    PubMed

    Sun, P; Zhang, Q; Zhang, Y; Wang, F; Wang, L; Yamamoto, R; Sugai, T; Kato, N

    2015-01-01

    It was previously shown that depression-like behavior is accompanied with suppression of the large-conductance calcium activated potassium (BK) channel in cingulate cortex pyramidal cells. To test whether BK channels are also involved in fear conditioning, we studied neuronal properties of amygdala principal cells in fear conditioned mice. After behavior, we made brain slices containing the amygdala, the structure critically relevant to fear memory. The resting membrane potential in lateral amygdala (LA) neurons obtained from fear conditioned mice (FC group) was more depolarized than in neurons from naïve controls. The frequencies of spikes evoked by current injections were higher in neurons from FC mice, demonstrating that excitability of LA neurons was elevated by fear conditioning. The depolarization in neurons from FC mice was shown to depend on BK channels by using the BK channel blocker charybdotoxin. Suppression of BK channels in LA neurons from the FC group was further confirmed on the basis of the spike width, since BK channels affect the descending phase of spikes. Spikes were broader in the FC group than those in the naïve control in a manner dependent on BK channels. Consistently, quantitative real-time PCR revealed a decreased expression of BK channel mRNA. The present findings suggest that emotional disorder manifested in the forms of fear conditioning is accompanied with BK channel suppression in the amygdala, the brain structure critical to this emotional disorder.

  20. Effective contractile response to voltage-gated Na+ channels revealed by a channel activator.

    PubMed

    Ho, W-S Vanessa; Davis, Alison J; Chadha, Preet S; Greenwood, Iain A

    2013-04-15

    This study investigated the molecular identity and impact of enhancing voltage-gated Na(+) (Na(V)) channels in the control of vascular tone. In rat isolated mesenteric and femoral arteries mounted for isometric tension recording, the vascular actions of the Na(V) channel activator veratridine were examined. Na(V) channel expression was probed by molecular techniques and immunocytochemistry. In mesenteric arteries, veratridine induced potent contractions (pEC(50) = 5.19 ± 0.20, E(max) = 12.0 ± 2.7 mN), which were inhibited by 1 μM TTX (a blocker of all Na(V) channel isoforms, except Na(V)1.5, Na(V)1.8, and Na(V)1.9), but not by selective blockers of Na(V)1.7 (ProTx-II, 10 nM) or Na(V)1.8 (A-80347, 1 μM) channels. The responses were insensitive to endothelium removal but were partly (~60%) reduced by chemical destruction of sympathetic nerves by 6-hydroxydopamine (2 mM) or antagonism at the α1-adrenoceptor by prazosin (1 μM). KB-R7943, a blocker of the reverse mode of the Na(+)/Ca(2+) exchanger (3 μM), inhibited veratridine contractions in the absence or presence of prazosin. T16A(inh)-A01, a Ca(2+)-activated Cl(-) channel blocker (10 μM), also inhibited the prazosin-resistant contraction to veratridine. Na(V) channel immunoreactivity was detected in freshly isolated mesenteric myocytes, with apparent colocalization with the Na(+)/Ca(2+) exchanger. Veratridine induced similar contractile effects in the femoral artery, and mRNA transcripts for Na(V)1.2 and Na(V)1.3 channels were evident in both vessel types. We conclude that, in addition to sympathetic nerves, NaV channels are expressed in vascular myocytes, where they are functionally coupled to the reverse mode of Na(+)/Ca(2+) exchanger and subsequent activation of Ca(2+)-activated Cl(-) channels, causing contraction. The TTX-sensitive Na(V)1.2 and Na(V)1.3 channels are likely involved in vascular control.

  1. Dihydroxyeicosatrienoic acids are potent activators of Ca2+-activated K+ channels in isolated rat coronary arterial myocytes

    PubMed Central

    Lu, Tong; Katakam, Prasad V G; VanRollins, Mike; Weintraub, Neal L; Spector, Arthur A; Lee, Hon-Chi

    2001-01-01

    Dihydroxyeicosatrienoic acids (DHETs), which are metabolites of arachidonic acid (AA) and epoxyeicosatrienoic acids (EETs), have been identified as highly potent endogenous vasodilators, but the mechanisms by which DHETs induce relaxation of vascular smooth muscle are unknown. Using inside-out patch clamp techniques, we examined the effects of DHETs on the large conductance Ca2+-activated K+ (BK) channels in smooth muscle cells from rat small coronary arteries (150–300 μm diameter). 11,12-DHET potently activated BK channels with an EC50 of 1.87 ± 0.57 nm (n = 5). Moreover, the three other regioisomers 5,6-, 8,9- and 14,15-DHET were equipotent with 11,12-DHET in activating BK channels. The efficacy of 11,12-DHET in opening BK channels was much greater than that of its immediate precursor 11,12-EET. In contrast, AA did not significantly affect BK channel activity. The voltage dependence of BK channels was dramatically modulated by 11,12-DHET. With physiological concentrations of cytoplasmic Ca2+ (200 nm), the voltage at which the channel open probability was half-maximal (V1/2) was shifted from a baseline of 115.6 ± 6.5 mV to 95.0 ± 10.1 mV with 5 nm 11,12-DHET, and to 60.0 ± 8.4 mV with 50 nm 11,12-DHET. 11,12-DHET also enhanced the sensitivity of BK channels to Ca2+ but did not activate the channels in the absence of Ca2+. 11,12-DHET (50 nm) reduced the Ca2+ EC50 of BK channels from a baseline of 1.02 ± 0.07 μm to 0.42 ± 0.11 μm. Single channel kinetic analysis indicated that 11,12-DHET did not alter BK channel conductance but did reduce the first latency of BK channel openings in response to a voltage step. 11,12-DHET dose-dependently increased the open dwell times, abbreviated the closed dwell times, and decreased the transition rates from open to closed states. We conclude that DHETs hyperpolarize vascular smooth muscle cells through modulation of the BK channel gating behaviour, and by enhancing the channel sensitivities to Ca2+ and voltage. Hence

  2. 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.

  3. An anion channel in Arabidopsis hypocotyls activated by blue light.

    PubMed Central

    Cho, M H; Spalding, E P

    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. PMID:8755616

  4. Solar activity affects avian timing of reproduction

    PubMed Central

    Visser, Marcel E.; Sanz, Juan José

    2009-01-01

    Avian timing of reproduction is strongly affected by ambient temperature. Here we show that there is an additional effect of sunspots on laying date, from five long-term population studies of great and blue tits (Parus major and Cyanistes caeruleus), demonstrating for the first time that solar activity not only has an effect on population numbers but that it also affects the timing of animal behaviour. This effect is statistically independent of ambient temperature. In years with few sunspots, birds initiate laying late while they are often early in years with many sunspots. The sunspot effect may be owing to a crucial difference between the method of temperature measurements by meteorological stations (in the shade) and the temperatures experienced by the birds. A better understanding of the impact of all the thermal components of weather on the phenology of ecosystems is essential when predicting their responses to climate change. PMID:19574283

  5. Collateral response to activation of potassium channels in vivo.

    PubMed

    Lamping, K G

    1998-04-01

    Activation of ATP-sensitive K+ channels is involved in the coronary vascular response to decreases in perfusion pressure and ischemia. Since activation of ATP-sensitive K+ channels in collateral vessels may be important in determining flow to collateral-dependent myocardium, the ability of collaterals to respond to activation of the channel was tested. In the beating heart of dogs, we compared responses of non-collaterals less than 100 microns in diameter to collaterals of similar size using computer-controlled stroboscopic epi-illumination of the left ventricle coupled to a microscope-video system. Aprikalim, a selective activator of ATP-sensitive K+ channels (0.1-10 microM) produced similar dose-dependent dilation of non-collaterals and collaterals. Relaxation was decreased by inhibition of ATP-sensitive K+ channels with glibenclamide, but not by inhibition of nitric oxide synthase with nitro-L-arginine. Bradykinin (10-100 microM) produced similar dilation of non-collaterals and collaterals which was decreased by nitro-L-arginine but not glibenclamide. Thus, in microvascular collaterals, relaxation to both nitric oxide and activation of ATP-sensitive K+ channels is similar to non-collaterals.

  6. Phosphatase inhibitors activate normal and defective CFTR chloride channels.

    PubMed Central

    Becq, F; Jensen, T J; Chang, X B; Savoia, A; Rommens, J M; Tsui, L C; Buchwald, M; Riordan, J R; Hanrahan, J W

    1994-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epithelial cells. We report that the alkaline phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophylline, and vanadate slow the rundown of CFTR channel activity in excised membrane patches and reduce dephosphorylation of CFTR protein in isolated membranes. It was also found that in unstimulated cells, CFTR channels can be activated by exposure to phosphatase inhibitors alone. Most importantly, exposure of mammalian cells to phosphatase inhibitors alone activates CFTR channels that have disease-causing mutations, provided the mutant channels are present in the plasma membrane (R117H, G551D, and delta F508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membrane-associated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis. Images PMID:7522329

  7. Activation of the human, intermediate-conductance, Ca2+-activated K+ channel by methylxanthines.

    PubMed

    Schrøder, R L; Jensen, B S; Strøbaek, D; Olesen, S P; Christophersen, P

    2000-10-01

    This study demonstrated that the methylxanthines, theophylline, IBMX and caffeine, activate the human, intermediate-conductance, Ca2+-activated K+ channel (hIK) stably expressed in HEK-293 cells. Whole-cell voltage-clamp experiments showed that the hIK current increased reversibly and voltage independently after the addition of methylxanthines. In current-clamp experiments, theophylline dose-dependently hyperpolarised the cell membrane from a resting potential of -18 mV to -56 mV. The methylxanthines did not affect large-conductance (BK) or small-conductance (SK2), Ca2+-activated K+ channels, demonstrating that the effects were not secondary to a rise in intracellular Ca2+. However, the activation of hIK by theophylline required an intracellular [Ca2+] above 30 nM. The hIK current was insensitive to 8-bromoadenosine cyclic 3',5'-monophosphate (8-bromo-cAMP), forskolin, 8-bromoguanosine cyclic 3',5'-monophosphate (8-bromo-cGMP) and sodium nitroprusside. Moreover, in the presence of inhibitors of protein kinase A (PKA) or protein kinase G (PKG) theophylline still activated the current. Finally, mutation of the putative PKA/PKG consensus phosphorylation site (Ser334) had no effect on the theophylline-induced activation of hIK. Since the observed activation is independent of changes in PKA/PKG-phosphorylation and of fluctuations in intracellular Ca2+, we suggest that the methylxanthines interact directly with the hIK protein.

  8. Smooth muscle BK channel activity influences blood pressure independent of vascular tone in mice

    PubMed Central

    Sachse, Gregor; Faulhaber, Jörg; Seniuk, Anika; Ehmke, Heimo; Pongs, Olaf

    2014-01-01

    The large conductance voltage- and Ca2+-activated K+ (BK) channel is an important determinant of vascular tone and contributes to blood pressure regulation. Both activities depend on the ancillary BKβ1 subunit. To determine the significance of smooth muscle BK channel activity for blood pressure regulation, we investigated the potential link between changes in arterial tone and altered blood pressure in BKβ1 knockout (BKβ1−/−) mice from three different genetically defined strains. While vascular tone was consistently increased in all BKβ1−/− mice independent of genetic background, BKβ1−/− strains exhibited increased (strain A), unaltered (strain B) or decreased (strain C) mean arterial blood pressures compared to their corresponding BKβ1+/+ controls. In agreement with previous data on aldosterone regulation by renal/adrenal BK channel function, BKβ1−/− strain A mice have increased plasma aldosterone and increased blood pressure. Consistently, blockade of mineralocorticoid receptors by spironolactone treatment reversibly restored the elevated blood pressure to the BKβ1+/+ strain A level. In contrast, loss of BKβ1 did not affect plasma aldosterone in strain C mice. Smooth muscle-restricted restoration of BKβ1 expression increased blood pressure in BKβ1−/− strain C mice, implying that impaired smooth muscle BK channel activity lowers blood pressure in these animals. We conclude that BK channel activity directly affects vascular tone but influences blood pressure independent of this effect via different pathways. PMID:24687584

  9. Cytogenetic and molecular localization of tipE: A gene affecting sodium channels in Drosophila melanogaster

    SciTech Connect

    Feng, G.; Deak, P.; Hall, L.M.

    1995-04-01

    Voltage-sensitive sodium channels play a key role in nerve cells where they are responsible for the increase in sodium permeability during the rising phase of action potentials. In Drosophila melanogaster a subset of temperature-sensitive paralytic mutations affect sodium channel function. One such mutation is temperature-induced paralysis locus E (tipE), which has been shown by electrophysiology and ligand binding studies to reduce sodium channel numbers. Three new {gamma}-ray-induced tipE alleles associated with either visible deletions in 64AB or a translocation breakpoint within 64B2 provide landmarks for positional cloning of tipE. Beginning with the flanking cloned gene Ras2, a 140-kb walk across the translocation breakpoint was completed. Germline transformation using a 42-kb cosmid clone and successively smaller subclones localized the tipE gene within a 7.4-kb genomic DNA segment. Although this chromosome region is rich in transcripts, only three overlapping mRNAs (5.4, 4.4, and 1.7 kb) lie completely within the smallest rescuing construct. The small sizes of the rescuing construct and transcripts suggests that tipE does not encode a standard sodium channel {alpha}-subunit with four homologous repeats. Sequencing these transcripts will elucidate the role of the tipE gene product in sodium channel functional regulation. 55 refs., 4 figs., 2 tabs.

  10. Cytogenetic and molecular localization of tipE: a gene affecting sodium channels in Drosophila melanogaster.

    PubMed

    Feng, G; Deák, P; Kasbekar, D P; Gil, D W; Hall, L M

    1995-04-01

    Voltage-sensitive sodium channels play a key role in nerve cells where they are responsible for the increase in sodium permeability during the rising phase of action potentials. In Drosophila melanogaster a subset of temperature-sensitive paralytic mutations affect sodium channel function. One such mutation is temperature-induced paralysis locus E (tipE), which has been shown by electrophysiology and ligand binding studies to reduce sodium channel numbers. Three new gamma-ray-induced tipE alleles associated with either visible deletions in 64AB or a translocation breakpoint within 64B2 provide landmarks for positional cloning of tipE. Beginning with the flanking cloned gene Ras2, a 140-kb walk across the translocation breakpoint was completed. Germline transformation using a 42-kb cosmid clone and successively smaller subclones localized the tipE gene within a 7.4-kb genomic DNA segment. Although this chromosome region is rich in transcripts, only three overlapping mRNAs (5.4, 4.4, and 1.7 kb) lie completely within the smallest rescuing construct. The small sizes of the rescuing construct and transcripts suggest that tipE does not encode a standard sodium channel alpha-subunit with four homologous repeats. Sequencing these transcripts will elucidate the role of the tipE gene product in sodium channel functional regulation.

  11. 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.

  12. Multi-channel fiber photometry for population neuronal activity recording.

    PubMed

    Guo, Qingchun; Zhou, Jingfeng; Feng, Qiru; Lin, Rui; Gong, Hui; Luo, Qingming; Zeng, Shaoqun; Luo, Minmin; Fu, Ling

    2015-10-01

    Fiber photometry has become increasingly popular among neuroscientists as a convenient tool for the recording of genetically defined neuronal population in behaving animals. Here, we report the development of the multi-channel fiber photometry system to simultaneously monitor neural activities in several brain areas of an animal or in different animals. In this system, a galvano-mirror modulates and cyclically couples the excitation light to individual multimode optical fiber bundles. A single photodetector collects excited light and the configuration of fiber bundle assembly and the scanner determines the total channel number. We demonstrated that the system exhibited negligible crosstalk between channels and optical signals could be sampled simultaneously with a sample rate of at least 100 Hz for each channel, which is sufficient for recording calcium signals. Using this system, we successfully recorded GCaMP6 fluorescent signals from the bilateral barrel cortices of a head-restrained mouse in a dual-channel mode, and the orbitofrontal cortices of multiple freely moving mice in a triple-channel mode. The multi-channel fiber photometry system would be a valuable tool for simultaneous recordings of population activities in different brain areas of a given animal and different interacting individuals.

  13. Active Integrated Filters for RF-Photonic Channelizers

    PubMed Central

    Nagdi, Amr El; 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. PMID:22319352

  14. Effect of Cytoskeletal Reagents on Stretch Activated Ion Channels

    DTIC Science & Technology

    1992-11-12

    transduction. Biophys J59: 1143-1145, 1991. 23. SACHS, F., W. SIGURDSON, A. RUKNUDIN, AND C. BOWMAN. Single- channel mechanosensitive currents. Science 253: 800... mechanosensitive ion channels . In: Advances in Comparative and Environmental Physiology, v0C, edited by F. Ito. Berlin: Springer-Verlag, 1992, p. 55-77. Report of Inventions: None 4 ...EFFECT OF CYTOSKELETAL REAGENTS ON STRETCH ACTIVATED ION CHANNELS b lfli..3-f-I’- o0*’t 6. AUTHOR(S) Dr.-Frederick Sachs DI 7. PERFORMING ORGANIZATION NAME

  15. ROMK1 channel activity is regulated by monoubiquitination.

    PubMed

    Lin, Dao-Hong; Sterling, Hyacinth; Wang, Zhijian; Babilonia, Elisa; Yang, Baofeng; Dong, Ke; Hebert, Steven C; Giebisch, Gerhard; Wang, Wen-Hui

    2005-03-22

    The ubiquitination of proteins can signal their degradation, modify their activity or target them to specific membranes or cellular organelles. Here, we show that monoubiquitination regulates the plasma membrane abundance and function of the potassium channel, ROMK. Immunoprecipitation of proteins obtained from renal cortex and outer medulla with ROMK antibody revealed that this channel was monoubiquitinated. To determine the ubiquitin binding site on ROMK1, all intracellular lysine (Lys) residues of ROMK1 were individually mutated to arginine (Arg), and a two-electrode voltage clamp was used to measure the ROMK1 channel activity in Xenopus oocytes. ROMK1 channel activity increased from 8.1 to 27.2 microA only when Lys-22 was mutated to Arg. Furthermore, Western blotting failed to detect the ubiquitinated ROMK1 in oocytes injected with R1K22R. Patch-clamp experiments showed that biophysical properties of R1K22R were identical to those of wild-type ROMK1. Although total protein expression levels of GFP-ROMK1 and GFP-R1K22R in oocytes were similar, confocal microscopy showed that the surface fluorescence intensity in oocytes injected with GFP-R1K22R was higher than that of GFP-ROMK1. In addition, biotin labeling of ROMK1 and R1K22R proteins expressed in HEK293 cells showed increased surface expression of the Lys-22 mutant channel. Finally, expression of R1K22R in COS7 cells significantly stimulated the surface expression of ROMK1. We conclude that ROMK1 can be monoubiquitinated and that Lys-22 is an ubiquitin-binding site. Thus, monoubiquitination of ROMK1 regulates channel activity by reducing the surface expression of channel protein. This finding implicates the linking of a single ubiquitin molecule to channels as an important posttranslational regulatory signal.

  16. ROMK1 channel activity is regulated by monoubiquitination

    PubMed Central

    Lin, Dao-Hong; Sterling, Hyacinth; Wang, Zhijian; Babilonia, Elisa; Yang, Baofeng; Dong, Ke; Hebert, Steven C.; Giebisch, Gerhard; Wang, Wen-Hui

    2005-01-01

    The ubiquitination of proteins can signal their degradation, modify their activity or target them to specific membranes or cellular organelles. Here, we show that monoubiquitination regulates the plasma membrane abundance and function of the potassium channel, ROMK. Immunoprecipitation of proteins obtained from renal cortex and outer medulla with ROMK antibody revealed that this channel was monoubiquitinated. To determine the ubiquitin binding site on ROMK1, all intracellular lysine (Lys) residues of ROMK1 were individually mutated to arginine (Arg), and a two-electrode voltage clamp was used to measure the ROMK1 channel activity in Xenopus oocytes. ROMK1 channel activity increased from 8.1 to 27.2 μA only when Lys-22 was mutated to Arg. Furthermore, Western blotting failed to detect the ubiquitinated ROMK1 in oocytes injected with R1K22R. Patch-clamp experiments showed that biophysical properties of R1K22R were identical to those of wild-type ROMK1. Although total protein expression levels of GFP-ROMK1 and GFP-R1K22R in oocytes were similar, confocal microscopy showed that the surface fluorescence intensity in oocytes injected with GFP-R1K22R was higher than that of GFP-ROMK1. In addition, biotin labeling of ROMK1 and R1K22R proteins expressed in HEK293 cells showed increased surface expression of the Lys-22 mutant channel. Finally, expression of R1K22R in COS7 cells significantly stimulated the surface expression of ROMK1. We conclude that ROMK1 can be monoubiquitinated and that Lys-22 is an ubiquitin-binding site. Thus, monoubiquitination of ROMK1 regulates channel activity by reducing the surface expression of channel protein. This finding implicates the linking of a single ubiquitin molecule to channels as an important posttranslational regulatory signal. PMID:15767585

  17. Designing Effective Persuasive Systems Utilizing the Power of Entanglement: Communication Channel, Strategy and Affect

    NASA Astrophysics Data System (ADS)

    Li, Haiqing; Chatterjee, Samir

    With rapid advances in information and communication technology, computer-mediated communication (CMC) technologies are utilizing multiple IT platforms such as email, websites, cell-phones/PDAs, social networking sites, and gaming environments. However, no studies have compared the effectiveness of a persuasive system using such alternative channels and various persuasive techniques. Moreover, how affective computing impacts the effectiveness of persuasive systems is not clear. This study proposes (1) persuasive technology channels in combination with persuasive strategies will have different persuasive effectiveness; (2) Adding positive emotion to a message that leads to a better overall user experience could increase persuasive effectiveness. The affective computing or emotion information was added to the experiment using emoticons. The initial results of a pilot study show that computer-mediated communication channels along with various persuasive strategies can affect the persuasive effectiveness to varying degrees. These results also shows that adding a positive emoticon to a message leads to a better user experience which increases the overall persuasive effectiveness of a system.

  18. Activation of purified calcium channels by stoichiometric protein phosphorylation

    SciTech Connect

    Nunoki, K.; Florio, V.; Catterall, W.A. )

    1989-09-01

    Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of {sup 45}Ca{sup 2+} uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of {sup 45}Ca{sup 2+} uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd{sup 2+}, Ni{sup 2+}, and Mg{sup 2+}. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.

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

    PubMed

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

    2014-01-01

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

  20. Block of a Ca(2+)-activated potassium channel by cocaine.

    PubMed

    Premkumar, L S

    2005-04-01

    The primary target for cocaine is believed to be monoamine transporters because of cocaine's high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K(+) channel in hippocampal neurons grown in culture (IC(50)=approximately 30 microM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca(2+)-activated K(+ )channels are 261+/-37 microM: (-1)s(-1) and 11451+/-1467 s(-1). The equilibrium dissociation constant (K(B)) for cocaine, determined from single-channel parameters, is 43 microM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca(2+)-dependent K(+) channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.

  1. Activation of the ATP-sensitive K+ channel by decavanadate in guinea-pig ventricular myocytes.

    PubMed

    Nakashima, H; Kakei, M; Tanaka, H

    1993-03-23

    To evaluate the effects of decavanadate on the ATP-sensitive K+ (KATP) channel, we applied the inside-out membrane patch-clamp technique to ventricular myocytes isolated from guinea-pig hearts. Decavanadate increased the probability of the KATP channel being open in a dose-dependent manner over the range of 0.1 to 5 mM in the presence of 0.3 mM ATP. Half-maximal activation occurred at 540 microM decavanadate and a Hill coefficient of 1.3 was obtained when the Hill equation was used to fit the dose-dependent activation for the channel by decavanadate. The half-maximum inhibition for the channel by ATP (K1/2) in the presence of 2 mM Mg2+ was 19 and 74 microM in its absence. In the presence of decavanadate, both curves shifted toward the higher concentration of ATP without a change in steepness of the slope (Hill coefficient = 2). The effect of decavanadate could be expressed by a model in which its binding prevents ATP binding from closing the channel. The estimated dissociation constant of decavanadate was 1.5 microM in the presence and 22.8 microM in the absence of Mg2+. Decavanadate reactivated the rundown channel in the absence of Mg2+ and ATP. Neither the single channel slope conductance nor the mean open and closed lifetime within the bursts of channel openings were affected by decavanadate. We conclude that internal Mg2+ is not required for the modulation produced by decavanadate, but this ion influences the channel and changes the dissociation constant of both ATP and decavanadate to the channel.

  2. Calcium activated potassium channel expression during human iPS cell-derived neurogenesis.

    PubMed

    Linta, Leonhard; Boeckers, Tobias M; Kleger, Alexander; Liebau, Stefan

    2013-07-01

    The family of calcium activated potassium channels of low and intermediate conductance, known as SK channels, consists of four members (SK1-4). These channels are widely expressed throughout the organism and involved in various cellular processes, such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date, the role of SK channels in developmental processes has been merely a marginal focus of investigation, although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently, several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein, we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells, neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly, we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro. The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis.

  3. Abscisic acid and 14-3-3 proteins control K channel activity in barley embryonic root.

    PubMed

    van den Wijngaard, Paul W J; Sinnige, Mark P; Roobeek, Ilja; Reumer, Annet; Schoonheim, Peter J; Mol, Jos N M; Wang, Mei; De Boer, Albertus H

    2005-01-01

    Germination of seeds proceeds in general in two phases, an initial imbibition phase and a subsequent growth phase. In grasses like barley, the latter phase is evident as the emergence of the embryonic root (radicle). The hormone abscisic acid (ABA) inhibits germination because it prevents the embryo from entering and completing the growth phase. Genetic and physiological studies have identified many steps in the ABA signal transduction cascade, but how it prevents radicle elongation is still not clear. For elongation growth to proceed, uptake of osmotically active substances (mainly K(+)) is essential. Therefore, we have addressed the question of how the activity of K(+) permeable ion channels in the plasma membrane of radicle cells is regulated under conditions of slow (+ABA) and rapid germination (+fusicoccin). We found that ABA arrests radicle growth, inhibits net K(+) uptake and reduces the activity of K(+) (in) channels as measured with the patch-clamp technique. In contrast, fusicoccin (FC), a well-known stimulator of germination, stimulates radicle growth, net K(+) uptake and reduces the activity of K(+) (out) channels. Both types of channels are under the control of 14-3-3 proteins, known as integral components of signal transduction pathways and instrumental in FC action. Intriguingly, 14-3-3 affected both channels in an opposite fashion: whereas K(+) (in) channel activity was fully dependent upon 14-3-3 proteins, K(+) (out) channel activity was reduced by 14-3-3 proteins by 60%. Together with previous data showing that 14-3-3 proteins control the activity of the plasma membrane H(+)-ATPase, this makes 14-3-3 a prime candidate for molecular master regulator of the cellular osmo-pump. Regulation of the osmo-pump activity by ABA and FC is an important mechanism in controlling the growth of the embryonic root during seed germination.

  4. Activation of peripheral KCNQ channels relieves gout pain

    PubMed Central

    Zheng, Yueming; Xu, Haiyan; Zhan, Li; Zhou, Xindi; Chen, Xueqin; Gao, Zhaobing

    2015-01-01

    Abstract Intense inflammatory pain caused by urate crystals in joints and other tissues is a major symptom of gout. Among therapy drugs that lower urate, benzbromarone (BBR), an inhibitor of urate transporters, is widely used because it is well tolerated and highly effective. We demonstrate that BBR is also an activator of voltage-gated KCNQ potassium channels. In cultured recombinant cells, BBR exhibited significant potentiation effects on KCNQ channels comparable to previously reported classical activators. In native dorsal root ganglion neurons, BBR effectively overcame the suppression of KCNQ currents, and the resultant neuronal hyperexcitability caused by inflammatory mediators, such as bradykinin (BK). Benzbromarone consistently attenuates BK-, formalin-, or monosodium urate–induced inflammatory pain in rat and mouse models. Notably, the analgesic effects of BBR are largely mediated through peripheral and not through central KCNQ channels, an observation supported both by pharmacokinetic studies and in vivo experiments. Moreover, multiple residues in the superficial part of the voltage sensing domain of KCNQ channels were identified critical for the potentiation activity of BBR by a molecular determinant investigation. Our data indicate that activation of peripheral KCNQ channels mediates the pain relief effects of BBR, potentially providing a new strategy for the development of more effective therapies for gout. PMID:25735002

  5. Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

    PubMed Central

    Blair, Nathaniel T.; Kaczmarek, J. Stefan

    2009-01-01

    TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation. PMID:19398778

  6. 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.

  7. Caffeine activates mouse TRPA1 channels but suppresses human TRPA1 channels

    PubMed Central

    Nagatomo, Katsuhiro; Kubo, Yoshihiro

    2008-01-01

    Caffeine has various well-characterized pharmacological effects, but in mammals there are no known plasma membrane receptors or ion channels activated by caffeine. We observed that caffeine activates mouse transient receptor potential A1 (TRPA1) in heterologous expression systems by Cai2+ imaging and electrophysiological analyses. These responses to caffeine were confirmed in acutely dissociated dorsal root ganglion sensory neurons from WT mice, which are known to express TRPA1, but were not seen in neurons from TRPA1 KO mice. Expression of TRPA1 was detected immunohistochemically in nerve fibers and bundles in the mouse tongue. Moreover, WT mice, but not KO mice, showed a remarkable aversion to caffeine-containing water. These results demonstrate that mouse TRPA1 channels expressed in sensory neurons cause an aversion to drinking caffeine-containing water, suggesting they mediate the perception of caffeine. Finally, we observed that caffeine does not activate human TRPA1; instead, it suppresses its activity. PMID:18988737

  8. Activation of Slo2.1 channels by niflumic acid

    PubMed Central

    Dai, Li; Garg, Vivek

    2010-01-01

    Slo2.1 channels conduct an outwardly rectifying K+ current when activated by high [Na+]i. Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na+. In Xenopus oocytes injected with <10 ng cRNA, heterologously expressed human Slo2.1 current was negligible, but rapidly activated by extracellular application of NFA (EC50 = 2.1 mM) or flufenamic acid (EC50 = 1.4 mM). Slo2.1 channels activated by 1 mM NFA exhibited weak voltage dependence. In high [K+]e, the conductance–voltage (G-V) relationship had a V1/2 of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V1/2 to more negative potentials (EC50 = 2.1 mM) and increased the minimum value of G/Gmax (EC50 = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V1/2 of the G-V relationship was shifted to more positive potentials when [K+]e was elevated from 1 to 300 mM (EC50 = 21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K+]e dependency (EC50 = 23.5 mM). Conductance was also [Na+]e dependent. Outward currents were reduced when Na+ was replaced with choline or mannitol, but unaffected by substitution with Rb+ or Li+. Neutralization of charged residues in the S1–S4 domains did not appreciably alter the voltage dependence of Slo2.1 activation. Thus, the weak voltage dependence of Slo2.1 channel activation is independent of charged residues in the S1–S4 segments. In contrast, mutation of R190 located in the adjacent S4–S5 linker to a neutral (Ala or Gln) or acidic (Glu) residue induced constitutive channel activity that was reduced by high [K+]e. Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K+]e and [Na+]e, and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na+. PMID:20176855

  9. Activation of Slo2.1 channels by niflumic acid.

    PubMed

    Dai, Li; Garg, Vivek; Sanguinetti, Michael C

    2010-03-01

    Slo2.1 channels conduct an outwardly rectifying K(+) current when activated by high [Na(+)](i). Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na(+). In Xenopus oocytes injected with <10 ng cRNA, heterologously expressed human Slo2.1 current was negligible, but rapidly activated by extracellular application of NFA (EC(50) = 2.1 mM) or flufenamic acid (EC(50) = 1.4 mM). Slo2.1 channels activated by 1 mM NFA exhibited weak voltage dependence. In high [K(+)](e), the conductance-voltage (G-V) relationship had a V(1/2) of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V(1/2) to more negative potentials (EC(50) = 2.1 mM) and increased the minimum value of G/G(max) (EC(50) = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V(1/2) of the G-V relationship was shifted to more positive potentials when [K(+)](e) was elevated from 1 to 300 mM (EC(50) = 21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K(+)](e) dependency (EC(50) = 23.5 mM). Conductance was also [Na(+)](e) dependent. Outward currents were reduced when Na(+) was replaced with choline or mannitol, but unaffected by substitution with Rb(+) or Li(+). Neutralization of charged residues in the S1-S4 domains did not appreciably alter the voltage dependence of Slo2.1 activation. Thus, the weak voltage dependence of Slo2.1 channel activation is independent of charged residues in the S1-S4 segments. In contrast, mutation of R190 located in the adjacent S4-S5 linker to a neutral (Ala or Gln) or acidic (Glu) residue induced constitutive channel activity that was reduced by high [K(+)](e). Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K(+)](e) and [Na(+)](e), and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na(+).

  10. Characterization of three novel mechanosensitive channel activities in Escherichia coli.

    PubMed

    Edwards, Michelle D; Black, Susan; Rasmussen, Tim; Rasmussen, Akiko; Stokes, Neil R; Stephen, Terri-Leigh; Miller, Samantha; Booth, Ian R

    2012-01-01

    Mechanosensitive channels sense elevated membrane tension that arises from rapid water influx occurring when cells move from high to low osmolarity environments (hypoosmotic shock). These non-specific channels in the cytoplasmic membrane release osmotically-active solutes and ions. The two major mechanosensitive channels in Escherichia coli are MscL and MscS. Deletion of both proteins severely compromises survival of hypoosmotic shock. However, like many bacteria, E. coli cells possess other MscS-type genes (kefA, ybdG, ybiO, yjeP and ynaI). Two homologs, MscK (kefA) and YbdG, have been characterized as mechanosensitive channels that play minor roles in maintaining cell integrity. Additional channel openings are occasionally observed in patches derived from mutants lacking MscS, MscK and MscL. Due to their rare occurrence, little is known about these extra pressure-induced currents or their genetic origins. Here we complete the identification of the remaining E. coli mechanosensitive channels YnaI, YbiO and YjeP. The latter is the major component of the previously described MscM activity (~300 pS), while YnaI (~100 pS) and YbiO (~1000 pS) were previously unknown. Expression of native YbiO is NaCl-specific and RpoS-dependent. A Δ7 strain was created with all seven E. coli mechanosensitive channel genes deleted. High level expression of YnaI, YbiO or YjeP proteins from a multicopy plasmid in the Δ7 strain (MJFGH) leads to substantial protection against hypoosmotic shock. Purified homologs exhibit high molecular masses that are consistent with heptameric assemblies. This work reveals novel mechanosensitive channels and discusses the regulation of their expression in the context of possible additional functions.

  11. 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).

  12. Science communication on YouTube: Factors that affect channel and video popularity.

    PubMed

    Welbourne, Dustin J; Grant, Will J

    2016-08-01

    YouTube has become one of the largest websites on the Internet. Among its many genres, both professional and amateur science communicators compete for audience attention. This article provides the first overview of science communication on YouTube and examines content factors that affect the popularity of science communication videos on the site. A content analysis of 390 videos from 39 YouTube channels was conducted. Although professionally generated content is superior in number, user-generated content was significantly more popular. Furthermore, videos that had consistent science communicators were more popular than those without a regular communicator. This study represents an important first step to understand content factors, which increases the channel and video popularity of science communication on YouTube.

  13. Bacteria-Based Analysis of HIV-1 Vpu Channel Activity

    PubMed Central

    Taube, Robert; Alhadeff, Raphael; Assa, Dror; Krugliak, Miriam; Arkin, Isaiah T.

    2014-01-01

    HIV-1 Vpu is a small, single-span membrane protein with two attributed functions that increase the virus' pathogenicity: degradation of CD4 and inactivation of BST-2. Vpu has also been shown to posses ion channel activity, yet no correlation has been found between this attribute and Vpu's role in viral release. In order to gain further insight into the channel activity of Vpu we devised two bacteria-based assays that can examine this function in detail. In the first assay Vpu was over-expressed, such that it was deleterious to bacterial growth due to membrane permeabilization. In the second and more sensitive assay, the channel was expressed at low levels in K+ transport deficient bacteria. Consequently, Vpu expression enabled the bacteria to grow at otherwise non permissive low K+ concentrations. Hence, Vpu had the opposite impact on bacterial growth in the two assays: detrimental in the former and beneficial in the latter. Furthermore, we show that channel blockers also behave reciprocally in the two assays, promoting growth in the first assay and hindering it in the second assay. Taken together, we investigated Vpu's channel activity in a rapid and quantitative approach that is amenable to high-throughput screening, in search of novel blockers. PMID:25272035

  14. 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.

  15. 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.

  16. Ion channel activity in lobster skeletal muscle membrane.

    PubMed

    Worden, M K; Rahamimoff, R; Kravitz, E A

    1993-09-01

    Ion channel activity in the sarcolemmal membrane of muscle fibers is critical for regulating the excitability, and therefore the contractility, of muscle. To begin the characterization of the biophysical properties of the sarcolemmal membrane of lobster exoskeletal muscle fibers, recordings were made from excised patches of membrane from enzymatically induced muscle fiber blebs. Blebs formed as evaginations of the muscle sarcolemmal membrane and were sufficiently free of extracellular debris to allow the formation of gigaohm seals. Under simple experimental conditions using bi-ionic symmetrical recording solutions and maintained holding potentials, a variety of single channel types with conductances in the range 32-380 pS were detected. Two of these ion channel species are described in detail, both are cation channels selective for potassium. They can be distinguished from each other on the basis of their single-channel conductance and gating properties. The results suggest that current flows through a large number of ion channels that open spontaneously in bleb membranes in the absence of exogenous metabolites or hormones.

  17. Ligand-Gated Ion Channels: Permeation and Activation1

    NASA Astrophysics Data System (ADS)

    Lynch, Joseph W.; Barry, Peter H.

    Ligand-gated ion channels (LGICs) are fast-responding channels in which the receptor, which binds the activating molecule (the ligand), and the ion channel are part of the same nanomolecular protein complex. This chapter will describe the properties and functions of the nicotinic acetylcholine LGIC superfamily, which play a critical role in the fast chemical transmission of electrical signals between nerve cells at synapses and between nerve and muscle cells at endplates. All the processing functions of the brain and the resulting behavioral output depend on chemical transmission across such neuronal interconnections. To describe the properties of the channels of this LGIC superfamily,we will mainly use two examples of this family of channels: the excitatory nicotinic acetylcholine receptor (nAChR) and the inhibitory glycine receptor (GlyR) channels. In the chemical transmission of electrical signals, the arrival of an electrical signal at the synaptic terminal of a nerve causes the release of a chemical signal—a neurotransmitter molecule (the ligand, also referred to as the agonist). The neurotransmitter rapidly diffuses across the very narrow 20-40 nm synaptic gap between the cells and binds to the LGIC receptors in the membrane of the target (postsynaptic) cell and generates a new electrical signal in that cell (e.g., Kandel et al., 2000). How this chemical signal is converted into an electrical one depends on the fundamental properties of LGICs and the ionic composition of the postsynaptic cell and its external solution.

  18. 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.

  19. Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels

    PubMed Central

    Maertens, Chantal; Wei, Lin; Tytgat, Jan; Droogmans, Guy; Nilius, Bernd

    2000-01-01

    It was the aim of this study to look for a high-affinity and selective polypeptide toxin, which could serve as a probe for the volume-regulated anion channel (VRAC) or the calcium-activated chloride channel (CaCC). We have partially purified chlorotoxin, including new and homologous short chain insectotoxins, from the crude venom of Leiurus quinquestriatus quinquestriatus (Lqq) by means of gel filtration chromatography. Material eluting between 280 and 420 min, corresponding to fractions 15–21, was lyophilized and tested on VRAC and CaCC, using the whole-cell patch-clamp technique. We have also tested the commercially available chlorotoxin on VRAC, CaCC, the cystic fibrosis transmembrane conductance regulator (CFTR) and on the glioma specific chloride channel (GCC). VRAC and the correspondent current, ICl,swell, was activated in Cultured Pulmonary Artery Endothelial (CPAE) cells by a 25% hypotonic solution. Neither of the fractions 16–21 significantly inhibited ICl,swell (n=4–5). Ca2+-activated Cl− currents, ICl,Ca, activated by loading T84 cells via the patch pipette with 1 μM free Ca2+, were not inhibited by any of the tested fractions (15–21), (n=2–5). Chlorotoxin (625 nM) did neither effect ICl,swell nor ICl,Ca (n=4–5). The CFTR channel, transiently transfected in COS cells and activated by a cocktail containing IBMX and forskolin, was not affected by 1.2 μM chlorotoxin (n=5). In addition, it did not affect currents through GCC. We conclude that submicromolar concentrations of chlorotoxin do not block volume-regulated, Ca2+-activated and CFTR chloride channels and that it can not be classified as a general chloride channel toxin. PMID:10683204

  20. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP.

    PubMed

    DiFrancesco, D; Tortora, P

    1991-05-09

    Cyclic AMP acts as a second messenger in the modulation of several ion channels that are typically controlled by a phosphorylation process. In cardiac pacemaker cells, adrenaline and acetylcholine regulate the hyperpolarization-activated current (if), but in opposite ways; this current is involved in the generation and modulation of pacemaker activity. These actions are mediated by cAMP and underlie control of spontaneous rate by neurotransmitters. Whether the cAMP modulation of if is mediated by channel phosphorylation is, however, still unknown. Here we investigate the action of cAMP on if in excised patches of cardiac pacemaker cells and find that cAMP activates if by a mechanism independent of phosphorylation, involving a direct interaction with the channels at their cytoplasmic side. Cyclic AMP activates if by shifting its activation curve to more positive voltages, in agreement with whole-cell results. This is the first evidence of an ion channel whose gating is dually regulated by voltage and direct cAMP binding.

  1. Understand spiciness: mechanism of TRPV1 channel activation by capsaicin.

    PubMed

    Yang, Fan; Zheng, Jie

    2017-03-01

    Capsaicin in chili peppers bestows the sensation of spiciness. Since the discovery of its receptor, transient receptor potential vanilloid 1 (TRPV1) ion channel, how capsaicin activates this channel has been under extensive investigation using a variety of experimental techniques including mutagenesis, patch-clamp recording, crystallography, cryo-electron microscopy, computational docking and molecular dynamic simulation. A framework of how capsaicin binds and activates TRPV1 has started to merge: capsaicin binds to a pocket formed by the channel's transmembrane segments, where it takes a "tail-up, head-down" configuration. Binding is mediated by both hydrogen bonds and van der Waals interactions. Upon binding, capsaicin stabilizes the open state of TRPV1 by "pull-and-contact" with the S4-S5 linker. Understanding the ligand-host interaction will greatly facilitate pharmaceutical efforts to develop novel analgesics targeting TRPV1.

  2. Identification of a dimerization domain in the TMEM16A calcium-activated chloride channel (CaCC)

    PubMed Central

    Tien, Jason; Lee, Hye Young; Minor, Daniel L.; Jan, Yuh Nung; Jan, Lily Yeh

    2013-01-01

    Transmembrane proteins with unknown function 16 (TMEM16A) is a calcium-activated chloride channel (CaCC) important for neuronal, exocrine, and smooth muscle functions. TMEM16A belongs to a family of integral membrane proteins that includes another CaCC, TMEM16B, responsible for controlling action potential waveform and synaptic efficacy, and a small-conductance calcium-activated nonselective cation channel, TMEM16F, linked to Scott syndrome. We find that these channels in the TMEM16 family share a homodimeric architecture facilitated by their cytoplasmic N termini. This dimerization domain is important for channel assembly in eukaryotic cells, and the in vitro association of peptides containing the dimerization domain is consistent with a homotypic protein–protein interaction. Amino acid substitutions in the dimerization domain affect functional TMEM16A-CaCC channel expression, as expected from its critical role in channel subunit assembly. PMID:23576756

  3. Role of small conductance Ca²⁺-activated K⁺ channels in controlling CA1 pyramidal cell excitability.

    PubMed

    Chen, Shmuel; Benninger, Felix; Yaari, Yoel

    2014-06-11

    Small-conductance Ca(2+)-activated K(+) (SK or K(Ca)2) channels are widely expressed in the CNS. In several types of neurons, these channels were shown to become activated during repetitive firing, causing early spike frequency adaptation. In CA1 pyramidal cells, SK channels in dendritic spines were shown to regulate synaptic transmission. However, the presence of functional SK channels in the somata and their role in controlling the intrinsic firing of these neurons has been controversial. Using whole-cell voltage-clamp and current-clamp recordings in acute hippocampal slices and focal applications of irreversible and reversible SK channel blockers, we provide evidence that functional SK channels are expressed in the somata and proximal dendrites of adult rat CA1 pyramidal cells. Although these channels can generate a medium duration afterhyperpolarizing current, they play only an auxiliary role in controlling the intrinsic excitability of these neurons, secondary to the low voltage-activating, noninactivating K(V)7/M channels. As long as K(V)7/M channels are operative, activation of SK channels during repetitive firing does not notably affect the spike output of CA1 pyramidal cells. However, when K(V)7/M channel activity is compromised, SK channel activation significantly and uniquely reduces spike output of these neurons. Therefore, proximal SK channels provide a "second line of defense" against intrinsic hyperexcitability, which may play a role in multiple conditions in which K(V)7/M channels activity is compromised, such as hyposmolarity.

  4. Nav channel mechanosensitivity: activation and inactivation accelerate reversibly with stretch.

    PubMed

    Morris, Catherine E; Juranka, Peter F

    2007-08-01

    Voltage-gated sodium channels (Nav) are modulated by many bilayer mechanical amphiphiles, but whether, like other voltage-gated channels (Kv, HCN, Cav), they respond to physical bilayer deformations is unknown. We expressed human heart Nav1.5 pore alpha-subunit in oocytes (where, unlike alphaNav1.4, alphaNav1.5 exhibits normal kinetics) and measured small macroscopic currents in cell-attached patches. Pipette pressure was used to reversibly stretch the membrane for comparison of I(Na)(t) before, during, and after stretch. At all voltages, and in a dose-dependent fashion, stretch accelerated the I(Na)(t) time course. The sign of membrane curvature was not relevant. Typical stretch stimuli reversibly accelerated both activation and inactivation by approximately 1.4-fold; normalization of peak I(Na)(t) followed by temporal scaling ( approximately 1.30- to 1.85-fold) resulted in full overlap of the stretch/no-stretch traces. Evidently the rate-limiting outward voltage sensor motion in the Nav1.5 activation path (as in Kv1) accelerated with stretch. Stretch-accelerated inactivation occurred even with activation saturated, so an independently stretch-modulated inactivation transition is also a possibility. Since Nav1.5 channel-stretch modulation was both reliable and reversible, and required stretch stimuli no more intense than what typically activates putative mechanotransducer channels (e.g., stretch-activated TRPC1-based currents), Nav channels join the ranks of putative mechanotransducers. It is noteworthy that at voltages near the activation threshold, moderate stretch increased the peak I(Na) amplitude approximately 1.5-fold. It will be important to determine whether stretch-modulated Nav current contributes to cardiac arrhythmias, to mechanosensory responses in interstitial cells of Cajal, to touch receptor responses, and to neuropathic (i.e., hypermechanosensitive) and/or normal pain reception.

  5. Light-Activated Ion Channels for Remote Control of Neural Activity

    PubMed Central

    Chambers, James J.; Kramer, Richard H.

    2009-01-01

    Light-activated ion channels provide a new opportunity to precisely and remotely control neuronal activity for experimental applications in neurobiology. In the past few years, several strategies have arisen that allow light to control ion channels and therefore neuronal function. Light-based triggers for ion channel control include caged compounds, which release active neurotransmitters when photolyzed with light, and natural photoreceptive proteins, which can be expressed exogenously in neurons. More recently, a third type of light trigger has been introduced: a photoisomerizable tethered ligand that directly controls ion channel activity in a light-dependent manner. Beyond the experimental applications for light-gated ion channels, there may be clinical applications in which these light-sensitive ion channels could prove advantageous over traditional methods. Electrodes for neural stimulation to control disease symptoms are invasive and often difficult to reposition between cells in tissue. Stimulation by chemical agents is difficult to constrain to individual cells and has limited temporal accuracy in tissue due to diffusional limitations. In contrast, ion channels that can be directly activated with light allow control with unparalleled spatial and temporal precision. The goal of this chapter is to describe light-regulated ion channels and how they have been tailored to control different aspects of neural activity, and how to use these channels to manipulate and better understand development, function, and plasticity of neurons and neural circuits. PMID:19195553

  6. Amphetamine activates calcium channels through dopamine transporter-mediated depolarization.

    PubMed

    Cameron, Krasnodara N; Solis, Ernesto; Ruchala, Iwona; De Felice, Louis J; Eltit, Jose M

    2015-11-01

    Amphetamine (AMPH) and its more potent enantiomer S(+)AMPH are psychostimulants used therapeutically to treat attention deficit hyperactivity disorder and have significant abuse liability. AMPH is a dopamine transporter (DAT) substrate that inhibits dopamine (DA) uptake and is implicated in DA release. Furthermore, AMPH activates ionic currents through DAT that modify cell excitability presumably by modulating voltage-gated channel activity. Indeed, several studies suggest that monoamine transporter-induced depolarization opens voltage-gated Ca(2+) channels (CaV), which would constitute an additional AMPH mechanism of action. In this study we co-express human DAT (hDAT) with Ca(2+) channels that have decreasing sensitivity to membrane depolarization (CaV1.3, CaV1.2 or CaV2.2). Although S(+)AMPH is more potent than DA in transport-competition assays and inward-current generation, at saturating concentrations both substrates indirectly activate voltage-gated L-type Ca(2+) channels (CaV1.3 and CaV1.2) but not the N-type Ca(2+) channel (CaV2.2). Furthermore, the potency to achieve hDAT-CaV electrical coupling is dominated by the substrate affinity on hDAT, with negligible influence of L-type channel voltage sensitivity. In contrast, the maximal coupling-strength (defined as Ca(2+) signal change per unit hDAT current) is influenced by CaV voltage sensitivity, which is greater in CaV1.3- than in CaV1.2-expressing cells. Moreover, relative to DA, S(+)AMPH showed greater coupling-strength at concentrations that induced relatively small hDAT-mediated currents. Therefore S(+)AMPH is not only more potent than DA at inducing hDAT-mediated L-type Ca(2+) channel currents but is a better depolarizing agent since it produces tighter electrical coupling between hDAT-mediated depolarization and L-type Ca(2+) channel activation.

  7. PKC-dependent activation of human K2P18.1 K+ channels

    PubMed Central

    Rahm, Ann-Kathrin; Gierten, Jakob; Kisselbach, Jana; Staudacher, Ingo; Staudacher, Kathrin; Schweizer, Patrick A; Becker, Rüdiger; Katus, Hugo A; Thomas, Dierk

    2012-01-01

    BACKGROUND AND PURPOSE Two-pore-domain K+ channels (K2P) mediate K+ background currents that modulate the membrane potential of excitable cells. K2P18.1 (TWIK-related spinal cord K+ channel) provides hyperpolarizing background currents in neurons. Recently, a dominant-negative loss-of-function mutation in K2P18.1 has been implicated in migraine, and activation of K2P18.1 channels was proposed as a therapeutic strategy. Here we elucidated the molecular mechanisms underlying PKC-dependent activation of K2P18.1 currents. EXPERIMENTAL APPROACH Human K2P18.1 channels were heterologously expressed in Xenopus laevis oocytes, and currents were recorded with the two-electrode voltage clamp technique. KEY RESULTS Stimulation of PKC using phorbol 12-myristate-13-acetate (PMA) activated the hK2P18.1 current by 3.1-fold in a concentration-dependent fashion. The inactive analogue 4α-PMA had no effect on channel activity. The specific PKC inhibitors bisindolylmaleimide I, Ro-32-0432 and chelerythrine reduced PMA-induced channel activation indicating that PKC is involved in this effect of PMA. Selective activation of conventional PKC isoforms with thymeleatoxin (100 nM) did not reproduce K2P18.1 channel activation. Current activation by PMA was not affected by pretreatment with CsA (calcineurin inhibitor) or KT 5720 (PKA inhibitor), ruling out a significant contribution of calcineurin or cross-talk with PKA to the PKC-dependent hK2P18.1 activation. Finally, mutation of putative PKC phosphorylation sites did not prevent PMA-induced K2P18.1 channel activation. CONCLUSIONS AND IMPLICATIONS We demonstrated that activation of hK2P18.1 (TRESK) by PMA is mediated by PKC stimulation. Hence, PKC-mediated activation of K2P18.1 background currents may serve as a novel molecular target for migraine treatment. PMID:22168364

  8. Human activities impact on mountain river channels (case study of Kamchatka peninsula rivers)

    NASA Astrophysics Data System (ADS)

    Ermakova, Aleksandra S.

    2010-05-01

    Human-induced driving factors along with natural environmental changes greatly impact on fluvial regime of rivers. On mountain and semi-mountain territories these processes are developed in the most complicated manner due to man-made activities diversity throughout river basins. Besides these processes are significantly enhanced because of the disastrous natural processes (like volcanic and mud-flow activity) frequent occurrences in mountainous regions. On of the most striking example on the matter is Kamchatka peninsula which is located at the North-West part of Russian Federation. This paper contributes to the study of human activities impact on fluvial systems in this volcanic mountain region. Human effects on rivers directly alter channel morphology and deformations, dynamics of water and sediment movement, aquatic communities or indirectly affect streams by altering the movement of water and sediment into the channel. In case study of Kamchatka peninsula human activities affect fluvial systems through engineering works including construction of bridges, dams and channel diversions and placer mining. These processes are characterized by spatial heterogeneity because of irregular population distribution. Due to specific natural conditions of the peninsula the most populated areas are the valleys of big rivers (rivers Kamchatka, Avacha, Bistraya (Bolshaya), etc) within piedmont and plain regions. These rivers are characterized by very unstable channels. Both with man-made activities this determines wide range of fluvial system changes. Firstly bridges construction leads to island and logjam formation directly near their piers and intensification of channels patterns shifts. Furthermore rivers of the peninsula are distinguished for high water flow velocities and water rate. Incorrect bridge constructions both with significant channel deformations lead to the destructions of the bridges themselves due to intensive bank erosion. Secondly, intensive water flow

  9. Computational study of a calcium release-activated calcium channel

    NASA Astrophysics Data System (ADS)

    Talukdar, Keka; Shantappa, Anil

    2016-05-01

    The naturally occurring proteins that form hole in membrane are commonly known as ion channels. They play multiple roles in many important biological processes. Deletion or alteration of these channels often leads to serious problems in the physiological processes as it controls the flow of ions through it. The proper maintenance of the flow of ions, in turn, is required for normal health. Here we have investigated the behavior of a calcium release-activated calcium ion channel with pdb entry 4HKR in Drosophila Melanogaster. The equilibrium energy as well as molecular dynamics simulation is performed first. The protein is subjected to molecular dynamics simulation to find their energy minimized value. Simulation of the protein in the environment of water and ions has given us important results too. The solvation energy is also found using Charmm potential.

  10. A cyclic AMP-activated K+ channel in Drosophila larval muscle is persistently activated in dunce.

    PubMed

    Delgado, R; Hidalgo, P; Diaz, F; Latorre, R; Labarca, P

    1991-01-15

    Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the presence of a potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2+. Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an increased probability of opening.

  11. A cyclic AMP-activated K+ channel in Drosophila larval muscle is persistently activated in dunce.

    PubMed Central

    Delgado, R; Hidalgo, P; Diaz, F; Latorre, R; Labarca, P

    1991-01-01

    Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the presence of a potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2+. Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an increased probability of opening. PMID:1846445

  12. Combined single channel and single molecule detection identifies subunit composition of STIM1-activated transient receptor potential canonical (TRPC) channels.

    PubMed

    Asanov, Alexander; Sampieri, Alicia; Moreno, Claudia; Pacheco, Jonathan; Salgado, Alfonso; Sherry, Ryan; Vaca, Luis

    2015-01-01

    Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.

  13. Pharmacological “cross-inhibition” of connexin hemichannels and swelling activated anion channels

    PubMed Central

    Ye, Zu-Cheng; Oberheim, NancyAnn; Kettenmann, Helmut; Ransom, Bruce R.

    2009-01-01

    The study of ion channels has relied heavily on the use of pharmacological blocking agents. However, many of these agents have multiple effects, which may compromise interpretation of results when the affected mechanisms/pathways mediate similar functions. Volume regulated anion channels (VRAC) and connexin hemichannels can both mediate the release of glutamate and taurine, although these channels have distinct activation stimuli and hemichannels, but not VRAC, are permeable to Lucifer yellow (LY). It has been reported that some anion channel blockers may inhibit connexin hemichannels. We further examined the effects of classic gap junction/hemichannel blockers and anion channel blockers on these channels. The typical VRAC blockers NPPB, IAA-94 and tamoxifen blocked low divalent cation-induced glutamate and taurine release and LY loading, presumed due to hemichannel opening. The blocking action of these compounds on hemichannels was concentration dependent and fell within the same range where the drugs classically block VRACs. Conversely, carbenoxolone (CBX), the most widely used gap junction/hemichannel blocker, was an effective blocker of VRAC mediated glutamate and taurine release, and blocked these channels at similar concentrations at which it blocked hemichannels. The CBX effect on VRACs was verified using astrocytes from connexin 43 knock out (Cx43 KO) animals. In these cells, the hypotonic induced amino acid flux was retained while the low divalent cation solution flux was lost. These results extend our knowledge about ‘cross-inhibition’ of VRACs and gap junctions/hemichannels by certain pharmacological agents. Given the overlap in function of these two types of channels, great care must be exerted in using pharmacological blockers to identify one channel from the other. PMID:18837047

  14. Variomics screen identifies the re-entrant loop of the calcium-activated chloride channel ANO1 that facilitates channel activation.

    PubMed

    Bill, Anke; Popa, M Oana; van Diepen, Michiel T; Gutierrez, Abraham; Lilley, Sarah; Velkova, Maria; Acheson, Kathryn; Choudhury, Hedaythul; Renaud, Nicole A; Auld, Douglas S; Gosling, Martin; Groot-Kormelink, Paul J; Gaither, L Alex

    2015-01-09

    The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases.

  15. Variomics Screen Identifies the Re-entrant Loop of the Calcium-activated Chloride Channel ANO1 That Facilitates Channel Activation*

    PubMed Central

    Bill, Anke; Popa, M. Oana; van Diepen, Michiel T.; Gutierrez, Abraham; Lilley, Sarah; Velkova, Maria; Acheson, Kathryn; Choudhury, Hedaythul; Renaud, Nicole A.; Auld, Douglas S.; Gosling, Martin; Groot-Kormelink, Paul J.; Gaither, L. Alex

    2015-01-01

    The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases. PMID:25425649

  16. Transmembrane helix straightening and buckling underlies activation of mechanosensitive and thermosensitive K(2P) channels.

    PubMed

    Lolicato, Marco; Riegelhaupt, Paul M; Arrigoni, Cristina; Clark, Kimberly A; Minor, Daniel L

    2014-12-17

    Mechanical and thermal activation of ion channels is central to touch, thermosensation, and pain. The TRAAK/TREK K(2P) potassium channel subfamily produces background currents that alter neuronal excitability in response to pressure, temperature, signaling lipids, and anesthetics. How such diverse stimuli control channel function is unclear. Here we report structures of K(2P)4.1 (TRAAK) bearing C-type gate-activating mutations that reveal a tilting and straightening of the M4 inner transmembrane helix and a buckling of the M2 transmembrane helix. These conformational changes move M4 in a direction opposite to that in classical potassium channel activation mechanisms and open a passage lateral to the pore that faces the lipid bilayer inner leaflet. Together, our findings uncover a unique aspect of K(2P) modulation, indicate a means for how the K(2P) C-terminal cytoplasmic domain affects the C-type gate which lies ∼40Å away, and suggest how lipids and bilayer inner leaflet deformations may gate the channel.

  17. Transmembrane Helix Straightening and Buckling Underlies Activation of Mechanosensitive and Thermosensitive K2P Channels

    PubMed Central

    Lolicato, Marco; Riegelhaupt, Paul M.; Arrigoni, Cristina; Clark, Kimberly A.; Minor, Daniel L.

    2014-01-01

    SUMMARY Mechanical and thermal activation of ion channels is central to touch, thermosensation, and pain. The TRAAK/TREK K2P potassium channel subfamily produces background currents that alter neuronal excitability in response to pressure, temperature, signaling lipids, and anesthetics. How such diverse stimuli control channel function is unclear. Here we report structures of K2P4.1 (TRAAK) bearing C-type gate-activating mutations that reveal a tilting and straightening of the M4 inner transmembrane helix and a buckling of the M2 transmembrane helix. These conformational changes move M4 in a direction opposite to that in classical potassium channel activation mechanisms and open a passage lateral to the pore that faces the lipid bilayer inner leaflet. Together, our findings uncover a unique aspect of K2P modulation, indicate a means for how the K2P C-terminal cytoplasmic domain affects the C-type gate which lies ~40Å away, and suggest how lipids and bilayer inner leaflet deformations may gate the channel. PMID:25500157

  18. Zinc activates damage-sensing TRPA1 ion channels

    PubMed Central

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

    2009-01-01

    Zinc is an essential biological trace element. It is required for the structure or function of over 300 proteins, and 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 novel 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 a major target for the sensory effects of zinc, and support an emerging role for zinc as a signaling molecule that can modulate sensory transmission. PMID:19202543

  19. Multi-ion occupancy alters gating in high-conductance, Ca(2+)-activated K+ channels

    PubMed Central

    1991-01-01

    In this study, single-channel recordings of high-conductance Ca(2+)- activated K+ channels from rat skeletal muscle inserted into planar lipid bilayer were used to analyze the effects of two ionic blockers, Ba2+ and Na+, on the channel's gating reactions. The gating equilibrium of the Ba(2+)-blocked channel was investigated through the kinetics of the discrete blockade induced by Ba2+ ions. Gating properties of Na(+)- blocked channels could be directly characterized due to the very high rates of Na+ blocking/unblocking reactions. While in the presence of K+ (5 mM) in the external solution Ba2+ is known to stabilize the open state of the blocked channel (Miller, C., R. Latorre, and I. Reisin. 1987. J. Gen. Physiol. 90:427-449), we show that the divalent blocker stabilizes the closed-blocked state if permeant ions are removed from the external solution (K+ less than 10 microM). Ionic substitutions in the outer solution induce changes in the gating equilibrium of the Ba(2+)-blocked channel that are tightly correlated to the inhibition of Ba2+ dissociation by external monovalent cations. In permeant ion-free external solutions, blockade of the channel by internal Na+ induces a shift (around 15 mV) in the open probability--voltage curve toward more depolarized potentials, indicating that Na+ induces a stabilization of the closed-blocked state, as does Ba2+ under the same conditions. A kinetic analysis of the Na(+)-blocked channel indicates that the closed- blocked state is favored mainly by a decrease in opening rate. Addition of 1 mM external K+ completely inhibits the shift in the activation curve without affecting the Na(+)-induced reduction in the apparent single-channel amplitude. The results suggest that in the absence of external permeant ions internal blockers regulate the permeant ion occupancy of a site near the outer end of the channel. Occupancy of this site appears to modulate gating primarily by speeding the rate of channel opening. PMID:2056305

  20. TRP channels activated by extracellular hypo-osmoticity in epithelia.

    PubMed

    Harteneck, C; Reiter, B

    2007-02-01

    TRP (transient receptor potential) channels comprise a superfamily of non-selective cation channels with at least seven subfamilies. The variety of subfamilies corresponds to the differences in the activation mechanisms and functions. TRPM3 (TRP melastatin 3) and TRPV4 (TRP vanilloid 3) have been characterized as cation channels activated by extracellular hypo-osmoticity. In addition, TRPV4 is activated by metabolites of arachidonic acid as well as alpha-isomers of phorbol esters known to be ineffective in stimulating proteins of the protein kinase C family. TRPM3 is responsive to sphingosine derivatives. The detection of splice variants with probably different activation mechanisms supports the idea that TRPM3 may have diverse cellular functions depending on the expression of a particular variant. The expression of TRPV4 in many epithelial cell types raised the question of the role of TRPV4 in epithelial physiology. Single-cell experiments as well as approaches using epithelial layers show that multiple cellular responses are triggered by TRPV4 activation and subsequent elevation of intracellular calcium. The TRPV4-induced responses increasing transcellular ion flux as well as paracellular permeability may allow the cells to adjust to changes in extracellular osmolarity. In summary, TRPV4 plays a central role in epithelial homoeostasis by modulating epithelial barrier function.

  1. Atomic basis for therapeutic activation of neuronal potassium channels

    PubMed Central

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

    2015-01-01

    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. PMID:26333338

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

    SciTech Connect

    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 inactivates 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.

  3. Differential effects of quercetin glycosides on GABAC receptor channel activity.

    PubMed

    Kim, Hyeon-Joong; Lee, Byung-Hwan; Choi, Sun-Hye; Jung, Seok-Won; Kim, Hyun-Sook; Lee, Joon-Hee; Hwang, Sung-Hee; Pyo, Mi-Kyung; Kim, Hyoung-Chun; Nah, Seung-Yeol

    2015-01-01

    Quercetin, a representative flavonoid, is a compound of low molecular weight found in various colored plants and vegetables. Quercetin shows a wide range of neuropharmacological activities. In fact, quercetin naturally exists as monomer-(quercetin-3-O-rhamnoside) (Rham1), dimer-(Rutin), or trimer-glycosides [quercetin-3-(2(G)-rhamnosylrutinoside)] (Rham2) at carbon-3 in fruits and vegetables. The carbohydrate components are removed after ingestion into gastrointestinal systems. The role of the glycosides attached to quercetin in the regulation of γ-aminobutyric acid class C (GABAC) receptor channel activity has not been determined. In the present study, we examined the effects of quercetin glycosides on GABAC receptor channel activity by expressing human GABAC alone in Xenopus oocytes using a two-electrode voltage clamp technique and also compared the effects of quercetin glycosides with quercetin. We found that GABA-induced inward current (I GABA ) was inhibited by quercetin or quercetin glycosides. The inhibitory effects of quercetin and its glycosides on I GABA were concentration-dependent and reversible in the order of Rutin ≈ quercetin ≈ Rham 1 > Rham 2. The inhibitory effects of quercetin and its glycosides on I GABA were noncompetitive and membrane voltage-insensitive. These results indicate that quercetin and its glycosides regulate GABAC receptor channel activity through interaction with a different site from that of GABA, and that the number of carbohydrate attached to quercetin might play an important role in the regulation of GABAC receptor channel activity.

  4. The complete structure of an activated open sodium channel

    PubMed Central

    Sula, Altin; Booker, Jennifer; Ng, Leo C. T.; Naylor, Claire E.; DeCaen, Paul G.; Wallace, B. A.

    2017-01-01

    Voltage-gated sodium channels (Navs) play essential roles in excitable tissues, with their activation and opening resulting in the initial phase of the action potential. The cycling of Navs through open, closed and inactivated states, and their closely choreographed relationships with the activities of other ion channels lead to exquisite control of intracellular ion concentrations in both prokaryotes and eukaryotes. Here we present the 2.45 Å resolution crystal structure of the complete NavMs prokaryotic sodium channel in a fully open conformation. A canonical activated conformation of the voltage sensor S4 helix, an open selectivity filter leading to an open activation gate at the intracellular membrane surface and the intracellular C-terminal domain are visible in the structure. It includes a heretofore unseen interaction motif between W77 of S3, the S4–S5 interdomain linker, and the C-terminus, which is associated with regulation of opening and closing of the intracellular gate. PMID:28205548

  5. The intrinsically liganded cyclic nucleotide-binding homology domain promotes KCNH channel activation.

    PubMed

    Zhao, Yaxian; Goldschen-Ohm, Marcel P; Morais-Cabral, João H; Chanda, Baron; Robertson, Gail A

    2017-02-01

    Channels in the ether-à-go-go or KCNH family of potassium channels are characterized by a conserved, C-terminal domain with homology to cyclic nucleotide-binding homology domains (CNBhDs). Instead of cyclic nucleotides, two amino acid residues, Y699 and L701, occupy the binding pocket, forming an "intrinsic ligand." The role of the CNBhD in KCNH channel gating is still unclear, however, and a detailed characterization of the intrinsic ligand is lacking. In this study, we show that mutating both Y699 and L701 to alanine, serine, aspartate, or glycine impairs human EAG1 channel function. These mutants slow channel activation and shift the conductance-voltage (G-V) relation to more depolarized potentials. The mutations affect activation and the G-V relation progressively, indicating that the gating machinery is sensitive to multiple conformations of the CNBhD. Substitution with glycine at both sites (GG), which eliminates the side chains that interact with the binding pocket, also reduces the ability of voltage prepulses to populate more preactivated states along the activation pathway (i.e., the Cole-Moore effect), as if stabilizing the voltage sensor in deep resting states. Notably, deletion of the entire CNBhD (577-708, ΔCNBhD) phenocopies the GG mutant, suggesting that GG is a loss-of-function mutation and the CNBhD requires an intrinsic ligand to exert its functional effects. We developed a kinetic model for both wild-type and ΔCNBhD mutant channels that describes all our observations on activation kinetics, the Cole-Moore shift, and G-V relations. These findings support a model in which the CNBhD both promotes voltage sensor activation and stabilizes the open pore. The intrinsic ligand is critical for these functional effects.

  6. Ca(2+)-activated anion channels and membrane depolarizations induced by blue light and cold in Arabidopsis seedlings

    NASA Technical Reports Server (NTRS)

    Lewis, B. D.; Karlin-Neumann, G.; Davis, R. W.; Spalding, E. P.; Evans, M. L. (Principal Investigator)

    1997-01-01

    The activation of an anion channel in the plasma membrane of Arabidopsis thaliana hypocotyls by blue light (BL) is believed to be a signal-transducing event leading to growth inhibition. Here we report that the open probability of this particular anion channel depends on cytoplasmic Ca2+ ([Ca2+]cyt) within the concentration range of 1 to 10 microM, raising the possibility that BL activates the anion channel by increasing [Ca2+]cyt. Arabidopsis seedlings cytoplasmically expressing aequorin were generated to test this possibility. Aequorin luminescence did not increase during or after BL, providing evidence that Ca2+ does not play a second-messenger role in the activation of anion channels. However, cold shock simultaneously triggered a large increase in [Ca2+]cyt and a 110-mV transient depolarization of the plasma membrane. A blocker of the anion channel, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, blocked 61% of the cold-induced depolarization without affecting the increase in [Ca2+]cyt. These data led us to propose that cold shock opens Ca2+ channels at the plasma membrane, allowing an inward, depolarizing Ca2+ current. The resulting large increase in [Ca2+]cyt activates the anion channel, which further depolarizes the membrane. Although an increase in [Ca2+]cyt may activate anion channels in response to cold, it appears that BL does so via a Ca(2+)-independent pathway.

  7. Allosteric coupling of the inner activation gate to the outer pore of a potassium channel.

    PubMed

    Peters, Christian J; Fedida, David; Accili, Eric A

    2013-10-23

    In potassium channels, functional coupling of the inner and outer pore gates may result from energetic interactions between residues and conformational rearrangements that occur along a structural path between them. Here, we show that conservative mutations of a residue near the inner activation gate of the Shaker potassium channel (I470) modify the rate of C-type inactivation at the outer pore, pointing to this residue as part of a pathway that couples inner gate opening to changes in outer pore structure and reduction of ion flow. Because they remain equally sensitive to rises in extracellular potassium, altered inactivation rates of the mutant channels are not secondary to modified binding of potassium to the outer pore. Conservative mutations of I470 also influence the interaction of the Shaker N-terminus with the inner gate, which separately affects the outer pore.

  8. Allosteric coupling of the inner activation gate to the outer pore of a potassium channel

    NASA Astrophysics Data System (ADS)

    Peters, Christian J.; Fedida, David; Accili, Eric A.

    2013-10-01

    In potassium channels, functional coupling of the inner and outer pore gates may result from energetic interactions between residues and conformational rearrangements that occur along a structural path between them. Here, we show that conservative mutations of a residue near the inner activation gate of the Shaker potassium channel (I470) modify the rate of C-type inactivation at the outer pore, pointing to this residue as part of a pathway that couples inner gate opening to changes in outer pore structure and reduction of ion flow. Because they remain equally sensitive to rises in extracellular potassium, altered inactivation rates of the mutant channels are not secondary to modified binding of potassium to the outer pore. Conservative mutations of I470 also influence the interaction of the Shaker N-terminus with the inner gate, which separately affects the outer pore.

  9. Swell activated chloride channel function in human neutrophils

    SciTech Connect

    Salmon, Michael D.; Ahluwalia, Jatinder

    2009-04-17

    Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I{sub e} has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I{sub e} must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.

  10. Fluctuation driven active molecular transport in passive channel proteins

    NASA Astrophysics Data System (ADS)

    Kosztin, Ioan

    2006-03-01

    Living cells interact with their extracellular environment through the cell membrane, which acts as a protective permeability barrier for preserving the internal integrity of the cell. However, cell metabolism requires controlled molecular transport across the cell membrane, a function that is fulfilled by a wide variety of transmembrane proteins, acting as either passive or active transporters. In this talk it is argued that, contrary to the general belief, in active cell membranes passive and spatially asymmetric channel proteins can act as active transporters by consuming energy from nonequilibrium fluctuations fueled by cell metabolism. This assertion is demonstrated in the case of the E. coli aquaglyceroporin GlpF channel protein, whose high resolution crystal structure is manifestly asymmetric. By calculating the glycerol flux through GlpF within the framework of a stochastic model, it is found that, as a result of channel asymmetry, glycerol uptake driven by a concentration gradient is enhanced significantly in the presence of non-equilibrium fluctuations. Furthermore, the enhancement caused by a ratchet-like mechanism is larger for the outward, i.e., from the cytoplasm to the periplasm, flux than for the inward one, suggesting that the same non-equilibrium fluctuations also play an important role in protecting the interior of the cell against poisoning by excess uptake of glycerol. Preliminary data on water and sugar transport through aquaporin and maltoporin channels, respectively, are indicative of the universality of the proposed nonequilibrium-fluctuation-driven active transport mechanism. This work was supported by grants from the Univ. of Missouri Research Board, the Institute for Theoretical Sciences and the Department of Energy (DOE Contract W-7405-ENG-36), and the National Science Foundation (FIBR-0526854).

  11. Activation of protein kinase C inhibits calcium-activated potassium channels in rat pituitary tumour cells.

    PubMed Central

    Shipston, M J; Armstrong, D L

    1996-01-01

    1. The regulation of large-conductance, calcium- and voltage-dependent potassium (BK) channels by protein kinase C (PKC) was investigated in clonal rat anterior pituitary cells (GH4C1), which were voltage clamped at -40 mV in a physiological potassium gradient through amphotericin-perforated patches. 2. Maximal activation of PKC by 100 nM phorbol 12, 13-dibutyrate (PdBu) almost completely inhibited the voltage-activated outward current through BK channels. In contrast PdBu had no significant effect on the residual outward current after block of BK channels with 2 mM TEA or 30 nM charybdotoxin. In single-channel recordings from cell-attached patches, PdBu reduced the open probability of BK channels more than eightfold with no significant effect on mean open lifetime or unitary conductance. 3. The effects of PdBu on BK channels were not mimicked by the 4 alpha-isomer, which does not activate PKC, and were blocked almost completely by 25 microM chelerythrine, a specific, noncompetitive PKC inhibitor. 4. PdBu had no significant effect on the amplitude of the pharmacologically isolated, high voltage-activated calcium current. 5. Inhibition of BK channel activity by PKC provides the first molecular mechanism linking hormonal activation of phospholipase C to sustained excitability in pituitary cells. PMID:8799890

  12. Increasing SK2 channel activity impairs associative learning

    PubMed Central

    McKay, Bridget M.; Oh, M. Matthew; Galvez, Roberto; Burgdorf, Jeffrey; Kroes, Roger A.; Weiss, Craig; Adelman, John P.; Moskal, Joseph R.

    2012-01-01

    Enhanced intrinsic neuronal excitability of hippocampal pyramidal neurons via reductions in the postburst afterhyperpolarization (AHP) has been hypothesized to be a biomarker of successful learning. This is supported by considerable evidence that pharmacologic enhancement of neuronal excitability facilitates learning. However, it has yet to be demonstrated that pharmacologic reduction of neuronal excitability restricted to the hippocampus can retard acquisition of a hippocampus-dependent task. Thus, the present study was designed to address this latter point using a small conductance potassium (SK) channel activator NS309 focally applied to the dorsal hippocampus. SK channels are important contributors to intrinsic excitability, as measured by the medium postburst AHP. NS309 increased the medium AHP and reduced excitatory postsynaptic potential width of CA1 neurons in vitro. In vivo, NS309 reduced the spontaneous firing rate of CA1 pyramidal neurons and impaired trace eyeblink conditioning in rats. Conversely, trace eyeblink conditioning reduced levels of SK2 channel mRNA and protein in the hippocampus. Therefore, the present findings indicate that modulation of SK channels is an important cellular mechanism for associative learning and further support postburst AHP reductions in hippocampal pyramidal neurons as a biomarker of successful learning. PMID:22552186

  13. Modulation of bone remodeling via mechanically activated ion channels

    NASA Technical Reports Server (NTRS)

    Duncan, Randall L. (Principal Investigator)

    1996-01-01

    A critical factor in the maintenance of bone mass is the physical forces imposed upon the skeleton. Removal of these forces, such as in a weightless environment, results in a rapid loss of bone, whereas application of exogenous mechanical strain has been shown to increase bone formation. Numerous flight and ground-based experiments indicate that the osteoblast is the key bone cell influenced by mechanical stimulation. Aside from early transient fluctuations in response to unloading, osteoclast number and activity seem unaffected by removal of strain. However, bone formation is drastically reduced in weightlessness and osteoblasts respond to mechanical strain with an increase in the activity of a number of second messenger pathways resulting in increased anabolic activity. Unfortunately, the mechanism by which the osteoblast converts physical stimuli into a biochemical message, a process we have termed biochemical coupling, remains elusive. Prior to the application of this grant, we had characterized a mechanosensitive, cation nonselective channel (SA-cat) in osteoblast-like osteosarcoma cells that we proposed is the initial signalling mechanism for mechanotransduction. During the execution of this grant, we have made considerable progress to further characterize this channel as well as to determine its role in the osteoblastic response to mechanical strain. To achieve these goals, we combined electrophysiologic techniques with cellular and molecular biology methods to examine the role of these channels in the normal function of the osteoblast in vitro.

  14. An Investigation of How the Channel of Input and Access to Test Questions Affect L2 Listening Test Performance

    ERIC Educational Resources Information Center

    Wagner, Elvis

    2013-01-01

    The use of video technology has become widespread in the teaching and testing of second-language (L2) listening, yet research into how this technology affects the learning and testing process has lagged. The current study investigated how the channel of input (audiovisual vs. audio-only) used on an L2 listening test affected test-taker…

  15. External copper inhibits the activity of the large-conductance calcium- and voltage-sensitive potassium channel from skeletal muscle.

    PubMed

    Morera, F J; Wolff, D; Vergara, C

    2003-03-01

    We have characterized the effect of external copper on the gating properties of the large-conductance calcium- and voltage-sensitive potassium channel from skeletal muscle, incorporated into artificial bilayers. The effect of Cu2+ was evaluated as changes in the gating kinetic properties of the channel after the addition of this ion. We found that, from concentrations of 20 microM and up, copper induced a concentration- and time-dependent decrease in channel open probability. The inhibition of channel activity by Cu2+ could not be reversed by washing or by addition of the copper chelator, bathocuproinedisulfonic acid. However, channel activity was appreciably restored by the sulfhydryl reducing agent dithiothreitol. The effect of copper was specific since other transition metal divalent cations such as Ni2+, Zn2+ or Cd2+ did not affect BK(Ca) channel activity in the same concentration range. These results suggest that external Cu2+-induced inhibition of channel activity was due to direct or indirect oxidation of key amino-acid sulfhydryl groups that might have a role in channel gating.

  16. Leptin excites POMC neurons via activation of TRPC channels

    PubMed Central

    Qiu, Jian; Fang, Yuan; Rønnekleiv, Oline K.; Kelly, Martin J.

    2010-01-01

    Leptin can exert its potent appetite-suppressing effects via activation of hypothalamic proopiomelanocortin (POMC) neurons. It depolarizes POMC neurons via activation of a yet unidentified non-selective cation current. Therefore, we sought to identify the conductance activated by leptin using whole cell recording in EGFP-POMC neurons from transgenic mice. The TRPC channel blockers SKF96365, FFA and 2-APB potently inhibited the leptin-induced current. Also, lanthanum (La3+) and intracellular Ca2+ potentiated the effects of leptin. Moreover, the DAG permeable analog OAG failed to activate any TRPC current. Using a Cs+-gluconate-based internal solution, leptin-activated current reversed near -20 mV. After replacement of external Na+ and K+ with Cs+, the reversal shifted to near 0 mV, and the I/V curve exhibited a negative slope conductance at voltages more negative than –40 mV. Based on scRT-PCR, TRPC1 and TRPC4-7 mRNA were expressed in POMC neurons with TRPC5 being the most prevalent. The leptin-induced current was blocked by Jak2 inhibitor AG490, the PI3 Kinase inhibitor wortmannin and the phospholipase C inhibitors, U73122 and ET-18-OCH3. Notably, we identified PLCγ1 transcripts in the majority of POMC neurons. Therefore, leptin through a Jak2-PI3 kinase-PLCγ pathway activates TRPC channels, and TRPC1, 4 and 5 appear to be the key channels mediating the depolarizing effects of leptin in POMC neurons. PMID:20107083

  17. Location of Release Sites and Calcium-Activated Chloride Channels Relative to Calcium Channels at the Photoreceptor Ribbon Synapse

    PubMed Central

    Mercer, A. J.; Rabl, K.; Riccardi, G. E.; Brecha, N. C.; Stella, S. L.

    2011-01-01

    Vesicle release from photoreceptor ribbon synapses is regulated by L-type Ca2+ channels, which are in turn regulated by Cl− moving through calcium-activated chloride [Cl(Ca)] channels. We assessed the proximity of Ca2+ channels to release sites and Cl(Ca) channels in synaptic terminals of salamander photoreceptors by comparing fast (BAPTA) and slow (EGTA) intracellular Ca2+ buffers. BAPTA did not fully block synaptic release, indicating some release sites are <100 nm from Ca2+ channels. Comparing Cl(Ca) currents with predicted Ca2+ diffusion profiles suggested that Cl(Ca) and Ca2+ channels average a few hundred nanometers apart, but the inability of BAPTA to block Cl(Ca) currents completely suggested some channels are much closer together. Diffuse immunolabeling of terminals with an antibody to the putative Cl(Ca) channel TMEM16A supports the idea that Cl(Ca) channels are dispersed throughout the presynaptic terminal, in contrast with clustering of Ca2+ channels near ribbons. Cl(Ca) currents evoked by intracellular calcium ion concentration ([Ca2+]i) elevation through flash photolysis of DM-nitrophen exhibited EC50 values of 556 and 377 nM with Hill slopes of 1.8 and 2.4 in rods and cones, respectively. These relationships were used to estimate average submembrane [Ca2+]i in photoreceptor terminals. Consistent with control of exocytosis by [Ca2+] nanodomains near Ca2+ channels, average submembrane [Ca2+]i remained below the vesicle release threshold (∼400 nM) over much of the physiological voltage range for cones. Positioning Ca2+ channels near release sites may improve fidelity in converting voltage changes to synaptic release. A diffuse distribution of Cl(Ca) channels may allow Ca2+ influx at one site to influence relatively distant Ca2+ channels. PMID:21084687

  18. Dynamitin affects cell-surface expression of voltage-gated sodium channel Nav1.5.

    PubMed

    Chatin, Benoît; Colombier, Pauline; Gamblin, Anne Laure; Allouis, Marie; Le Bouffant, Françoise

    2014-11-01

    The major cardiac voltage-gated sodium channel Nav1.5 associates with proteins that regulate its biosynthesis, localization, activity and degradation. Identification of partner proteins is crucial for a better understanding of the channel regulation. Using a yeast two-hybrid screen, we identified dynamitin as a Nav1.5-interacting protein. Dynamitin is part of the microtubule-binding multiprotein complex dynactin. When overexpressed it is a potent inhibitor of dynein/kinesin-mediated transport along the microtubules by disrupting the dynactin complex and dissociating cargoes from microtubules. The use of deletion constructs showed that the C-terminal domain of dynamitin is essential for binding to the first intracellular interdomain of Nav1.5. Co-immunoprecipitation assays confirmed the association between Nav1.5 and dynamitin in mouse heart extracts. Immunostaining experiments showed that dynamitin and Nav1.5 co-localize at intercalated discs of mouse cardiomyocytes. The whole-cell patch-clamp technique was applied to test the functional link between Nav1.5 and dynamitin. Dynamitin overexpression in HEK-293 (human embryonic kidney 293) cells expressing Nav1.5 resulted in a decrease in sodium current density in the membrane with no modification of the channel-gating properties. Biotinylation experiments produced similar information with a reduction in Nav1.5 at the cell surface when dynactin-dependent transport was inhibited. The present study strongly suggests that dynamitin is involved in the regulation of Nav1.5 cell-surface density.

  19. A highly polarized excitable cell separates sodium channels from sodium-activated potassium channels by more than a millimeter

    PubMed Central

    Smith, Benjamin E.; Markham, Michael R.

    2015-01-01

    The bioelectrical properties and resulting metabolic demands of electrogenic cells are determined by their morphology and the subcellular localization of ion channels. The electric organ cells (electrocytes) of the electric fish Eigenmannia virescens generate action potentials (APs) with Na+ currents >10 μA and repolarize the AP with Na+-activated K+ (KNa) channels. To better understand the role of morphology and ion channel localization in determining the metabolic cost of electrocyte APs, we used two-photon three-dimensional imaging to determine the fine cellular morphology and immunohistochemistry to localize the electrocytes' ion channels, ionotropic receptors, and Na+-K+-ATPases. We found that electrocytes are highly polarized cells ∼1.5 mm in anterior-posterior length and ∼0.6 mm in diameter, containing ∼30,000 nuclei along the cell periphery. The cell's innervated posterior region is deeply invaginated and vascularized with complex ultrastructural features, whereas the anterior region is relatively smooth. Cholinergic receptors and Na+ channels are restricted to the innervated posterior region, whereas inward rectifier K+ channels and the KNa channels that terminate the electrocyte AP are localized to the anterior region, separated by >1 mm from the only sources of Na+ influx. In other systems, submicrometer spatial coupling of Na+ and KNa channels is necessary for KNa channel activation. However, our computational simulations showed that KNa channels at a great distance from Na+ influx can still terminate the AP, suggesting that KNa channels can be activated by distant sources of Na+ influx and overturning a long-standing assumption that AP-generating ion channels are restricted to the electrocyte's posterior face. PMID:25925327

  20. Downregulation of transient K+ channels in dendrites of hippocampal CA1 pyramidal neurons by activation of PKA and PKC.

    PubMed

    Hoffman, D A; Johnston, D

    1998-05-15

    We have reported recently a high density of transient A-type K+ channels located in the distal dendrites of CA1 hippocampal pyramidal neurons and shown that these channels shape EPSPs, limit the back-propagation of action potentials, and prevent dendritic action potential initiation (). Because of the importance of these channels in dendritic signal propagation, their modulation by protein kinases would be of significant interest. We investigated the effects of activators of cAMP-dependent protein kinase (PKA) and the Ca2+-dependent phospholipid-sensitive protein kinase (PKC) on K+ channels in cell-attached patches from the distal dendrites of hippocampal CA1 pyramidal neurons. Inclusion of the membrane-permeant PKA activators 8-bromo-cAMP (8-br-cAMP) or forskolin in the dendritic patch pipette resulted in a depolarizing shift in the activation curve for the transient channels of approximately 15 mV. Activation of PKC by either of two phorbol esters also resulted in a 15 mV depolarizing shift of the activation curve. Neither PKA nor PKC activation affected the sustained or slowly inactivating component of the total outward current. This downregulation of transient K+ channels in the distal dendrites may be responsible for some of the frequently reported increases in cell excitability found after PKA and PKC activation. In support of this hypothesis, we found that activation of either PKA or PKC significantly increased the amplitude of back-propagating action potentials in distal dendrites.

  1. Low intravascular pressure activates endothelial cell TRPV4 channels, local Ca2+ events, and IKCa channels, reducing arteriolar tone

    PubMed Central

    Bagher, Pooneh; Beleznai, Timea; Kansui, Yasuo; Mitchell, Ray; Garland, Christopher J.; Dora, Kim A.

    2012-01-01

    Endothelial cell (EC) Ca2+-activated K channels (SKCa and IKCa channels) generate hyperpolarization that passes to the adjacent smooth muscle cells causing vasodilation. IKCa channels focused within EC projections toward the smooth muscle cells are activated by spontaneous Ca2+ events (Ca2+ puffs/pulsars). We now show that transient receptor potential, vanilloid 4 channels (TRPV4 channels) also cluster within this microdomain and are selectively activated at low intravascular pressure. In arterioles pressurized to 80 mmHg, ECs generated low-frequency (∼2 min−1) inositol 1,4,5-trisphosphate receptor-based Ca2+ events. Decreasing intraluminal pressure below 50 mmHg increased the frequency of EC Ca2+ events twofold to threefold, an effect blocked with the TRPV4 antagonist RN1734. These discrete events represent both TRPV4-sparklet- and nonsparklet-evoked Ca2+ increases, which on occasion led to intracellular Ca2+ waves. The concurrent vasodilation associated with increases in Ca2+ event frequency was inhibited, and basal myogenic tone was increased, by either RN1734 or TRAM-34 (IKCa channel blocker), but not by apamin (SKCa channel blocker). These data show that intraluminal pressure influences an endothelial microdomain inversely to alter Ca2+ event frequency; at low pressures the consequence is activation of EC IKCa channels and vasodilation, reducing the myogenic tone that underpins tissue blood-flow autoregulation. PMID:23071308

  2. Unfolding of a temperature-sensitive domain controls voltage-gated channel activation

    PubMed Central

    Arrigoni, Cristina; Rohaim, Ahmed; Shaya, David; Findeisen, Felix; Stein, Richard A.; Nurva, Shailika Reddy; Mishra, Smriti; Mchaourab, Hassane S.; Minor, Daniel L.

    2016-01-01

    Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNaV) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNaV CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNaV CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNaV voltage dependencies, and demonstrate that a discrete domain can encode the temperature dependent response of a channel. PMID:26919429

  3. Selective potentiation of 2-APB-induced activation of TRPV1–3 channels by acid

    PubMed Central

    Gao, Luna; Yang, Pu; Qin, Peizhong; Lu, Yungang; Li, Xinxin; Tian, Quan; Li, Yang; Xie, Chang; Tian, Jin-bin; Zhang, Chengwei; Tian, Changlin; Zhu, Michael X.; Yao, Jing

    2016-01-01

    Temperature-sensitive TRP channels are important for responses to pain and inflammation, to both of which tissue acidosis is a major contributing factor. However, except for TRPV1, acid-sensing by other ThermoTRP channels remains mysterious. We show here that unique among TRPV1–3 channels, TRPV3 is directly activated by protons from cytoplasmic side. This effect is very weak and involves key cytoplasmic residues L508, D512, S518, or A520. However, mutations of these residues did not affect a strong proton induced potentiation of TRPV3 currents elicited by the TRPV1–3 common agonist, 2-aminoethoxydiphenyl borate (2-APB), no matter if the ligand was applied from extracellular or cytoplasmic side. The acid potentiation was common among TRPV1–3 and only seen with 2-APB-related ligands. Using 1H-nuclear magnetic resonance to examine the solution structures of 2-APB and its analogs, we observed striking structural differences of the boron-containing compounds at neutral/basic as compared to acidic pH, suggesting that a pH-dependent configuration switch of 2-APB-based drugs may underlie their functionality. Supporting this notion, protons also enhanced the inhibitory action of 2-APB on TRPM8. Collectively, our findings reveal novel insights into 2-APB action on TRP channels, which should facilitate the design of new drugs for these channels. PMID:26876731

  4. Mutations affecting enzymatic activity in liver arginase

    SciTech Connect

    Vockley, J.G.; Tabor, D.E.; Goodman, B.K.

    1994-09-01

    The hydrolysis of arginine to ornithine and urea is catalyzed by arginase in the last step of the urea cycle. We examined a group of arginase deficient patients by PCR-SSCP analysis to characterize the molecular basis of this disorder. A heterogeneous population of nonsense mutations, microdeletions, and missense mutations has been identified in our cohort. Microdeletions which introduce premature stop codons downstream of the deletion and nonsense mutations result in no arginase activity. These mutations occur randomly along the gene. The majority of missense mutations identified appear to occur in regions of high cross-species homology. To test the effect of these missense mutations on arginase activity, site-directed mutagenesis was used to re-create the patient mutations for in vivo expression studies in a prokaryotic fusion-protein expression system. Of 4 different missense mutations identified in 6 individuals, only one was located outside of a conserved region. The three substitution mutations within the conserved regions had a significant effect on enzymatic activity (0-3.1 nmole/30min, normal is 1300-1400 nmoles/30min, as determined by in vitro arginase assay), while the fourth mutation, a T to S substitution, did not. In addition, site-directed mutagenesis was utilized to create mutations not in residues postulated to play a significant role in the enzymatic function or active site formation in manganese-binding proteins such as arginase. We have determined that the substitution of glycine for a histidine residue, located in a very highly conserved region of exon 3, and the substitution of a histidine and an aspartic acid residue within a similarly conserved region in exon 4, totally abolishes enzymatic activity. Mutations substituting glycine for an additional histidine and aspartic acid residue in exon 4 and two aspartic acid residues in exon 7 have also been created. We are currently in the process of characterizing these mutations.

  5. Ca(2+)-activated K(+) channels as therapeutic targets for myocardial and vascular protection.

    PubMed

    Clements, Richard T; Terentyev, Dmitry; Sellke, Frank W

    2015-01-01

    Small- and large-conductance Ca(2+)-activated K(+)channels (SKCa and BKCa, respectively) may be important targets for therapeutic interventions in a variety of cardiac conditions. In cardiomyocytes, BKCa channels are localized to mitochondria where they beneficially modulate reactive oxygen species, mitochondrial Ca(2+), and respiration. In vascular smooth muscle cells, BKCa channels regulate vascular tone and promote vasodilation. Activation of BKCa channels has demonstrated significant cardioprotection following ischemic injury, including improved function and reduced infarct size. SKCa channels are expressed in both the membrane and mitochondria of cardiomyocytes. Modulation of cardiomyocyte SKCa channels may be beneficial for arrhythmia, heart failure, and ischemia. Mitochondrial SKCa channels may provide similar benefit to BKCa channels. In addition, activation of SKCa channels on the endothelium promotes vasodilation. This mini-review focuses on the modulation of cardiomyocyte BKCa and SKCa channels for cardioprotection and briefly address associated potential therapeutic benefits in the coronary circulation.

  6. Activation of KCNN3/SK3/K(Ca)2.3 channels attenuates enhanced calcium influx and inflammatory cytokine production in activated microglia.

    PubMed

    Dolga, Amalia M; Letsche, Till; Gold, Maike; Doti, Nunzianna; Bacher, Michael; Chiamvimonvat, Nipavan; Dodel, Richard; Culmsee, Carsten

    2012-12-01

    In neurons, small-conductance calcium-activated potassium (KCNN/SK/K(Ca)2) channels maintain calcium homeostasis after N-methyl-D-aspartate (NMDA) receptor activation, thereby preventing excitotoxic neuronal death. So far, little is known about the function of KCNN/SK/K(Ca)2 channels in non-neuronal cells, such as microglial cells. In this study, we addressed the question whether KCNN/SK/K(Ca)2 channels activation affected inflammatory responses of primary mouse microglial cells upon lipopolysaccharide (LPS) stimulation. We found that N-cyclohexyl-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine (CyPPA), a positive pharmacological activator of KCNN/SK/K(Ca)2 channels, significantly reduced LPS-stimulated activation of microglia in a concentration-dependent manner. The general KCNN/SK/K(Ca)2 channel blocker apamin reverted these effects of CyPPA on microglial proliferation. Since calcium plays a central role in microglial activation, we further addressed whether KCNN/SK/K(Ca)2 channel activation affected the changes of intracellular calcium levels, [Ca(2+)](i), in microglial cells. Our data show that LPS-induced elevation of [Ca(2+)](i) was attenuated following activation of KCNN2/3/K(Ca)2.2/K(Ca)2.3 channels by CyPPA. Furthermore, CyPPA reduced downstream events including tumor necrosis factor alpha and interleukin 6 cytokine production and nitric oxide release in activated microglia. Further, we applied specific peptide inhibitors of the KCNN/SK/K(Ca)2 channel subtypes to identify which particular channel subtype mediated the observed anti-inflammatory effects. Only inhibitory peptides targeting KCNN3/SK3/K(Ca)2.3 channels, but not KCNN2/SK2/K(Ca)2.2 channel inhibition, reversed the CyPPA-effects on LPS-induced microglial proliferation. These findings revealed that KCNN3/SK3/K(Ca)2.3 channels can modulate the LPS-induced inflammatory responses in microglial cells. Thus, KCNN3/SK3/K(Ca)2.3 channels may serve as a therapeutic target for reducing microglial

  7. Skeletal muscle sodium channel is affected by an epileptogenic beta1 subunit mutation.

    PubMed

    Moran, O; Conti, F

    2001-03-23

    The syndrome of generalized epilepsy with febrile seizures plus type 1 (GEFS+) has been associated to the gene SCN1B coding for the sodium channel beta1 subunit (Wallace, R. H. et al. (1998) Nature Genetics 19, 366-370). In patients, a mutation of the cysteine 121 to trpyptophane (C121W) would cause a lack of modulatory activity of the beta1 subunit on sodium channels expressed in the brain, rendering neurons hyperexcitable. We have confirmed that the normal beta1-modulation of type-IIA adult brain alpha subunits (BIIA) expressed in frog oocytes is defective in C121W. We observed that the mixture of wild-type and mutant beta1 subunits is less effective than wild-type alone, suggesting that the mutant beta1 subunit does bind the alpha subunit. However, we also observed a similar lack of modulation by C121W of the in adult skeletal muscle alpha subunit (SkM1). This finding is in contrast with the simple idea that the mutational effect observed in the oocyte expression system is the principal physiopathological correlate of GEFS+, because no skeletal muscle symptoms have been reported in GEFS+ patients. We conclude that the manifestation of the pathological phenotype is conditioned by the presence of susceptibility genes and/or that the frog oocyte expression system is inadequate for the study of the mutant beta1 subunit physiopathology.

  8. 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

  9. Phosphoinositide interacting regulator of TRP (Pirt) enhances TRPM8 channel activity in vitro via increasing channel conductance

    PubMed Central

    Tang, Min; Wu, Guang-yi; Dong, Xin-zhong; Tang, Zong-xiang

    2016-01-01

    Aim: Pirt is a two-transmembrane domain protein that regulates the function of a variety of ion channels. Our previous study indicated that Pirt acts as a positive endogenous regulator of the TRPM8 channel. The aim of this study was to investigate the mechanism underlying the regulation of TRPM8 channel by Pirt. Methods: HEK293 cells were transfected with TRPM8+Pirt or TRPM8 alone. Menthol (1 mmol/L) was applied through perfusion to induce TRPM8-mediated voltage-dependent currents, which were recorded using a whole-cell recording technique. PIP2 (10 μmol/L) was added into the electrode pipettes (PI was taken as a control). Additionally, cell-attached single-channel recordings were conducted in CHO cells transfected with TRPM8+Pirt or TRPM8 alone, and menthol (1 mmol/L) was added into the pipette solution. Results: Either co-transfection with Pirt or intracellular application of PIP2 (but not PI) significantly enhanced menthol-induced TRPM8 currents. Furthermore, Pirt and PIP2 synergistically modulated menthol-induced TRPM8 currents. Single-channel recordings revealed that co-transfection with Pirt significantly increased the single channel conductance. Conclusion: Pirt and PIP2 synergistically enhance TRPM8 channel activity, and Pirt regulates TRPM8 channel activity by increasing the single channel conductance. PMID:26657057

  10. Tissue kallikrein activation of the epithelial Na channel

    PubMed Central

    Patel, Ankit B.; Chao, Julie

    2012-01-01

    Epithelial Na Channels (ENaC) are responsible for the apical entry of Na+ in a number of different epithelia including the renal connecting tubule and cortical collecting duct. Proteolytic cleavage of γ-ENaC by serine proteases, including trypsin, furin, elastase, and prostasin, has been shown to increase channel activity. Here, we investigate the ability of another serine protease, tissue kallikrein, to regulate ENaC. We show that excretion of tissue kallikrein, which is secreted into the lumen of the connecting tubule, is stimulated following 5 days of a high-K+ or low-Na+ diet in rats. Urinary proteins reconstituted in a low-Na buffer activated amiloride-sensitive currents (INa) in ENaC-expressing oocytes, suggesting an endogenous urinary protease can activate ENaC. We next tested whether tissue kallikrein can directly cleave and activate ENaC. When rat ENaC-expressing oocytes were exposed to purified tissue kallikrein from rat urine (RTK), ENaC currents increased threefold in both the presence and absence of a soybean trypsin inhibitor (SBTI). RTK and trypsin both decreased the apparent molecular mass of cleaved cell-surface γ-ENaC, while immunodepleted RTK produced no shift in apparent molecular mass, demonstrating the specificity of the tissue kallikrein. A decreased effect of RTK on Xenopus ENaC, which has variations in the putative prostasin cleavage sites in γ-ENaC, suggests these sites are important in RTK activation of ENaC. Mutating the prostasin site in mouse γ-ENaC (γRKRK186QQQQ) abolished ENaC activation and cleavage by RTK while wild-type mouse ENaC was activated and cleaved similar to that of the rat. We conclude that tissue kallikrein can be a physiologically relevant regulator of ENaC activity. PMID:22622459

  11. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

    PubMed Central

    Kheradpezhouh, E.; Barritt, G.J.; Rychkov, G.Y.

    2015-01-01

    Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca2+ homeostasis, resulting in a sustained elevation of the free cytosolic Ca2+ concentration ([Ca2+]c) in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca2+ entry through Transient Receptor Potential Melastatin 2 (TRPM2) channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5 µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca2+]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50 nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels. PMID:26609559

  12. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes.

    PubMed

    Kheradpezhouh, E; Barritt, G J; Rychkov, G Y

    2016-04-01

    Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca(2+) homeostasis, resulting in a sustained elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)]c) in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca(2+) entry through Transient Receptor Potential Melastatin 2 (TRPM2) channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca(2+)]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels.

  13. BAX channel activity mediates lysosomal disruption linked to Parkinson disease.

    PubMed

    Bové, Jordi; Martínez-Vicente, Marta; Dehay, Benjamin; Perier, Celine; Recasens, Ariadna; Bombrun, Agnes; Antonsson, Bruno; Vila, Miquel

    2014-05-01

    Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.

  14. Biologically active extracts with kidney affections applications

    NASA Astrophysics Data System (ADS)

    Pascu (Neagu), Mihaela; Pascu, Daniela-Elena; Cozea, Andreea; Bunaciu, Andrei A.; Miron, Alexandra Raluca; Nechifor, Cristina Aurelia

    2015-12-01

    This paper is aimed to select plant materials rich in bioflavonoid compounds, made from herbs known for their application performances in the prevention and therapy of renal diseases, namely kidney stones and urinary infections (renal lithiasis, nephritis, urethritis, cystitis, etc.). This paper presents a comparative study of the medicinal plant extracts composition belonging to Ericaceae-Cranberry (fruit and leaves) - Vaccinium vitis-idaea L. and Bilberry (fruit) - Vaccinium myrtillus L. Concentrated extracts obtained from medicinal plants used in this work were analyzed from structural, morphological and compositional points of view using different techniques: chromatographic methods (HPLC), scanning electronic microscopy, infrared, and UV spectrophotometry, also by using kinetic model. Liquid chromatography was able to identify the specific compounds of the Ericaceae family, present in all three extracts, arbutosid, as well as specific components of each species, mostly from the class of polyphenols. The identification and quantitative determination of the active ingredients from these extracts can give information related to their therapeutic effects.

  15. Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels.

    PubMed

    Reyes, J P; Huanosta-Gutiérrez, A; López-Rodríguez, A; Martínez-Torres, A

    2015-01-01

    We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na(+) permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na(+). Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.

  16. Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

    PubMed Central

    Reyes, JP; Huanosta-Gutiérrez, A; López-Rodríguez, A; Martínez-Torres, A

    2015-01-01

    We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na+ permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na+. Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade. PMID:25853341

  17. Ryanodine modification of RyR1 retrogradely affects L-type Ca(2+) channel gating in skeletal muscle.

    PubMed

    Bannister, R A; Beam, K G

    2009-01-01

    In skeletal muscle, there is bidirectional signalling between the L-type Ca(2+) channel (1,4-dihydropyridine receptor; DHPR) and the type 1 ryanodine-sensitive Ca(2+) release channel (RyR1) of the sarcoplasmic reticulum (SR). In the case of "orthograde signalling" (i.e., excitation-contraction coupling), the conformation of RyR1 is controlled by depolarization-induced conformational changes of the DHPR resulting in Ca(2+) release from the SR. "Retrograde coupling" is manifested as enhanced L-type current. The nature of this retrograde signal, and its dependence on RyR1 conformation, are poorly understood. Here, we have examined L-type currents in normal myotubes after an exposure to ryanodine (200 microM, 1 h at 37 degrees C) sufficient to lock RyR1 in a non-conducting, inactivated, conformational state. This treatment caused an increase in L-type current at less depolarized test potentials in comparison to myotubes similarly exposed to vehicle as a result of a approximately 5 mV hyperpolarizing shift in the voltage-dependence of activation. Charge movements of ryanodine-treated myotubes were also shifted to more hyperpolarizing potentials (approximately 13 mV) relative to vehicle-treated myotubes. Enhancement of the L-type current by ryanodine was absent in dyspedic (RyR1 null) myotubes, indicating that ryanodine does not act directly on the DHPR. Our findings indicate that in retrograde signaling, the functional state of RyR1 influences conformational changes of the DHPR involved in activation of L-type current. This raises the possibility that physiological regulators of the conformational state of RyR1 (e.g., Ca(2+), CaM, CaMK, redox potential) may also affect DHPR gating.

  18. Activation of volume-regulated Cl− channels by ACh and ATP in Xenopus follicles

    PubMed Central

    Pérez-Samartín, Alberto L; Miledi, Ricardo; Arellano, Rogelio O

    2000-01-01

    Osmolarity-dependent ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using electrophysiological techniques. Whole follicle currents were monitored using a two-electrode voltage clamp and single-channel activity was measured using the patch-clamp technique.In follicles held at -60 mV two chloride currents were activated in external hyposmotic solutions. One was the habitual volume-regulated current elicited by external hyposmolarity (ICl,swell), and the second was a slow and smooth current (Sin) generated by ACh or ATP application.In follicles, the permeability ratios for different anions with respect to Cl− were similar for both ICl,swell and Sin, with a sequence of: SCN− > I− > Br−≥ NO3−≥ Cl− > gluconate ≥ cyclamate > acetate > SO42−.Extracellular ATP blocked the outward component of Sin. Also, extracellular pH modulated the inactivation kinetics of Sin elicited by ACh; e.g. inactivation at +80 mV was ∼100% slower at pH 8.0 compared with that at pH 6.0.Lanthanides inhibited ICl,swell and Sin. La3+ completely inhibited ICl,swell with a half-maximal inhibitory concentration (IC50) of 17 ± 1.9 μm, while Sin was blocked up to 55% with an apparent IC50 of 36 ± 2.6 μm.Patch-clamp recordings in follicular cells showed that hyposmotic challenge opened inward single-channel currents. The single channel conductance (4.7 ± 0.4 pS) had a linear current-voltage relationship with a reversal membrane potential close to −20 mV. This single-channel activity was increased by application of ACh or ATP.The ICl,swell generation was not affected by pirenzepine or metoctramine, and did not affect the purinergic activation of the chloride current named Fin. Thus, ICl,swell was not generated via neurotransmitters released during cellular swelling.All together, equal discrimination for different anions, similar modulatory effects by extracellular pH, the blocking effects by ATP and La3+, and the same single-channel activity

  19. [Activation and regulation of nociceptive transient receptor potential (TRP) channels, TRPV1 and TRPA1].

    PubMed

    Tominaga, Makoto

    2010-03-01

    TRP channels are well recognized for their contributions to sensory transduction, responding to a wide variety of stimuli including temperature, nociceptive stimuli, touch, osmolarity and pheromones. In particular, the involvement of TRP channels in nociception has been extensively studied following the cloning of the capsaicin receptor, TRPV1. Painful diabetic peripheral neuropathy is described as a superficial burning pain, and it is one of the most commonly encountered neuropathic pain syndromes in clinical practice. We found that hypoxic and high glucose conditions commonly observed in diabetes potentiate TRPV1 activity without affecting TRPV1 expression both in native rat sensory neurons and HEK293 cells expressing rat TRPV1. The potentiation seems to be caused by phosphorylation of the serine residues of TRPV1 by PKC. These data indicate that PKC-dependent potentiation of TRPV1 activities under hypoxia and hyperglycemia might be involved in early diabetic neuropathy. Mechanisms for the detection of alkaline pH by sensory neurons are not well understood, although it is well accepted that acidic pH monitoring can be attributed to several ion channels, including TRPV1 and ASICs. We found that alkaline pH activates TRPA1 and that the TRPA1 activation is involved in nociception, using Ca(2+)-imaging and patch-clamp methods. In addition, intracellular alkalization activated TRPA1 at the whole-cell level, and single-channel openings were observed in the inside-out configuration. Furthermore, intraplantar injection of ammonium chloride into the mouse hind paw caused pain-related behaviors, which were not observed in TRPA1-deficient mice. These results suggest that alkaline pH causes pain sensation through activation of TRPA1.

  20. Disruption of the IS6-AID Linker Affects Voltage-gated Calcium Channel Inactivation and Facilitation

    PubMed Central

    Findeisen, Felix

    2009-01-01

    Two processes dominate voltage-gated calcium channel (CaV) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The CaVβ/CaVα1-I-II loop and Ca2+/calmodulin (CaM)/CaVα1–C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6–α-interaction domain (AID) linker provides a rigid connection between the pore and CaVβ/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate CaV1.2 (L-type) and CaV2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt CaVβ/I-II association sharply decelerate CDI and reduce a second Ca2+/CaM/CaVα1–C-terminal–mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, CaVβ and the IS6-AID linker, are essential for calcium-dependent modulation, and that both CaVβ-dependent and CaM-dependent components couple to the pore by a common mechanism requiring CaVβ and an intact IS6-AID linker. PMID:19237593

  1. Synchronization of active atomic clocks via quantum and classical channels

    NASA Astrophysics Data System (ADS)

    Roth, Alexander; Hammerer, Klemens

    2016-10-01

    Superradiant lasers based on atomic ensembles exhibiting ultranarrow optical transitions can emit light of unprecedented spectral purity and may serve as active atomic clocks. We consider two frequency-detuned active atomic clocks, which are coupled in a cascaded setup, i.e., as master and slave lasers, and study the synchronization of the slave to the master clock. In a setup where both atomic ensembles are coupled to a common cavity mode, such synchronization phenomena have been predicted by Xu et al. [M. Xu, D. A. Tieri, E. C. Fine, J. K. Thompson, and M. J. Holland, Phys. Rev. Lett. 113, 154101 (2014)., 10.1103/PhysRevLett.113.154101] and experimentally observed by Weiner et al. (J. M. Weiner et al., arXiv:1503.06464). Here we demonstrate that synchronization still occurs in cascaded setups but exhibits distinctly different phase diagrams. We study the characteristics of synchronization in comparison to the case of coupling through a common cavity. We also consider synchronization through a classical channel where light of the master laser is measured phase sensitively and the slave laser is injection locked by feedback and compare to the results achievable by coupling through quantum channels.

  2. Nicotine activates the chemosensory cation channel TRPA1.

    PubMed

    Talavera, Karel; Gees, Maarten; Karashima, Yuji; Meseguer, Víctor M; Vanoirbeek, Jeroen A J; Damann, Nils; Everaerts, Wouter; Benoit, Melissa; Janssens, Annelies; Vennekens, Rudi; Viana, Félix; Nemery, Benoit; Nilius, Bernd; Voets, Thomas

    2009-10-01

    Topical application of nicotine, as used in nicotine replacement therapies, causes irritation of the mucosa and skin. This reaction has been attributed to activation of nicotinic acetylcholine receptors (nAChRs) in chemosensory neurons. In contrast with this view, we found that the chemosensory cation channel transient receptor potential A1 (TRPA1) is crucially involved in nicotine-induced irritation. We found that micromolar concentrations of nicotine activated heterologously expressed mouse and human TRPA1. Nicotine acted in a membrane-delimited manner, stabilizing the open state(s) and destabilizing the closed state(s) of the channel. In the presence of the general nAChR blocker hexamethonium, nociceptive neurons showed nicotine-induced responses that were strongly reduced in TRPA1-deficient mice. Finally, TRPA1 mediated the mouse airway constriction reflex to nasal instillation of nicotine. The identification of TRPA1 as a nicotine target suggests that existing models of nicotine-induced irritation should be revised and may facilitate the development of smoking cessation therapies with less adverse effects.

  3. Sodium channel activation augments NMDA receptor function and promotes neurite outgrowth in immature cerebrocortical neurons

    PubMed Central

    George, Joju; Dravid, Shashank M.; Prakash, Anand; Xie, Jun; Peterson, Jennifer; Jabba, Sairam V.; Baden, Daniel G.; Murray, Thomas F.

    2009-01-01

    A range of extrinsic signals, including afferent activity, affect neuronal growth and plasticity. Neuronal activity regulates intracellular Ca2+ and activity-dependent calcium signaling has been shown to regulate dendritic growth and branching (Konur and Ghosh, 2005). NMDA receptor (NMDAR) stimulation of Ca2+/calmodulin-dependent protein kinase signaling cascades has moreover been demonstrated to regulate neurite/axonal outgrowth (Wayman et al., 2004). We used a sodium channel activator, brevetoxin (PbTx-2), to explore the relationship between intracellular [Na+] and NMDAR-dependent development. PbTx-2 alone, at a concentration of 30 nM, did not affect Ca2+ dynamics in DIV-2 cerebrocortical neurons; however, this treatment robustly potentiated NMDA-induced Ca2+ influx. The 30 nM PbTx-2 treatment produced a maximum [Na+]i of 16.9 ± 1.5 mM representing an increment of 8.8 ± 1.8 mM over basal. The corresponding membrane potential change produced by 30 nM PbTx-2 was modest and therefore insufficient to relieve the voltage-dependent Mg2+ block of NMDARs. To unambiguously demonstrate the enhancement of NMDA receptor function by PbTx-2, we recorded single-channel currents from cell-attached patches. PbTx-2 treatment was found to increase both the mean open time and open probability of NMDA receptors. These effects of PbTx-2 on NMDA receptor function were dependent on extracellular Na+ and activation of Src kinase. The functional consequences of PbTx-2-induced enhancement of NMDAR function were evaluated in immature cerebrocortical neurons. PbTx-2 concentrations between 3 and 300 nM enhanced neurite outgrowth. Voltage-gated sodium channel activators may accordingly represent a novel pharmacologic strategy to regulate neuronal plasticity through an NMDA receptor and Src family kinase-dependent mechanism. PMID:19279266

  4. Voltage-dependent antagonist/agonist actions of taurine on Ca(2+)-activated potassium channels of rat skeletal muscle fibers.

    PubMed

    Tricarico, D; Barbieri, M; Conte Camerino, D

    2001-09-01

    Emerging evidence supports the idea that taurine exerts some of its actions through inhibition of inward rectifier K(+) channels, ATP-sensitive K(+) channels, and voltage-dependent K(+) channels. However, to date not much is known about the effects of this sulfonic amino acid on Ca(2+)-activated K(+) (K(Ca(2+))) channels, which are widely expressed in various tissues, including skeletal muscle. In the present work, the effects of taurine on K(Ca(2+)) channels of rat skeletal muscle fibers were investigated using the patch-clamp technique. The application of the amino acid to the internal side of the excised macropatches induced a dose-dependent decrease in the outward K(Ca(2+)) currents recorded at positive membrane potentials in the presence of 8 to 16 microM concentrations of free Ca(2+) ions in the bath with an IC(50) of 31.9. 10(-3) +/- 1 M (slope factor = 1.2) (n = 11 patches). In contrast, at negative membrane potentials taurine caused an enhancement of the muscular inward K(Ca(2+)) currents with a DE(50) (drug concentration needed to enhance the current by 50%) of 46.7. 10(-3) +/- 2 M (slope factor = 1.3) (n = 9 patches). Single channel analysis revealed that this effect was mediated by changes in the reversal potential of the K(Ca(2+)) channel for K(+) ions with no changes in the gating properties or in the sensitivity of the channel to Ca(2+) ions. Taurine also did not affect the single channel conductance. In conclusion, taurine shows a voltage-dependent dualistic action on K(Ca(2+)) channels, being an inhibitor of the channel at positive membrane potentials and an activator at negative membrane potentials.

  5. Interaction between 2 extracellular loops influences the activity of the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    Broadbent, Steven D; Wang, Wuyang; Linsdell, Paul

    2014-10-01

    Activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is thought to be controlled by cytoplasmic factors. However, recent evidence has shown that overall channel activity is also influenced by extracellular anions that interact directly with the extracellular loops (ECLs) of the CFTR protein. Very little is known about the structure of the ECLs or how substances interacting with these ECLs might affect CFTR function. We used patch-clamp recording to investigate the accessibility of cysteine-reactive reagents to cysteines introduced throughout ECL1 and 2 key sites in ECL4. Furthermore, interactions between ECL1 and ECL4 were investigated by the formation of disulfide crosslinks between cysteines introduced into these 2 regions. Crosslinks could be formed between R899C (in ECL4) and a number of sites in ECL1 in a manner that was dependent on channel activity, suggesting that the relative orientation of these 2 loops changes on activation. Formation of these crosslinks inhibited channel function, suggesting that relative movement of these ECLs is important to normal channel function. Implications of these findings for the effects of mutations in the ECLs that are associated with cystic fibrosis and interactions with extracellular substances that influence channel activity are discussed.

  6. 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.

  7. Small conductance Ca2+-activated K+ channels regulate firing properties and excitability in parasympathetic cardiac motoneurons in the nucleus ambiguus.

    PubMed

    Lin, Min; Hatcher, Jeff T; Chen, Qin-Hui; Wurster, Robert D; Cheng, Zixi Jack

    2010-12-01

    Small conductance Ca(2+)-activated K(+) channels (SK) regulate action potential (AP) firing properties and excitability in many central neurons. However, the functional roles of SK channels of parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus have not yet been well characterized. In this study, the tracer X-rhodamine-5 (and 6)-isothiocyanate (XRITC) was injected into the pericardial sac to retrogradely label PCMNs in FVB mice at postnatal days 7-9. Two days later, XRITC-labeled PCMNs in brain stem slices were identified. With the use of whole cell current clamp, single APs and spike trains of different frequencies were evoked by current injections. We found that 1) PCMNs have two different firing patterns: the majority of PCMNs (90%) exhibited spike frequency adaptation (SFA) and the rest (10%) showed less or no adaptation; 2) application of the specific SK channel blocker apamin significantly increased spike half-width in single APs and trains and reduced the spike frequency-dependent AP broadening in trains; 3) SK channel blockade suppressed afterhyperpolarization (AHP) amplitude following single APs and trains and abolished spike-frequency dependence of AHP in trains; and 4) SK channel blockade increased the spike frequency but did not alter the pattern of SFA. Using whole cell voltage clamp, we measured outward currents and afterhyperpolarization current (I(AHP)). SK channel blockade revealed that SK-mediated outward currents had both transient and persistent components. After bath application of apamin and Ca(2+)-free solution, we found that apamin-sensitive and Ca(2+)-sensitive I(AHP) were comparable, confirming that SK channels may contribute to a major portion of Ca(2+)-activated K(+) channel-mediated I(AHP). These results suggest that PCMNs have SK channels that significantly regulate AP repolarization, AHP, and spike frequency but do not affect SFA. We conclude that activation of SK channels underlies one of the mechanisms for negative

  8. Pre- and postsynaptic activation of M-channels by a novel opener dampens neuronal firing and transmitter release.

    PubMed

    Peretz, Asher; Sheinin, Anton; Yue, Cuiyong; Degani-Katzav, Nurit; Gibor, Gilad; Nachman, Rachel; Gopin, Anna; Tam, Eyal; Shabat, Doron; Yaari, Yoel; Attali, Bernard

    2007-01-01

    The M-type K(+) current (M-current), encoded by Kv7.2/3 (KCNQ2/3) K(+) channels, plays a critical role in regulating neuronal excitability because it counteracts subthreshold depolarizations. Here we have characterized the functions of pre- and postsynaptic M-channels using a novel Kv7.2/3 channel opener, NH6, which we synthesized as a new derivative of N-phenylanthranilic acid. NH6 exhibits a good selectivity as it does not affect Kv7.1 and I(KS) K(+) currents as well as NR1/NR2B, AMPA, and GABA(A) receptor-mediated currents. Superfusion of NH6 increased recombinant Kv7.2/3 current amplitude (EC(50) = 18 muM) by causing a hyperpolarizing shift of the voltage activation curve and by markedly slowing the deactivation kinetics. Activation of native M-currents by NH6 robustly reduced the number of evoked and spontaneous action potentials in cultured cortical, hippocampal and dorsal root ganglion neurons. In hippocampal slices, NH6 decreased somatically evoked spike after depolarization of CA1 pyramidal neurons and induced regular firing in bursting neurons. Activation of M-channels by NH6, potently reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents. Activation of M-channels also decreased the frequency of miniature excitatory (mEPSC) and inhibitory (mIPSC) postsynaptic currents without affecting their amplitude and waveform, thus suggesting that M-channels presynaptically inhibit glutamate and GABA release. Our results suggest a role of presynaptic M-channels in the release of glutamate and GABA. They also indicate that M-channels act pre- and postsynaptically to dampen neuronal excitability.

  9. Threshold bedrock channels in tectonically active mountains with frequent mass wasting

    NASA Astrophysics Data System (ADS)

    Korup, O.; Hayakawa, Y. S.; Codilean, A.; Oguchi, T.

    2013-12-01

    determining the fraction of landslide-affected terrain throughout Japan. We infer that neither average nor extreme bedrock channel steepness is a sufficiently sensitive measure for gauging landslide activity in terms of volumes mobilized as opposed to bedrock river sinuosity. This new and largely underexplored channel planform metric may reflect spacing of discontinuities in rock mass that constrains the susceptibility to large rock-slope failures, and may contain valuable compound information on both fluvial and hillslope erosion dynamics in tectonically active landscapes.

  10. Src Tyrosine Kinase Alters Gating of Hyperpolarization-Activated HCN4 Pacemaker Channel through Tyr531

    PubMed Central

    Li, Chen-Hong; Zhang, Qi; Teng, Bunyen; Mustafa, S. Jamal; Huang, Jian-Ying; Yu, Han-Gang

    2009-01-01

    We recently discovered that the constitutively active Src tyrosine kinase can enhance the HCN4 channel activity by binding to the channel protein. To investigate the mechanism of modulation by Src of HCN channels, we studied the effects of a selective inhibitor of Src tyrosine kinase, PP2, on HCN4 and its mutant channels ex pressed in HEK293 cells using whole-cell patch clamp technique. We found that PP2 can inhibit HCN4 currents by negatively shifting the voltage dependence of channel activation, decreasing the whole-cell channel conductance, and slowing activation and deactivation kinetics. Screening putative tyrosine residues subject to phosphorylation yielded two candidates: Tyr531 and Tyr554. Substituting HCN4-Tyr531 with phenylalanine largely abolished the effects of PP2 on HCN4 channels. Replacing HCN4-Tyr554 by phenylalanine did not abolish the effects of PP2 on voltage-dependent activation, but did eliminate PP2-induced slowing of channel kinetics. The inhibitory effects of HCN channels associated with reduced Src tyrosine activity is confirmed in HL-1 cardiomyocytes. Finally, we found that PP2 can decrease the heart rate in a mouse model. These results demonstrate that Src tyrosine kinase enhances HCN4 currents by shifting their activation to more positive potentials and increasing the whole-cell channel conductance as well as speeding the channel kinetics. The tyrosine residue that mediates most of Src actions on HCN4 channels is Tyr531. PMID:17977941

  11. 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

  12. Effects of microgravity on liposome-reconstituted cardiac gap junction channeling activity

    NASA Technical Reports Server (NTRS)

    Claassen, D. E.; Spooner, B. S.

    1989-01-01

    Effects of microgravity on cardiac gap junction channeling activity were investigated aboard NASA zero-gravity aircraft. Liposome-reconstituted gap junctions were assayed for channel function during free-fall, and the data were compared with channeling at 1 g. Control experiments tested for 0 g effects on the structural stability of liposomes, and on the enzyme-substrate signalling system of the assay. The results demonstrate that short periods of microgravity do not perturb reconstituted cardiac gap junction channeling activity.

  13. Fluoride affects calcium homeostasis and osteogenic transcription factor expressions through L-type calcium channels in osteoblast cell line.

    PubMed

    Duan, Xiao-Qin; Zhao, Zhi-Tao; Zhang, Xiu-Yun; Wang, Ying; Wang, Huan; Liu, Da-Wei; Li, Guang-Sheng; Jing, Ling

    2014-12-01

    Osteoblast L-type voltage-dependent calcium channels (VDCC) play important roles in maintaining intracellular homeostasis and influencing multiple cellular processes. In particular, they contribute to the activities and functions of osteoblasts (OBs). In order to study how L-type VDCC modulate calcium ion (Ca(2+)) homeostasis and the expression of osteogenic transcription factors in OBs exposed to fluoride, MC3T3-E1 cells were exposed to a gradient of concentrations of fluoride (0, 2.0, 5.0, 10.0 mg/L) in combination with 10 μM nifedipine, a specific inhibitor of VDCC, for 48 h. We examined messenger RNA (mRNA) and protein levels of Cav1.2, the main subunit of VDCC, and c-fos, c-jun, runt-related transcription factor 2 (Runx2), osterix (OSX), and intracellular free Ca(2+) ([Ca(2+)]i) concentrations in MC3T3-E1 cells. Our results showed that [Ca(2+)]i levels increased in a dose-dependent manner with increase in concentration of fluoride. Meantime, results indicated that lower concentrations of fluoride (less than 5 mg/L, especially 2 mg/L) can lead to high expression of Cav1.2 and enhance osteogenic function, while high concentration of fluoride (10 mg/L) can induce decreased Cav1.2 and osteogenic transcriptional factors in MC3T3E1 cells exposed to fluoride. However, the levels of [Ca(2+)]i, Cav1.2, c-fos, c-jun, Runx2, and OSX induced by fluoride were significantly altered and even reversed in the presence of nifedipine. These results demonstrate that L-type calcium channels play a crucial role in Ca(2+) homeostasis and they affect the expression of osteogenic transcription factors in fluoride-treated osteoblasts.

  14. [Activation of ATP-sensitive K+ channels by ADP and K+ channel openers: homology model of sulfonylurea receptor carboxyl-termini].

    PubMed

    Fujita, A; Matsuoka, T; Matsushita, K; Kurachi, Y

    2001-09-01

    The ATP-sensitive K+ channels (KATP) are composed of Kir6.0 subunits and sulfonylurea receptors (SUR1, 2A and 2B). SUR2A and SUR2B are splice variants and differ only in the C-terminal 42 amino acid residue (C42). SURs are supposed to be the subunit that determines the different response of KATPs to intracellular nucleotides, K+ channel openers and inhibitors. In this study, we report that C42 of SURs plays critical roles in differential activation of various KATPs by ADP and K+ channel openers such as diazoxide and nicorandil. KATPs containing distinct SURs and Kir6.2 were reconstructed on HEK293T cells. Much higher concentrations of ADP were necessary to activate channels which SUR1 or SUR2B. In all KATPs containing different SUR, diazoxide increased the potency of ADP for channel activity without affecting its efficacy. From the electrophysiological data obtained from C-terminal chimeras and point mutants in the second nucleotide binding domain (NBDs), we developed the homology model of each SUR-NBD2 based on the crystallgraphically determined structure of HisP, a member of the ABC protein superfamily. In this model, C42 is located just beneath the Walker A motif of NBD2 and regulates the binding of nucleotide to NBD2 by affecting the 3-D construct of NBD2. This homology model well explains the different response of KATPs to ADP. Based on this model, it will be possible to develop new ligands for KATPs.

  15. Activation of the epithelial Na+ channel in the collecting duct by vasopressin contributes to water reabsorption.

    PubMed

    Bugaj, Vladislav; Pochynyuk, Oleh; Stockand, James D

    2009-11-01

    We used patch-clamp electrophysiology on isolated, split-open murine collecting ducts (CD) to test the hypothesis that regulation of epithelial sodium channel (ENaC) activity is a physiologically important effect of vasopressin. Surprisingly, this has not been tested directly before. We ask whether vasopressin affects ENaC activity distinguishing between acute and chronic effects, as well as, parsing the cellular signaling pathway and molecular mechanism of regulation. In addition, we quantified possible synergistic regulation of ENaC by vasopressin and aldosterone associating this with a requirement for distal nephron Na+ reabsorption during water conservation vs. maintenance of Na+ balance. We find that vasopressin significantly increases ENaC activity within 2-3 min by increasing open probability (P(o)). This activation was dependent on adenylyl cyclase (AC) and PKA. Water restriction (18-24 h) and pretreatment of isolated CD with vasopressin (approximately 30 min) resulted in a similar increase in P(o). In addition, this also increased the number (N) of active ENaC in the apical membrane. Similar to P(o), increases in N were sensitive to inhibitors of AC. Stressing animals with water and salt restriction separately and jointly revealed an important effect of vasopressin: conservation of water and Na+ each independently increased ENaC activity and jointly had a synergistic effect on channel activity. These results demonstrate a quantitatively important action of vasopressin on ENaC suggesting that distal nephron Na+ reabsorption mediated by this channel contributes to maintenance of water reabsorption. In addition, our results support that the combined actions of vasopressin and aldosterone are required to achieve maximally activated ENaC.

  16. Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit from the Endoplasmic Reticulum*

    PubMed Central

    Alaimo, Alessandro; Gómez-Posada, Juan Camilo; Aivar, Paloma; Etxeberría, Ainhoa; Rodriguez-Alfaro, Jose Angel; Areso, Pilar; Villarroel, Alvaro

    2009-01-01

    The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K+ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an “active” conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca2+ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca2+ sensor, conferring Ca2+ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function. PMID:19494108

  17. Mechanosensitive channels of Escherichia coli: the MscL gene, protein, and activities

    NASA Technical Reports Server (NTRS)

    Sukharev, S. I.; Blount, P.; Martinac, B.; Kung, C.

    1997-01-01

    Although mechanosensory responses are ubiquitous and diverse, the molecular bases of mechanosensation in most cases remain mysterious MscL, a mechanosensitive channel of large conductance of Escherichia coli and its bacterial homologues are the first and currently only channel molecules shown to directly sense mechanical stretch of the membrane. In response to the tension conveyed via the lipid bilayer, MscL increases its open probability by several orders of magnitude. In the present review we describe the identification, cloning, and first sets of biophysical and structural data on this simplest mechanosensory molecule. We discovered a 2.5-ns mechanosensitive conductance in giant E. coli spheroplasts. Using chromatographies to enrich the target and patch clamp to assay the channel activity in liposome-reconstituted fractions, we identified the MscL protein and cloned the mscL gene. MscL comprises 136 amino acid residues (15 kDa), with two highly hydrophobic regions, and resides in the inner membrane of the bacterium. PhoA-fusion experiments indicate that the protein spans the membrane twice with both termini in the cytoplasm. Spectroscopic techniques show that it is highly helical. Expression of MscL tandems and covalent cross-linking suggest that the active channel complex is a homo-hexamer. We have identified several residues, which when deleted or substituted, affect channel kinetics or mechanosensitivity. Although unique when discovered, highly conserved MscL homologues in both gram-negative and gram-positive bacteria have been found, suggesting their ubiquitous importance among bacteria.

  18. Analysis of glycine-activated inhibitory post-synaptic channels in brain-stem neurones of the lamprey.

    PubMed Central

    Gold, M R; Martin, A R

    1983-01-01

    Voltage-clamp techniques were used to measure fluctuations in membrane current produced by the application of glycine to Müller cells in the brain stem of the lamprey. The power density spectrum of the glycine-induced current 'noise' was consistent with the hypothesis that glycine activated a single population of conductance channels with open times determined by first-order kinetics. In normal bathing solution the channel conductance was 73 +/- 12 pS (mean +/- S.D.) and the channel open time 34 +/- 6 msec at 5 degrees C. The reversal potential for the response was 66 +/- 5 mV. Neither channel conductance nor mean open time was voltage-dependent. Replacement of Cl- in the bathing solution by isethionate and sulphate reversibly abolished the response to glycine. Increasing intracellular Cl-, either by using Cl- -filled micropipettes or by raising extracellular K+, decreased channel conductance. This unexpected decrease was a direct effect of intracellular Cl- and was not related to coincident changes in reversal potential. Channel open time was unaffected by intracellular Cl- concentration. Reducing extracellular Cl- concentration from 126.5 to 31 mM reduced channel conductance at all levels of intracellular Cl- without affecting open time. Increasing the temperature of the preparation resulted in increase in channel conductance and a decrease in mean open time. Q10S for the effects were of the order of 1.3 and -2.3 respectively in the range 4-14 degrees C. PMID:6313912

  19. Impact of calcium-activated potassium channels on NMDA spikes in cortical layer 5 pyramidal neurons

    PubMed Central

    Bock, Tobias

    2016-01-01

    Active electrical events play an important role in shaping signal processing in dendrites. As these events are usually associated with an increase in intracellular calcium, they are likely to be under the control of calcium-activated potassium channels. Here, we investigate the impact of calcium-activated potassium channels on N-methyl-d-aspartate (NMDA) receptor-dependent spikes, or NMDA spikes, evoked by glutamate iontophoresis onto basal dendrites of cortical layer 5 pyramidal neurons. We found that small-conductance calcium-activated potassium channels (SK channels) act to reduce NMDA spike amplitude but at the same time, also decrease the iontophoretic current required for their generation. This SK-mediated decrease in NMDA spike threshold was dependent on R-type voltage-gated calcium channels and indicates a counterintuitive, excitatory effect of SK channels on NMDA spike generation, whereas the capacity of SK channels to suppress NMDA spike amplitude is in line with the expected inhibitory action of potassium channels on dendritic excitability. Large-conductance calcium-activated potassium channels had no significant impact on NMDA spikes, indicating that these channels are either absent from basal dendrites or not activated by NMDA spikes. These experiments reveal complex and opposing interactions among NMDA receptors, SK channels, and voltage-gated calcium channels in basal dendrites of cortical layer 5 pyramidal neurons during NMDA spike generation, which are likely to play an important role in regulating the way these neurons integrate the thousands of synaptic inputs they receive. PMID:26936985

  20. Observations of the Behavior and Distribution of Fish in Relation to the Columbia River Navigation Channel and Channel Maintenance Activities

    SciTech Connect

    Carlson, Thomas J.; Ploskey, Gene R.; Johnson, R. L.; Mueller, Robert P.; Weiland, Mark A.; Johnson, P. N.

    2001-10-19

    This report is a compilation of 7 studies conducted for the U.S. Army Corps of Engineers between 1995 and 1998 which used hydroacoustic methods to study the behavior of migrating salmon in response to navigation channel maintenance activities in the lower Columbia River near river mile 45. Differences between daytime and nighttime behavior and fish densities were noted. Comparisons were made of fish distribution across the river (in the channel, channel margin or near shore) and fish depth upstream and downstream of dikes, dredges, and pile driving areas.

  1. Probability distribution functions of turbulence in seepage-affected alluvial channel

    NASA Astrophysics Data System (ADS)

    Sharma, Anurag; Kumar, Bimlesh

    2017-02-01

    The present experimental study is carried out on the probability distribution functions (PDFs) of turbulent flow characteristics within near-bed-surface and away-from-bed surfaces for both no seepage and seepage flow. Laboratory experiments were conducted in the plane sand bed for no seepage (NS), 10% seepage (10%S) and 15% seepage (15%) cases. The experimental calculation of the PDFs of turbulent parameters such as Reynolds shear stress, velocity fluctuations, and bursting events is compared with theoretical expression obtained by Gram-Charlier (GC)-based exponential distribution. Experimental observations follow the computed PDF distributions for both no seepage and seepage cases. Jensen-Shannon divergence (JSD) method is used to measure the similarity between theoretical and experimental PDFs. The value of JSD for PDFs of velocity fluctuation lies between 0.0005 to 0.003 while the JSD value for PDFs of Reynolds shear stress varies between 0.001 to 0.006. Even with the application of seepage, the PDF distribution of bursting events, sweeps and ejections are well characterized by the exponential distribution of the GC series, except that a slight deflection of inward and outward interactions is observed which may be due to weaker events. The value of JSD for outward and inward interactions ranges from 0.0013 to 0.032, while the JSD value for sweep and ejection events varies between 0.0001 to 0.0025. The theoretical expression for the PDF of turbulent intensity is developed in the present study, which agrees well with the experimental observations and JSD lies between 0.007 and 0.015. The work presented is potentially applicable to the probability distribution of mobile-bed sediments in seepage-affected alluvial channels typically characterized by the various turbulent parameters. The purpose of PDF estimation from experimental data is that it provides a complete numerical description in the areas of turbulent flow either at a single or finite number of points.

  2. Vehicle type affects filling of fractional laser-ablated channels imaged by optical coherence tomography.

    PubMed

    Olesen, Uffe Høgh; Mogensen, Mette; Haedersdal, Merete

    2017-04-01

    Ablative fractional laser (AFXL) is an emerging method that enhances topical drug delivery. Penetrating the skin in microscopic, vertical channels, termed microscopic treatment zones (MTZs), the fractional technique circumvents the skin barrier and allows increased uptake of topically applied drugs. This study aims to elucidate the impact of vehicle type on the filling of MTZs from application of liquid, gel, and cream vehicles. Ex vivo pig skin was exposed to 10,600 nm fractional CO2 laser at 5% density, 120 μm beam diameter, and fluences of 40 and 80 mJ/microbeam (mJ/mb). Six repetitions were performed for each of six interventions (2 fluences and 3 vehicle types, n = 36). MTZ dimensions and filling by vehicle type were evaluated by optical coherence tomography, using blue tissue dye as a contrast-enhancing agent. Outcome measure was degree of MTZ filling assessed as percentages of empty, partially filled, and completely filled MTZs (108-127 MTZs/intervention analyzed) and evaluated statistically using Kruskal-Wallis and Dunn's tests. MTZs reached mid-dermal levels of 225 μm (40 mJ/mb) and 375 μm (80 mJ/mb) penetration depths (p < 0.0001). Filling of MTZs depended on type of applied vehicle. At 80 mJ/mb, liquid (67% completely filled, p < 0.01) and gel (60%, p < 0.05) formulations filled MTZs significantly better than cream formulation (31%). At 40 mJ/mb, liquid and gel formulations filled 90% (p < 0.05) and 77% (p > 0.05) of MTZs completely versus 55% for cream formulation. Thus, filling was overall greater for more superficial MTZs. In conclusion, vehicle type affects filling of MTZs, which may be of importance for AFXL-assisted drug delivery.

  3. Substituted N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamides: potent anticonvulsants that affect frequency (use) dependence and slow inactivation of sodium channels.

    PubMed

    Lee, Hyosung; Park, Ki Duk; Torregrosa, Robert; Yang, Xiao-Fang; Dustrude, Erik T; Wang, Yuying; Wilson, Sarah M; Barbosa, Cindy; Xiao, Yucheng; Cummins, Theodore R; Khanna, Rajesh; Kohn, Harold

    2014-07-24

    We prepared 13 derivatives of N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamide that differed in type and placement of a R-substituent in the terminal aryl unit. We demonstrated that the R-substituent impacted the compound's whole animal and cellular pharmacological activities. In rodents, select compounds exhibited excellent anticonvulsant activities and protective indices (PI=TD50/ED50) that compared favorably with clinical antiseizure drugs. Compounds with a polar, aprotic R-substituent potently promoted Na+ channel slow inactivation and displayed frequency (use) inhibition of Na+ currents at low micromolar concentrations. The possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed.

  4. 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

  5. Calcium ions open a selectivity filter gate during activation of the MthK potassium channel.

    PubMed

    Posson, David J; Rusinova, Radda; Andersen, Olaf S; Nimigean, Crina M

    2015-09-23

    Ion channel opening and closing are fundamental to cellular signalling and homeostasis. Gates that control K(+) channel activity were found both at an intracellular pore constriction and within the selectivity filter near the extracellular side but the specific location of the gate that opens Ca(2+)-activated K(+) channels has remained elusive. Using the Methanobacterium thermoautotrophicum homologue (MthK) and a stopped-flow fluorometric assay for fast channel activation, we show that intracellular quaternary ammonium blockers bind to closed MthK channels. Since the blockers are known to bind inside a central channel cavity, past the intracellular entryway, the gate must be within the selectivity filter. Furthermore, the blockers access the closed channel slower than the open channel, suggesting that the intracellular entryway narrows upon pore closure, without preventing access of either the blockers or the smaller K(+). Thus, Ca(2+)-dependent gating in MthK occurs at the selectivity filter with coupled movement of the intracellular helices.

  6. Calcium ions open a selectivity filter gate during activation of the MthK potassium channel

    NASA Astrophysics Data System (ADS)

    Posson, David J.; Rusinova, Radda; Andersen, Olaf S.; Nimigean, Crina M.

    2015-09-01

    Ion channel opening and closing are fundamental to cellular signalling and homeostasis. Gates that control K+ channel activity were found both at an intracellular pore constriction and within the selectivity filter near the extracellular side but the specific location of the gate that opens Ca2+-activated K+ channels has remained elusive. Using the Methanobacterium thermoautotrophicum homologue (MthK) and a stopped-flow fluorometric assay for fast channel activation, we show that intracellular quaternary ammonium blockers bind to closed MthK channels. Since the blockers are known to bind inside a central channel cavity, past the intracellular entryway, the gate must be within the selectivity filter. Furthermore, the blockers access the closed channel slower than the open channel, suggesting that the intracellular entryway narrows upon pore closure, without preventing access of either the blockers or the smaller K+. Thus, Ca2+-dependent gating in MthK occurs at the selectivity filter with coupled movement of the intracellular helices.

  7. Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes*

    PubMed Central

    Ramos Gomes, Fernanda; Romaniello, Vincenzo; Sánchez, Araceli; Weber, Claudia; Narayanan, Pratibha; Psol, Maryna; Pardo, Luis A.

    2015-01-01

    KV10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of KV10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of KV10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length KV10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of KV10.1, which need to be considered when ion channels are targeted for cancer therapy. PMID:26518875

  8. Hippocampal hypoglycaemia-activated K+ channels: single-channel analysis of glucose and voltage dependence.

    PubMed

    Tromba, C; Salvaggio, A; Racagni, G; Volterra, A

    1994-11-01

    The effect of glucose on kinetics and the voltage-dependent characteristics of glucose-sensitive channels in hippocampal neurons were examined with the cell-attached mode of the patch-clamp technique. Recordings of a 100-pS K+ channel in the presence or absence of glucose demonstrate that the increase in channel open state probability (Po) induced by glucose deprivation (40- to 400-times the control in high-glucose medium) was largely due to a decrease in the global amount of time spent by the channel in a long-lived closed state. The Po value of the same 100-pS channel was also found to increase (by approx. 80-times) following a depolarization of 40 mV from rest, the main factor responsible for this being a dramatic shortening of the long closed-times on depolarization. Another glucose-sensitive channel of smaller conductance (approx. 10 pS) showed a similar dependence of Po on glucose, but different dependence on voltage, with long openings at the same hyperpolarized potentials where the 100-pS channel was almost always closed. Our results indicate that the action of glucose on the kinetics of hippocampal channels closely resembles that of ATP-sensitive channels in pancreatic beta-cells. Furthermore, they indicate that the two types of glucose-sensitive channels found in hippocampal neurons, differing not only in their single-channel conductance but also in the dependence on voltage, could play different roles in the responses of these cells to modified energetic supply.

  9. CALCINEURIN ENHANCES L-TYPE Ca2+ CHANNEL ACTIVITY IN HIPPOCAMPAL NEURONS: INCREASED EFFECT WITH AGE IN CULTURE

    PubMed Central

    NORRIS, C. M.; BLALOCK, E. M.; CHEN, K.-C.; PORTER, N. M.; LANDFIELD, P. W.

    2006-01-01

    The Ca2+/calmodulin-dependent protein phosphatase, calcineurin, modulates a number of key Ca2+ signaling pathways in neurons, and has been implicated in Ca2+-dependent negative feedback inactivation of N-methyl-d-aspartate receptors and voltage-sensitive Ca2+ channels. In contrast, we report here that three mechanistically disparate calcineurin inhibitors, FK-506, cyclosporin A, and the calcineurin autoinhibitory peptide, inhibited high-voltage-activated Ca2+ channel currents by up to 40% in cultured hippocampal neurons, suggesting that calcineurin acts to enhance Ca2+ currents. This effect occurred with Ba2+ or Ca2+ as charge carrier, and with or without intracellular Ca2+ buffered by EGTA. Ca2+-dependent inactivation of Ca2+ channels was not affected by FK-506. The immunosuppressant, rapamycin, and the protein phosphatase 1/2A inhibitor, okadaic acid, did not decrease Ca2+ channel current, showing specificity for effects on calcineurin. Blockade of L-type Ca2+ channels with nimodipine fully negated the effect of FK-506 on Ca2+ channel current, while blockade of N-, and P-/Q-type Ca2+ channels enhanced FK-506-mediated inhibition of the remaining L-type-enriched current. FK-506 also inhibited substantially more Ca2+ channel current in 4-week-old vs. 2-week-old cultures, an effect paralleled by an increase in calcineurin A mRNA levels. These studies provide the first evidence that calcineurin selectively enhances L-type Ca2+ channel activity in neurons. Moreover, this action appears to be increased concomitantly with the well-characterized increase in L-type Ca2+ channel availability in hippocampal neurons with age-in-culture. PMID:11958864

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

    PubMed Central

    1988-01-01

    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. PMID:3171535

  11. Diacylglycerols Activate Mitochondrial Cationic Channel(s) and Release Sequestered Ca2+

    PubMed Central

    Chinopoulos, Christos; Starkov, Anatoly A.; Grigoriev, Sergey; Dejean, Laurent M.; Kinnally, Kathleen W.; Liu, Xibao; Ambudkar, Indu S.; Fiskum, Gary

    2008-01-01

    Mitochondria contribute to cytosolic Ca2+ homeostasis through several uptake and release pathways. Here we report that 1,2-sn-diacylglycerols (DAGs) induce Ca2+ release from Ca2+-loaded mammalian mitochondria. Release is not mediated by the uniporter or the Na+/Ca2+ exchanger, nor is it attributed to putative catabolites. DAGs-induced Ca2+ efflux is biphasic. Initial release is rapid and transient, insensitive to permeability transition inhibitors, and not accompanied by mitochondrial swelling. Following initial rapid release of Ca2+ and relatively slowreuptake, a secondary progressive release of Ca2+ occurs, associated with swelling, and mitigated by permeability transition inhibitors. The initial peak of DAGs-induced Ca2+ efflux is abolished by La3+ (1mM) and potentiated by protein kinase C inhibitors. Phorbol esters, 1,3-diacylglycerols and 1-monoacylglycerols do not induce mitochondrial Ca2+ efflux. Ca2+-loaded mitoplasts devoid of outer mitochondrial membrane also exhibit DAGsinduced Ca2+ release, indicating that this mechanism resides at the inner mitochondrial membrane. Patch clamping brainmitoplasts reveal DAGs-induced slightly cation-selective channel activity that is insensitive to bongkrekic acid and abolished by La3+. The presence of a second messenger-sensitive Ca2+ release mechanism in mitochondria could have an important impact on intracellular Ca2+ homeostasis. PMID:16167179

  12. Modulation of voltage-gated sodium channels hyperpolarizes the voltage threshold for activation in spinal motoneurones.

    PubMed

    Power, Kevin E; Carlin, Kevin P; Fedirchuk, Brent

    2012-03-01

    Previous work has shown that motoneurone excitability is enhanced by a hyperpolarization of the membrane potential at which an action potential is initiated (V(th)) at the onset, and throughout brainstem-evoked fictive locomotion in the adult decerebrate cat and neonatal rat. Modeling work has suggested the modulation of Na(+) conductance as a putative mechanism underlying this state-dependent change in excitability. This study sought to determine whether modulation of voltage-gated sodium channels could induce V(th) hyperpolarization. Whole-cell patch-clamp recordings were made from antidromically identified lumbar spinal motoneurones in an isolated neonatal rat spinal cord preparation. Recordings were made with and without the bath application of veratridine, a plant alkaloid neurotoxin that acts as a sodium channel modulator. As seen in HEK 293 cells expressing Nav1.2 channels, veratridine-modified channels demonstrated a hyperpolarizing shift in their voltage-dependence of activation and a slowing of inactivation that resulted in an enhanced inward current in response to voltage ramp stimulations. In the native rat motoneurones, veratridine-modified sodium channels induced a hyperpolarization of V(th) in all 29 neonatal rat motoneurones examined (mean hyperpolarization: -6.6 ± 4.3 mV). V(th) hyperpolarization was not due to the effects on Ca(2+) and/or K(+) channels as blockade of these currents did not alter V(th). Veratridine also significantly increased the amplitude of persistent inward currents (PICs; mean increase: 72.5 ± 98.5 pA) evoked in response to slow depolarizing current ramps. However, the enhancement of the PIC amplitude had a slower time course than the hyperpolarization of V(th), and the PIC onset voltage could be either depolarized or hyperpolarized, suggesting that PIC facilitation did not mediate the V(th) hyperpolarization. We therefore suggest that central neuronal circuitry in mammals could affect V(th) in a mechanism similar to that of

  13. The Sodium-Activated Potassium Channel Slack Is Required for Optimal Cognitive Flexibility in Mice

    ERIC Educational Resources Information Center

    Bausch, Anne E.; Dieter, Rebekka; Nann, Yvette; Hausmann, Mario; Meyerdierks, Nora; Kaczmarek, Leonard K.; Ruth, Peter; Lukowski, Robert

    2015-01-01

    "Kcnt1" encoded sodium-activated potassium channels (Slack channels) are highly expressed throughout the brain where they modulate the firing patterns and general excitability of many types of neurons. Increasing evidence suggests that Slack channels may be important for higher brain functions such as cognition and normal intellectual…

  14. Requirement of calcium-activated chloride channels in the activation of mouse vomeronasal neurons

    PubMed Central

    Kim, SangSeong; Ma, Limei; Yu, C. Ron

    2011-01-01

    In terrestrial vertebrates, the vomeronasal organ (VNO) detects and transduces pheromone signals. VNO activation is thought to be mediated by the transient receptor potential C2 (TRPC2) channel. The aberrant behavioural phenotypes observed in TRPC2−/− mice are generally attributed to the lost VNO function. Recently, calcium-activated chloride channels have been shown to contribute to VNO activation. Here we show that CACCs can be activated in VNO slice preparations from the TRPC2−/− mice and this activation is blocked by pharmacological agents that inhibit intracellular Ca2+ release. Urine-evoked Cl− current is sufficient to drive spiking changes in VNO neurons from both wild-type (WT) and TRPC2−/− mice. Moreover, blocking Cl− conductance essentially abolishes VNO activation in WT neurons. These results suggest a TRPC2-independent signalling pathway in the VNO and the requirement of calcium-activated chloride channels currents to mediate pheromone activation. Our data further suggest that TRPC2−/− mice retain partial VNO function. PMID:21694713

  15. Activation of Ca(2+) -activated Cl(-) channel ANO1 by localized Ca(2+) signals.

    PubMed

    Jin, Xin; Shah, Sihab; Du, Xiaona; Zhang, Hailin; Gamper, Nikita

    2016-01-01

    Ca(2+)-activated chloride channels (CaCCs) regulate numerous physiological processes including epithelial transport, smooth muscle contraction and sensory processing. Anoctamin-1 (ANO1, TMEM16A) is a principal CaCC subunit in many cell types, yet our understanding of the mechanisms of ANO1 activation and regulation are only beginning to emerge. Ca(2+) sensitivity of ANO1 is rather low and at negative membrane potentials the channel requires several micromoles of intracellular Ca(2+) for activation. However, global Ca(2+) levels in cells rarely reach such levels and, therefore, there must be mechanisms that focus intracellular Ca(2+) transients towards the ANO1 channels. Recent findings indeed indicate that ANO1 channels often co-localize with sources of intracellular Ca(2+) signals. Interestingly, it appears that in many cell types ANO1 is particularly tightly coupled to the Ca(2+) release sites of the intracellular Ca(2+) stores. Such preferential coupling may represent a general mechanism of ANO1 activation in native tissues.

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

    SciTech Connect

    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 not 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.

  17. A novel BK channel-targeted peptide suppresses sound evoked activity in the mouse inferior colliculus

    PubMed Central

    Scott, L. L.; Brecht, E. J.; Philpo, A.; Iyer, S.; Wu, N. S.; Mihic, S. J.; Aldrich, R. W.; Pierce, J.; Walton, J. P.

    2017-01-01

    Large conductance calcium-activated (BK) channels are broadly expressed in neurons and muscle where they modulate cellular activity. Decades of research support an interest in pharmaceutical applications for modulating BK channel function. Here we report a novel BK channel-targeted peptide with functional activity in vitro and in vivo. This 9-amino acid peptide, LS3, has a unique action, suppressing channel gating rather than blocking the pore of heterologously expressed human BK channels. With an IC50 in the high picomolar range, the apparent affinity is higher than known high affinity BK channel toxins. LS3 suppresses locomotor activity via a BK channel-specific mechanism in wild-type or BK channel-humanized Caenorhabditis elegans. Topical application on the dural surface of the auditory midbrain in mouse suppresses sound evoked neural activity, similar to a well-characterized pore blocker of the BK channel. Moreover, this novel ion channel-targeted peptide rapidly crosses the BBB after systemic delivery to modulate auditory processing. Thus, a potent BK channel peptide modulator is open to neurological applications, such as preventing audiogenic seizures that originate in the auditory midbrain. PMID:28195225

  18. Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

    PubMed Central

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

    2015-01-01

    The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates. PMID:25867140

  19. Voltage-dependent potassium channels in activated rat microglia.

    PubMed Central

    Nörenberg, W; Gebicke-Haerter, P J; Illes, P

    1994-01-01

    equimolar concentration of Cs+, and the extracellular application of tetraethylammonium and quinine inhibited both currents. 7. An increase of extracellular Ca2+ from 2 to 20 mM resulted in outwardly rectifying K+ channels activating at more positive potentials. Omission of Ca2+ from the extracellular medium had the opposite effect. When the intracellular free Ca2+ was increased from 0.01 to 1 microM, the outward current amplitudes were depressed. The Ca2+ ionophore A23187 had a similar effect. 8. LPS-treated microglial cells possess inwardly and outwardly rectifying K+ channels. The physiological and pharmacological characteristics of these two channel populations are markedly different.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7514664

  20. In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing.

    PubMed

    Nicolas, Jonathan; Hendriksen, Peter J M; de Haan, Laura H J; Koning, Rosella; Rietjens, Ivonne M C M; Bovee, Toine F H

    2015-03-01

    The present study investigated if and to what extent murine stem cell-derived beating cardiomyocytes within embryoid bodies can be used as a broad screening in vitro assay for neurotoxicity testing, replacing for example in vivo tests for marine neurotoxins. Effect of nine model compounds, acting on either the Na(+), K(+), or Ca(2+) channels or the Na(+)/K(+) ATP-ase pump, on the beating was assessed. Diphenhydramine, veratridine, isradipine, verapamil and ouabain induced specific beating arrests that were reversible and none of the concentrations tested induced cytotoxicity. Three K(+) channel blockers, amiodarone, clofilium and sematilide, and the Na(+)/K(+) ATPase pump inhibitor digoxin had no specific effect on the beating. In addition, two marine neurotoxins i.e. saxitoxin and tetrodotoxin elicited specific beating arrests in cardiomyocytes. Comparison of the results obtained with cardiomyocytes to those obtained with the neuroblastoma neuro-2a assay revealed that the cardiomyocytes were generally somewhat more sensitive for the model compounds affecting Na(+) and Ca(2+) channels, but less sensitive for the compounds affecting K(+) channels. The stem cell-derived cardiomyocytes were not as sensitive as the neuroblastoma neuro-2a assay for saxitoxin and tetrodotoxin. It is concluded that the murine stem cell-derived beating cardiomyocytes provide a sensitive model for detection of specific neurotoxins and that the neuroblastoma neuro-2a assay may be a more promising cell-based assay for the screening of marine biotoxins.

  1. Glycosylation-dependent activation of epithelial sodium channel by solnatide.

    PubMed

    Shabbir, Waheed; Tzotzos, Susan; Bedak, Minela; Aufy, Mohammad; Willam, Anita; Kraihammer, Martin; Holzner, Alexander; Czikora, Istvan; Scherbaum-Hazemi, Parastoo; Fischer, Hendrik; Pietschmann, Helmut; Fischer, Bernhard; Lucas, Rudolf; Lemmens-Gruber, Rosa

    2015-12-15

    Dysfunction of the epithelial sodium channel (ENaC), which regulates salt and water homeostasis in epithelia, causes several human pathological conditions, including pulmonary oedema. This is a potentially lethal complication of acute lung injury at least partially caused by dysfunctional alveolar liquid clearance, which in turn impairs alveolar gas exchange. Solnatide (named TIP-peptide, AP301), a 17 residue peptide mimicking the lectin-like domain of TNF has been shown to activate ENaC in several experimental animal models of acute lung injury and is being evaluated as a potential therapy for pulmonary oedema. The peptide has recently completed phase 1 and 2a clinical trials. In this study, we identify a glycosylation-dependent mechanism that preserves ENaC function and expression. Since our previous data suggested that the pore-forming subunits of ENaC are essential for maximal current activation by solnatide, we performed single- and multi-N-glycosylation site mutations in αN232,293,312,397,511Q- and δN166,211,384Q-subunits, in order to identify crucial residues for interaction with solnatide within the extracellular loop of the channel. Additionally, we generated αL576X and αN232,293,312,397,511Q,L576X deletion mutants of ENaC-α, since we have previously demonstrated that the carboxy terminal domain of this subunit is also involved in its interaction with solnatide. In cells expressing αN232,293,312,397,511Q,L576Xβγ-hENaC or δN166,311,384Q,D552Xβγ-hENaC activation by solnatide, as measured in whole cell patch clamp mode, was completely abolished, whereas it was attenuated in αL576Xβγ-hENaC- and δD552Xβγ-hENaC-expressing cells. Taken together, our findings delineate an N-glycan dependent interaction between the TIP-peptide and ENaC leading to normalization of both sodium and fluid absorption in oedematous alveoli to non-oedematous levels.

  2. Pupil Size Changes as an Active Information Channel for Biofeedback Applications.

    PubMed

    Ehlers, Jan; Strauch, Christoph; Georgi, Juliane; Huckauf, Anke

    2016-09-01

    Pupil size is usually regarded as a passive information channel that provides insight into cognitive and affective states but defies any further control. However, in a recent study (Ehlers et al. 2015) we demonstrate that sympathetic activity indexed by pupil dynamics allows strategic interference by means of simple cognitive techniques. Utilizing positive/negative imaginings, subjects were able to expand pupil diameter beyond baseline variations; albeit with varying degrees of success and only over brief periods. The current study provides a comprehensive replication on the basis of considerable changes to the experimental set-up. Results show that stricter methodological conditions (controlled baseline settings and specified user instructions) strengthen the reported effect, whereas overall performance increases by one standard deviation. Effects are thereby not restricted to pupillary level. Parallel recordings of skin conductance changes prove a general enhancement of induced autonomic arousal. Considering the stability of the results across studies, we conclude that pupil size information exceeds affective monitoring and may constitute an active input channel in human-computer interaction. Furthermore, since variations in pupil diameter reliably display self-induced changes in sympathetic arousal, the relevance of this parameter is strongly indicated for future approaches in clinical biofeedback.

  3. Effects of N-glycosylation on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

    PubMed

    Li, Mo; Tonggu, Lige; Tang, Lan; Wang, Liguo

    2015-02-15

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization and conduct an inward cation current, which contributes to rhythmic electrical activity of neural and cardiac pacemaker cells. HCN channels have been shown to undergo N-linked glycosylation, and the N-glycosylation has been shown to be required for membrane trafficking and possibly function. In this study, recombinant wild-type (WT) and glycosylation-defective N380Q HCN2 channels were individually or co-expressed in HEK-293 cells. We demonstrate that glycosylation is required for trafficking to the plasma membrane and for the stability of HCN channels in the cell. Interestingly, the heteromeric HCN2 channels of WT and glycosylation-defective N380Q have been observed on cell membranes, indicating that not all four subunits of a tetrameric HCN2 channel need to be glycosylated for HCN2 channels to traffic to plasma membranes. Subsequently, we investigate the effect of N-glycosylation on the function of HCN2 channels. We developed a fluorescence-based flux assay, which makes it possible to establish a negative potential inside liposomes to open HCN2 channels. Using this flux assay, we demonstrate that glycosylation-defective N380Q HCN2 channels reconstituted into liposomes function similarly to WT HCN2 channels. This suggests that N-glycosylation is not required for HCN2 channels to function.

  4. A Leucine Zipper Motif Essential for Gating of Hyperpolarization-activated Channels*

    PubMed Central

    Wemhöner, Konstantin; Silbernagel, Nicole; Marzian, Stefanie; Netter, Michael F.; Rinné, Susanne; Stansfeld, Phillip J.; Decher, Niels

    2012-01-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are pacemakers in cardiac myocytes and neurons. Although their membrane topology closely resembles that of voltage-gated K+ channels, the mechanism of their unique gating behavior in response to hyperpolarization is still poorly understood. We have identified a highly conserved leucine zipper motif in the S5 segment of HCN family members. In order to study the role of this motif for channel function, the leucine residues of the zipper were individually mutated to alanine, arginine, or glutamine residues. Leucine zipper mutants traffic to the plasma membrane, but the channels lose their sensitivity to open upon hyperpolarization. Thus, our data indicate that the leucine zipper is an important molecular determinant for hyperpolarization-activated channel gating. Residues of the leucine zipper interact with the adjacent S6 segment of the channel. This interaction is essential for voltage-dependent gating of the channel. The lower part of the leucine zipper, at the intracellular mouth of the channel, is important for stabilizing the closed state. Mutations at these sites increase current amplitudes or result in channels with deficient closing and increased min-Po. Our data are further supported by homology models of the open and closed state of the HCN2 channel pore. Thus, we conclude that the leucine zipper of HCN channels is a major determinant for hyperpolarization-activated channel gating. PMID:23048023

  5. Activation and deactivation of vibronic channels in intact phycocyanin rods.

    PubMed

    Nganou, C; David, L; Meinke, R; Adir, N; Maultzsch, J; Mkandawire, M; Pouhè, D; Thomsen, C

    2014-02-28

    We investigated the excitation modes of the light-harvesting protein phycocyanin (PC) from Thermosynechococcus vulcanus in the crystalline state using UV and near-infrared Raman spectroscopy. The spectra revealed the absence of a hydrogen out-of-plane wagging (HOOP) mode in the PC trimer, which suggests that the HOOP mode is activated in the intact PC rod, while it is not active in the PC trimer. Furthermore, in the PC trimer an intense mode at 984 cm(-1) is assigned to the C-C stretching vibration while the mode at 454 cm(-1) is likely due to ethyl group torsion. In contrast, in the similar chromophore phytochromobilin the C5,10,15-D wag mode at 622 cm(-1) does not come from a downshift of the HOOP. Additionally, the absence of modes between 1200 and 1300 cm(-1) rules out functional monomerization. A correlation between phycocyanobilin (PCB) and phycoerythrobilin (PEB) suggests that the PCB cofactors of the PC trimer appear in a conformation similar to that of PEB. The conformation of the PC rod is consistent with that of the allophycocyanin (APC) trimer, and thus excitonic flow is facilitated between these two independent light-harvesting compounds. This excitonic flow from the PC rod to APC appears to be modulated by the vibration channels during HOOP wagging, C = C stretching, and the N-H rocking in-plan vibration.

  6. Activation and deactivation of vibronic channels in intact phycocyanin rods

    NASA Astrophysics Data System (ADS)

    Nganou, C.; David, L.; Meinke, R.; Adir, N.; Maultzsch, J.; Mkandawire, M.; Pouhè, D.; Thomsen, C.

    2014-02-01

    We investigated the excitation modes of the light-harvesting protein phycocyanin (PC) from Thermosynechococcus vulcanus in the crystalline state using UV and near-infrared Raman spectroscopy. The spectra revealed the absence of a hydrogen out-of-plane wagging (HOOP) mode in the PC trimer, which suggests that the HOOP mode is activated in the intact PC rod, while it is not active in the PC trimer. Furthermore, in the PC trimer an intense mode at 984 cm-1 is assigned to the C-C stretching vibration while the mode at 454 cm-1 is likely due to ethyl group torsion. In contrast, in the similar chromophore phytochromobilin the C5,10,15-D wag mode at 622 cm-1 does not come from a downshift of the HOOP. Additionally, the absence of modes between 1200 and 1300 cm-1 rules out functional monomerization. A correlation between phycocyanobilin (PCB) and phycoerythrobilin (PEB) suggests that the PCB cofactors of the PC trimer appear in a conformation similar to that of PEB. The conformation of the PC rod is consistent with that of the allophycocyanin (APC) trimer, and thus excitonic flow is facilitated between these two independent light-harvesting compounds. This excitonic flow from the PC rod to APC appears to be modulated by the vibration channels during HOOP wagging, C = C stretching, and the N-H rocking in-plan vibration.

  7. GlialCAM, a CLC-2 Cl(-) channel subunit, activates the slow gate of CLC chloride channels.

    PubMed

    Jeworutzki, Elena; Lagostena, Laura; Elorza-Vidal, Xabier; López-Hernández, Tania; Estévez, Raúl; Pusch, Michael

    2014-09-02

    GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl(-) channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction.

  8. Ca2+ influx through L-type Ca2+ channels and transient receptor potential channels activate pathological hypertrophy signaling

    PubMed Central

    Gao, Hui; Wang, Fang; Wang, Wei; Makarewich, Catherine A.; Zhang, Hongyu; Kubo, Hajime; Berretta, Remus M.; Barr, Larry A.; Molkentin, Jeffrey D.; Houser, Steven R.

    2012-01-01

    Common cardiovascular diseases such as hypertension and myocardial infarction require that myocytes develop greater than normal force to maintain cardiac pump function. This requires increases in [Ca2+]. These diseases induce cardiac hypertrophy and increases in [Ca2+] are known to be an essential proximal signal for activation of hypertrophic genes. However, the source of “hypertrophic” [Ca2+] is not known and is the topic of this study. The role of Ca2+ influx through L-type Ca2+ channels (LTCC), T-type Ca2+ channels (TTCC) and transient receptor potential (TRP) channels on the activation of Calcineurin (Cn) – Nuclear Factor of Activated T cells (NFAT) signaling and myocyte hypertrophy was studied. Neonatal rat (NRVMs) and adult feline (AFVM) ventricular myocytes were infected with an adenovirus containing NFAT-GFP, to determine factors that could induce NFAT nuclear translocation. Four millimolar Ca2+ or pacing induced NFAT nuclear translocation. This effect was blocked by Cn inhibitors. In NRVMs Nifedipine (Nif, LTCC antagonist) blocked high Ca2+-induced NFAT nuclear translocation while SKF-96365 (TRP channel antagonist) and Nickel (Ni, TTCC antagonist) were less effective. The relative potency of these antagonists against Ca2+ induced NFAT nuclear translocation (Nif>SKF-96365>Ni) was similar to their effects on Ca2+ transients and the LTCC current. Infection of NRVM with viruses containing TRP channels also activated NFAT-GFP nuclear translocation and caused myocyte hypertrophy. TRP effects were reduced by SKF-96365, but were more effectively antagonized by Nif. These experiments suggest that Ca2+ influx through LTCCs is the primary source of Ca2+ to activate Cn-NFAT signaling in NRVMs and AFVMs. While TRP channels cause hypertrophy, they appear to do so through a mechanism involving Ca2+ entry via LTCCs. PMID:22921230

  9. Phentolamine relaxes human corpus cavernosum by a nonadrenergic mechanism activating ATP-sensitive K+ channel.

    PubMed

    Silva, L F G; Nascimento, N R F; Fonteles, M C; de Nucci, G; Moraes, M E; Vasconcelos, P R L; Moraes, M O

    2005-01-01

    To investigate the pharmacodynamics of phentolamine in human corpus cavernosum (HCC) with special attention to the role of the K+ channels. Strips of HCC precontracted with nonadrenergic stimuli and kept in isometric organ bath immersed in a modified Krebs-Henseleit solution enriched with guanethidine and indomethacine were used in order to study the mechanism of the phentolamine-induced relaxation. Phentolamine caused relaxation (approximately 50%) in HCC strips precontracted with K+ 40 mM. This effect was not blocked by tetrodotoxin (1 microM) (54.6+/-4.6 vs 48.9+/-6.4%) or (atropine (10 microM) (52.7+/-6.5 vs 58.6+/-5.6%). However, this relaxation was significantly attenuated by L-NAME (100 microM) (59.7+/-5.8 vs 27.8+/-7.1%; P<0.05; n = 8) and ODQ (100 microM) (62.7+/-5.1 vs 26.8+/-3.9%; P<0.05; n = 8). Charybdotoxin and apamin (K(Ca)-channel blockers) did not affect the phentolamine relaxations (54.6+/-4.6 vs 59.3+/-5.2%). Glibenclamide (100 microM), an inhibitor of K(ATP)-channel, caused a significant inhibition (56.7+/-6.3 vs 11.3+/-2.3%; P<0.05; n = 8) of the phentolamine-induced relaxation. In addition, the association of glibenclamide and L-NAME almost abolished the phentolamine-mediated relaxation (54.6+/-5.6 vs 5.7+/-1.4%; P<0.05; n = 8). The results suggest that phentolamine relaxes HCC by a nonadrenergic-noncholinergic mechanism dependent on nitric oxide synthase activity and activation of K(ATP)-channel.

  10. Chloride Channel 3 Channels in the Activation and Migration of Human Blood Eosinophils in Allergic Asthma.

    PubMed

    Gaurav, Rohit; Bewtra, Againdra K; Agrawal, Devendra K

    2015-08-01

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is responsible for respiratory burst in immune cells. Chloride channel 3 (CLC3) has been linked to the respiratory burst in eosinophils and neutrophils. The effect of cytokines and the involvement of CLC3 in the regulation of NADPH-dependent oxidative stress and on cytokine-mediated migration of eosinophils are not known. Human peripheral blood eosinophils were isolated from healthy individuals and from individuals with asthma by negative selection. Real-time PCR was used to detect the expression of NADPH oxidases in eosinophils. Intracellular reactive oxygen species (ROS) measurement was done with flow cytometry. Superoxide generation was measured with transforming growth factor (TGF)-β, eotaxin, and CLC3 blockers. CLC3 dependence of eosinophils in TGF-β- and eotaxin-induced migration was also examined. The messenger RNA (mRNA) transcripts of NADPH oxidase (NOX) 2, dual oxidase (DUOX) 1, and DUOX2 were detected in blood eosinophils, with very low expression of NOX1, NOX3, and NOX5 and no NOX4 mRNA. The level of NOX2 mRNA transcripts increased with disease severity in the eosinophils of subjects with asthma compared with healthy nonatopic volunteers. Change in granularity and size in eosinophils, but no change in intracellular ROS, was observed with phorbol myristate acetate (PMA). PMA, TGF-β, and eotaxin used the CLC3-dependent pathway to increase superoxide radicals. TGF-β and eotaxin induced CLC3-dependent chemotaxis of eosinophils. These findings support the requirement of CLC3 in the activation and migration of human blood eosinophils and may provide a potential novel therapeutic target to regulate eosinophil hyperactivity in allergic airway inflammation in asthma.

  11. 4-isopropylcyclohexanol has potential analgesic effects through the inhibition of anoctamin 1, TRPV1 and TRPA1 channel activities

    PubMed Central

    Takayama, Yasunori; Furue, Hidemasa; Tominaga, Makoto

    2017-01-01

    Interactions between calcium-activated chloride channel anoctamin 1 (ANO1) and transient receptor potential vanilloid 1 (TRPV1) enhance pain sensations in mice, suggesting that ANO1 inhibition could have analgesic effects. Here we show that menthol and the menthol analogue isopropylcyclohexane (iPr-CyH) inhibited ANO1 channels in mice. The iPr-CyH derivative 4-isopropylcyclohexanol (4-iPr-CyH-OH) inhibited mouse ANO1 currents more potently than iPr-CyH. Moreover, 4-iPr-CyH-OH inhibited the activities of TRPV1, TRP ankyrin 1 (TRPA1), TRP melastatin 8 (TRPM8) and TRPV4. Single-channel analysis revealed that 4-iPr-CyH-OH reduced TRPV1 and TRPA1 current open-times without affecting unitary amplitude or closed-time, suggesting that it affected gating rather than blocking the channel pore. The ability of 4-iPr-CyH-OH to inhibit action potential generation and reduce pain-related behaviors induced by capsaicin in mice suggests that 4-iPr-CyH-OH could have analgesic applications. Thus, 4-iPr-CyH-OH is a promising base chemical to develop novel analgesics that target ANO1 and TRP channels. PMID:28225032

  12. 4-isopropylcyclohexanol has potential analgesic effects through the inhibition of anoctamin 1, TRPV1 and TRPA1 channel activities.

    PubMed

    Takayama, Yasunori; Furue, Hidemasa; Tominaga, Makoto

    2017-02-22

    Interactions between calcium-activated chloride channel anoctamin 1 (ANO1) and transient receptor potential vanilloid 1 (TRPV1) enhance pain sensations in mice, suggesting that ANO1 inhibition could have analgesic effects. Here we show that menthol and the menthol analogue isopropylcyclohexane (iPr-CyH) inhibited ANO1 channels in mice. The iPr-CyH derivative 4-isopropylcyclohexanol (4-iPr-CyH-OH) inhibited mouse ANO1 currents more potently than iPr-CyH. Moreover, 4-iPr-CyH-OH inhibited the activities of TRPV1, TRP ankyrin 1 (TRPA1), TRP melastatin 8 (TRPM8) and TRPV4. Single-channel analysis revealed that 4-iPr-CyH-OH reduced TRPV1 and TRPA1 current open-times without affecting unitary amplitude or closed-time, suggesting that it affected gating rather than blocking the channel pore. The ability of 4-iPr-CyH-OH to inhibit action potential generation and reduce pain-related behaviors induced by capsaicin in mice suggests that 4-iPr-CyH-OH could have analgesic applications. Thus, 4-iPr-CyH-OH is a promising base chemical to develop novel analgesics that target ANO1 and TRP channels.

  13. Mutating three residues in the bovine rod cyclic nucleotide-activated channel can switch a nucleotide from inactive to active.

    PubMed Central

    Scott, S P; Cummings, J; Joe, J C; Tanaka, J C

    2000-01-01

    Cyclic nucleotide-gated (CNG) channels, which were initially studied in retina and olfactory neurons, are activated by cytoplasmic cGMP or cAMP. Detailed comparisons of nucleotide-activated currents using nucleotide analogs and mutagenesis revealed channel-specific residues in the nucleotide-binding domain that regulate the binding and channel-activation properties. Of particular interest are N(1)-oxide cAMP, which does not activate bovine rod channels, and Rp-cGMPS, which activates bovine rod, but not catfish, olfactory channels. Previously, we showed that four residues coordinate the purine interactions in the binding domain and that three of these residues vary in the alpha subunits of the bovine rod, catfish, and rat olfactory channels. Here we show that both N(1)-oxide cAMP and Rp-cGMPS activate rat olfactory channels. A mutant of the bovine rod alpha subunit, substituted with residues from the rat olfactory channel at the three variable positions, was weakly activated by N(1)-oxide cAMP, and a catfish olfactory-like bovine rod mutant lost activation by Rp-cGMPS. These experiments underscore the functional importance of purine contacts with three residues in the cyclic nucleotide-binding domain. Molecular models of nucleotide analogs in the binding domains, constructed with AMMP, showed differences in the purine contacts among the channels that might account for activation differences. PMID:10777730

  14. Long-range forces affecting equilibrium inertial focusing behavior in straight high aspect ratio microfluidic channels

    NASA Astrophysics Data System (ADS)

    Reece, Amy E.; Oakey, John

    2016-04-01

    The controlled and directed focusing of particles within flowing fluids is a problem of fundamental and technological significance. Microfluidic inertial focusing provides passive and precise lateral and longitudinal alignment of small particles without the need for external actuation or sheath fluid. The benefits of inertial focusing have quickly enabled the development of miniaturized flow cytometers, size-selective sorting devices, and other high-throughput particle screening tools. Straight channel inertial focusing device design requires knowledge of fluid properties and particle-channel size ratio. Equilibrium behavior of inertially focused particles has been extensively characterized and the constitutive phenomena described by scaling relationships for straight channels of square and rectangular cross section. In concentrated particle suspensions, however, long-range hydrodynamic repulsions give rise to complex particle ordering that, while interesting and potentially useful, can also dramatically diminish the technique's effectiveness for high-throughput particle handling applications. We have empirically investigated particle focusing behavior within channels of increasing aspect ratio and have identified three scaling regimes that produce varying degrees of geometrical ordering between focused particles. To explore the limits of inertial particle focusing and identify the origins of these long-range interparticle forces, we have explored equilibrium focusing behavior as a function of channel geometry and particle concentration. Experimental results for highly concentrated particle solutions identify equilibrium thresholds for focusing that scale weakly with concentration and strongly with channel geometry. Balancing geometry mediated inertial forces with estimates for interparticle repulsive forces now provide a complete picture of pattern formation among concentrated inertially focused particles and enhance our understanding of the fundamental limits of

  15. Long-range forces affecting equilibrium inertial focusing behavior in straight high aspect ratio microfluidic channels.

    PubMed

    Reece, Amy E; Oakey, John

    2016-04-01

    The controlled and directed focusing of particles within flowing fluids is a problem of fundamental and technological significance. Microfluidic inertial focusing provides passive and precise lateral and longitudinal alignment of small particles without the need for external actuation or sheath fluid. The benefits of inertial focusing have quickly enabled the development of miniaturized flow cytometers, size-selective sorting devices, and other high-throughput particle screening tools. Straight channel inertial focusing device design requires knowledge of fluid properties and particle-channel size ratio. Equilibrium behavior of inertially focused particles has been extensively characterized and the constitutive phenomena described by scaling relationships for straight channels of square and rectangular cross section. In concentrated particle suspensions, however, long-range hydrodynamic repulsions give rise to complex particle ordering that, while interesting and potentially useful, can also dramatically diminish the technique's effectiveness for high-throughput particle handling applications. We have empirically investigated particle focusing behavior within channels of increasing aspect ratio and have identified three scaling regimes that produce varying degrees of geometrical ordering between focused particles. To explore the limits of inertial particle focusing and identify the origins of these long-range interparticle forces, we have explored equilibrium focusing behavior as a function of channel geometry and particle concentration. Experimental results for highly concentrated particle solutions identify equilibrium thresholds for focusing that scale weakly with concentration and strongly with channel geometry. Balancing geometry mediated inertial forces with estimates for interparticle repulsive forces now provide a complete picture of pattern formation among concentrated inertially focused particles and enhance our understanding of the fundamental limits of

  16. Selectivity filter gating in large-conductance Ca(2+)-activated K+ channels.

    PubMed

    Thompson, Jill; Begenisich, Ted

    2012-03-01

    Membrane voltage controls the passage of ions through voltage-gated K (K(v)) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of K(v) channels is well established, it is not clear if such a cytoplasmic gate exists in all K(+) channels. Some studies on large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker "ball" peptide (BP) on BK channels with either K(+) or Rb(+) as the permeant ion. When tested in K(+) solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb(+) replaced K(+) as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these K(v) channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating.

  17. Selectivity filter gating in large-conductance Ca2+-activated K+ channels

    PubMed Central

    Thompson, Jill

    2012-01-01

    Membrane voltage controls the passage of ions through voltage-gated K (Kv) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of Kv channels is well established, it is not clear if such a cytoplasmic gate exists in all K+ channels. Some studies on large-conductance, voltage- and Ca2+-activated K+ (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker “ball” peptide (BP) on BK channels with either K+ or Rb+ as the permeant ion. When tested in K+ solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb+ replaced K+ as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these Kv channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating. PMID:22371364

  18. Melanocortin 4 receptor constitutive activity inhibits L-type voltage-gated calcium channels in neurons.

    PubMed

    Agosti, F; Cordisco Gonzalez, S; Martinez Damonte, V; Tolosa, M J; Di Siervi, N; Schioth, H B; Davio, C; Perello, M; Raingo, J

    2017-03-27

    The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that is expressed in several brain nuclei playing a crucial role in the regulation of energy balance controlling the homeostasis of the organism. It displays both agonist-evoked and constitutive activity, and moreover, it can couple to different G proteins. Most of the research on MC4R has been focused on agonist-induced activity, while the molecular and cellular basis of MC4R constitutive activity remains scarcely studied. We have previously shown that neuronal N-type voltage-gated calcium channels (CaV2.2) are inhibited by MC4R agonist-dependent activation, while the CaV subtypes that carry L- and P/Q-type current are not. Here, we tested the hypothesis that MC4R constitutive activity can affect CaV, with focus on the channel subtypes that can control transcriptional activity coupled to depolarization (L-type, CaV1.2/1.3) and neurotransmitter release (N- and P/Q-type, CaV2.2 and CaV2.1). We found that MC4R constitutive activity inhibits specifically CaV1.2/1.3 and CaV2.1 subtypes of CaV. We also explored the signaling pathways mediating this inhibition, and thus propose that agonist-dependent and basal MC4R activation modes signal differentially through Gs and Gi/o pathways to impact on different CaV subtypes. In addition, we found that chronic incubation with MC4R endogenous inverse agonist, agouti and agouti-related peptide (AgRP), occludes CaV inhibition in a cell line and in amygdaloid complex cultured neurons as well. Thus, we define new mechanisms of control of the main mediators of depolarization-induced calcium entry into neurons by a GPCR that displays constitutive activity.

  19. Small Conductance Ca2+-Activated K+ Channel Knock-Out Mice Reveal the Identity of Calcium-Dependent Afterhyperpolarization Currents

    PubMed Central

    Bond, Chris T.; Herson, Paco S.; Strassmaier, Timothy; Hammond, Rebecca; Stackman, Robert; Maylie, James; Adelman, John P.

    2010-01-01

    Action potentials in many central neurons are followed by a prolonged afterhyperpolarization (AHP) that influences firing frequency and affects neuronal integration. In hippocampal CA1 pyramidal neurons, the current ascribed to the AHP (IAHP) has three kinetic components. The IfastAHP is predominantly attributable to voltage-dependent K+ channels, whereas Ca2+-dependent and voltage-independent K+ channels contribute to the ImediumAHP (ImAHP) and IslowAHP (IsAHP). Apamin, which selectively suppresses a component of the mAHP, increases neuronal excitability and facilitates the induction of synaptic plasticity at Schaffer collateral synapses and hippocampal-dependent learning. The Ca2+-dependent components of the AHP have been attributed to the activity of small conductance Ca2+-activated K+(SK) channels. Examination of transgenic mice, each lacking one of the three SK channel genes expressed in the CNS, reveals that mice without the SK2 subunit completely lack the apamin-sensitive component of the ImAHP in CA1 neurons, whereas the IsAHP is not different in any of the SK transgenic mice. In each of the transgenic lines, the expression levels of the remaining SK genes are not changed. The results demonstrate that only SK2 channels are necessary for the ImAHP, and none of the SK channels underlie the IsAHP. PMID:15190101

  20. Effects of Active Subsidence Vs. Existing Basin Geometry on Fluviodeltaic Channels and Stratal Architecture

    NASA Astrophysics Data System (ADS)

    Liang, M.; Kim, W.; Passalacqua, P.

    2015-12-01

    Tectonic subsidence and basin topography, both determining the accommodation, are fundamental controls on the basin filling processes. Their effects on the fluvial organization and the resultant subsurface patterns remain difficult to predict due to the lack of understanding about interaction between internal dynamics and external controls. Despite the intensive studies on tectonic steering effects on alluvial architecture, how the self-organization of deltaic channels, especially the distributary channel network, respond to tectonics and basin geometry is mostly unknown. Recently physical experiments and field studies have hinted dramatic differences in fluviodeltaic evolution between ones associated with active differential subsidence and existing basin depth. In this work we designed a series of numerical experiments using a reduced-complexity channel-resolving model for delta formation, and tested over a range of localized subsidence rates and topographic depression in basin geometry. We also used a set of robust delta metrics to analyze: i) shoreline planform asymmetry, ii) channel and lobe geometry, iii) channel network pattern, iv) autogenic timescales, and v) subsurface structure. The modeling results show that given a similar final thickness, active subsidence enhances channel branching with smaller channel sand bodies that are both laterally and vertically connected, whereas existing topographic depression causes more large-scale channel avulsions with larger channel sand bodies. In general, both subsidence and existing basin geometry could steer channels and/or lock channels in place but develop distinct channel patterns and thus stratal architecture.

  1. Voltage-induced slow activation and deactivation of mechanosensitive channels in Xenopus oocytes.

    PubMed Central

    Silberberg, S D; Magleby, K L

    1997-01-01

    1. The relationship between stretch and voltage activation of mechanosensitive (MS) channels from Xenopus oocytes was studied in excised patches of membrane using the patch clamp technique. 2. As is characteristic of MS channels to oocytes, stretching the membrane by applying negative pressure to the patch pipette at -50 mV activated the MS channels rapidly. The channels then deactivated rapidly when the stretch was removed. The stretch-activated MS channels entered a main conductance level (45 pS) and one or more subconductance levels in the range of about 75-90% of the main conductance level. 3. In the absence of stretch, a depolarizing step from -50 to +50 mV activated apparent MS channels after long delays of typically 1-20 s (range, 100 ms to 6 min). Upon repolarization, the channels deactivated slowly with a single exponential (mean time constant of 4 s) or double exponential (mean time constants of 0.8 and 3 s) time course. 4. Delayed activation with depolarization and slow deactivation upon repolarization were also observed for apparent MS channels in on-cell patches. 5. The voltage-activated channels were cation selective and had the same selectivity and conductance levels as the stretch activated MS channels. Applying stretch during voltage-induced channel activity did not activate any additional channels, and the same maximal number of channels were typically activated by either stretch or by voltage. These observations suggest that voltage activates the same MS channels that are activated by stretch. 6. The opening of MS channels following steps to +50 mV occurred in an apparently co-operative manner in 70% of the excised patches containing multiple MS channels. 7. In the absence of stretch, the opening frequency and open probability of MS channels increased with depolarization in the examined voltage range of -60 to -20 mV. 8. Applying a brief stretch during the delay to activation at +50 mV activated the MS channels rapidly, which then remained active

  2. Function and regulation of large conductance Ca(2+)-activated K+ channel in vascular smooth muscle cells.

    PubMed

    Hu, Xiang-Qun; Zhang, Lubo

    2012-09-01

    Large conductance Ca(2+)-activated K(+) (BK(Ca)) channels are abundantly expressed in vascular smooth muscle cells. Activation of BK(Ca) channels leads to hyperpolarization of cell membrane, which in turn counteracts vasoconstriction. Therefore, BK(Ca) channels have an important role in regulation of vascular tone and blood pressure. The activity of BK(Ca) channels is subject to modulation by various factors. Furthermore, the function of BK(Ca) channels are altered in both physiological and pathophysiological conditions, such as pregnancy, hypertension and diabetes, which has dramatic impacts on vascular tone and hemodynamics. Consequently, compounds and genetic manipulation that alter activity and expression of the channel might be of therapeutic interest.

  3. Corrugator activity confirms immediate negative affect in surprise

    PubMed Central

    Topolinski, Sascha; Strack, Fritz

    2015-01-01

    The emotion of surprise entails a complex of immediate responses, such as cognitive interruption, attention allocation to, and more systematic processing of the surprising stimulus. All these processes serve the ultimate function to increase processing depth and thus cognitively master the surprising stimulus. The present account introduces phasic negative affect as the underlying mechanism responsible for this switch in operating mode. Surprising stimuli are schema-discrepant and thus entail cognitive disfluency, which elicits immediate negative affect. This affect in turn works like a phasic cognitive tuning switching the current processing mode from more automatic and heuristic to more systematic and reflective processing. Directly testing the initial elicitation of negative affect by surprising events, the present experiment presented high and low surprising neutral trivia statements to N = 28 participants while assessing their spontaneous facial expressions via facial electromyography. High compared to low surprising trivia elicited higher corrugator activity, indicative of negative affect and mental effort, while leaving zygomaticus (positive affect) and frontalis (cultural surprise expression) activity unaffected. Future research shall investigate the mediating role of negative affect in eliciting surprise-related outcomes. PMID:25762956

  4. Pre-treating channel catfish with copper sulfate affects susceptibility to columnaris disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Columnaris disease is one of the most important bacterial diseases of channel catfish, Ictalurus punctatus, commercially grown in the US. This disease can greatly diminish the profitability of aquaculture operations by large-scale mortality events, particularly in the fingerling production phase. ...

  5. Designing Effective Persuasive Systems Utilizing the Power of Entanglement: Communication Channel, Strategy & Affect

    ERIC Educational Resources Information Center

    Li, Haiqing

    2010-01-01

    With rapid advancements in information and communication technologies, computer-mediated communication channels such as email, web, mobile smart-phones with SMS, social networking websites (Facebook), multimedia websites, and OEM devices provide users with multiple technology choices to seek information. However, no study has compared the…

  6. Membrane stretch affects gating modes of a skeletal muscle sodium channel.

    PubMed Central

    Tabarean, I V; Juranka, P; Morris, C E

    1999-01-01

    The alpha subunit of the human skeletal muscle Na(+) channel recorded from cell-attached patches yielded, as expected for Xenopus oocytes, two current components that were stable for tens of minutes during 0.2 Hz stimulation. Within seconds of applying sustained stretch, however, the slower component began decreasing and, depending on stretch intensity, disappeared in 1-3 min. Simultaneously, the faster current increased. The resulting fast current kinetics and voltage sensitivity were indistinguishable from the fast components 1) left after 10 Hz depolarizations, and 2) that dominated when alpha subunit was co-expressed with human beta1 subunit. Although high frequency depolarization-induced loss of slow current was reversible, the stretch-induced slow-to-fast conversion was irreversible. The conclusion that stretch converted a single population of alpha subunits from an abnormal slow to a bona fide fast gating mode was confirmed by using gigaohm seals formed without suction, in which fast gating was originally absent. For brain Na(+) channels, co-expressing G proteins with the channel alpha subunit yields slow gating. Because both stretch and beta1 subunits induced the fast gating mode, perhaps they do so by minimizing alpha subunit interactions with G proteins or with other regulatory molecules available in oocyte membrane. Because of the possible involvement of oocyte molecules, it remains to be determined whether the Na(+) channel alpha subunit was directly or secondarily susceptible to bilayer tension. PMID:10423424

  7. Src tyrosine kinase alters gating of hyperpolarization-activated HCN4 pacemaker channel through Tyr531.

    PubMed

    Li, Chen-Hong; Zhang, Qi; Teng, Bunyen; Mustafa, S Jamal; Huang, Jian-Ying; Yu, Han-Gang

    2008-01-01

    We recently discovered that the constitutively active Src tyrosine kinase can enhance hyperpolarization-activated, cyclic nucleotide-gated (HCN) 4 channel activity by binding to the channel protein. To investigate the mechanism of modulation by Src of HCN channels, we studied the effects of a selective inhibitor of Src tyrosine kinase, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), on HCN4 and its mutant channels expressed in HEK 293 cells by using a whole cell patch-clamp technique. We found that PP2 can inhibit HCN4 currents by negatively shifting the voltage dependence of channel activation, decreasing the whole cell channel conductance, and slowing activation and deactivation kinetics. Screening putative tyrosine residues subject to phosphorylation yielded two candidates: Tyr(531) and Tyr(554). Substituting HCN4-Tyr(531) with phenylalanine largely abolished the effects of PP2 on HCN4 channels. Replacing HCN4-Tyr(554) with phenylalanine did not abolish the effects of PP2 on voltage-dependent activation but did eliminate PP2-induced slowing of channel kinetics. The inhibitory effects of HCN channels associated with reduced Src tyrosine activity is confirmed in HL-1 cardiomyocytes. Finally, we found that PP2 can decrease the heart rate in a mouse model. These results demonstrate that Src tyrosine kinase enhances HCN4 currents by shifting their activation to more positive potentials and increasing the whole cell channel conductance as well as speeding the channel kinetics. The tyrosine residue that mediates most of Src's actions on HCN4 channels is Tyr(531).

  8. X-ray irradiation activates K+ channels via H2O2 signaling.

    PubMed

    Gibhardt, Christine S; Roth, Bastian; Schroeder, Indra; Fuck, Sebastian; Becker, Patrick; Jakob, Burkhard; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

    2015-09-09

    Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca2+ and eventually an activation of hIK channels.

  9. X-ray irradiation activates K+ channels via H2O2 signaling

    PubMed Central

    Gibhardt, Christine S.; Roth, Bastian; Schroeder, Indra; Fuck, Sebastian; Becker, Patrick; Jakob, Burkhard; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

    2015-01-01

    Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca2+ and eventually an activation of hIK channels. PMID:26350345

  10. Hysteresis of KcsA potassium channel's activation- deactivation gating is caused by structural changes at the channel's selectivity filter.

    PubMed

    Tilegenova, Cholpon; Cortes, D Marien; Cuello, Luis G

    2017-03-21

    Mode-shift or hysteresis has been reported in ion channels. Voltage-shift for gating currents is well documented for voltage-gated cation channels (VGCC), and it is considered a voltage-sensing domain's (VSD) intrinsic property. However, uncoupling the Shaker K(+) channel's pore domain (PD) from the VSD prevented the mode-shift of the gating currents. Consequently, it was proposed that an open-state stabilization of the PD imposes a mechanical load on the VSD, which causes its mode-shift. Furthermore, the mode-shift displayed by hyperpolarization-gated cation channels is likely caused by structural changes at the channel's PD similar to those underlying C-type inactivation. To demonstrate that the PD of VGCC undergoes hysteresis, it is imperative to study its gating process in the absence of the VSD. A back-door strategy is to use KcsA (a K(+) channel from the bacteria Streptomyces lividans) as a surrogate because it lacks a VSD and exhibits an activation coupled to C-type inactivation. By directly measuring KcsA's activation gate opening and closing in conditions that promote or halt C-type inactivation, we have found (i) that KcsA undergoes mode-shift of gating when having K(+) as the permeant ion; (ii) that Cs(+) or Rb(+), known to halt C-inactivation, prevented mode-shift of gating; and (iii) that, in the total absence of C-type inactivation, KcsA's mode-shift was prevented. Finally, our results demonstrate that an allosteric communication causes KcsA's activation gate to "remember" the conformation of the selectivity filter, and hence KcsA requires a different amount of energy for opening than for closing.

  11. A large-conductance calcium-activated potassium channel in potato (Solanum tuberosum) tuber mitochondria.

    PubMed

    Koszela-Piotrowska, Izabela; Matkovic, Karolina; Szewczyk, Adam; Jarmuszkiewicz, Wieslawa

    2009-11-11

    In the present study, we describe the existence of a novel potassium channel in the plant [potato (Solanum tuberosum) tuber] mitochondrial inner membrane. We found that substances known to modulate large-conductance calcium-activated potassium channel activity influenced the bioenergetics of potato tuber mitochondria. In isolated mitochondria, Ca2+ and NS1619 {1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-ben-zimidazole-2-one; a potassium channel opener} were found to depolarize the mitochondrial membrane potential and to stimulate resting respiration. These effects were blocked by iberiotoxin (a potassium channel inhibitor) in a potassium-dependent manner. Additionally, the electrophysiological properties of the large-conductance potassium channel present in the potato tuber inner mitochondrial membrane are described in a reconstituted system, using planar lipid bilayers. After incorporation in 50/450 mM KCl gradient solutions, we recorded large-conductance potassium channel activity with conductance from 502+/-15 to 615+/-12 pS. The probability of channel opening was increased by Ca2+ and reduced by iberiotoxin. Immunological analysis with antibodies raised against the mammalian plasma-membrane large-conductance Ca2+-dependent K+ channel identified a pore-forming alpha subunit and an auxiliary beta2 subunit of the channel in potato tuber mitochondrial inner membrane. These results suggest that a large-conductance calcium-activated potassium channel similar to that of mammalian mitochondria is present in potato tuber mitochondria.

  12. Activation of olfactory-type cyclic nucleotide-gated channels is highly cooperative

    PubMed Central

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

    2005-01-01

    Cyclic nucleotide-gated (CNG) ion channels play a key role in the sensory transduction of vision and olfaction. The channels are opened by the binding of cyclic nucleotides. Native olfactory CNG channels are heterotetramers of CNGA2, CNGA4, and CNGB1b subunits. Upon heterologous expression, only CNGA2 subunits can form functional homotetrameric channels. It is presently not known how the binding of the ligands to the four subunits is translated to channel opening. We studied activation of olfactory CNG channels by photolysis-induced jumps of cGMP or cAMP, two cyclic nucleotides with markedly different apparent affinity. It is shown that at equal degree of activation, the activation time course of homotetrameric channels is similar with cGMP and cAMP and it is also similar in homo- and heterotetrameric channels with the same cyclic nucleotide. Kinetic models were globally fitted to activation time courses of homotetrameric channels. While all models containing equivalent binding sites failed, a model containing three binding sites with a ligand affinity high–low–high described the data adequately. Only the second binding step switches from a very low to a very high open probability. We propose a unique gating mechanism for homotetrameric and heterotetrameric channels that involves only three highly cooperative binding steps. PMID:16081488

  13. Cell membrane stretch activates intermediate-conductance Ca2+-activated K+ channels in arterial smooth muscle cells.

    PubMed

    Hayabuchi, Yasunobu; Nakaya, Yutaka; Mawatari, Kazuaki; Inoue, Miki; Sakata, Miho; Kagami, Shoji

    2011-01-01

    The aim of this study is to determine the signal transduction of membrane stretch on intermediate-conductance Ca(2+)-activated K(+) (IKca) channels in rat aorta smooth muscle cells using the patch-clamp technique. To stretch the cell membrane, both suction to the rear end of patch pipette and hypotonic shock were used. In cell-attached and inside-out patch configurations, the open probability of IKca channels increased when 20- to 45-mmHg suction was applied. Hyposmotic swelling efficiently increased IKca channel current. When the Ca(2+)-free solution was superfused, the activation of IKca current by the hyposmotic swelling was reduced. Furthermore, gadolinium (Gd(3+)) attenuated the activation of IKca channels induced by hyposmotic swelling, whereas nicardipine did not. In the experiments with Ca(2+)-free bath solution, pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely abolished the stretch-induced activation of IKca currents. The stretch-induced activation of IKca channels was strongly inhibited by cytochalasin D, indicating a role for the F-actin in modulation of IKca channels by changes in cell stretching. These data suggest that cell membrane stretch activates IKca channels. In addition, the activation is associated with extracellular Ca(2+) influx through stretch-activated nonselective cation channels, and is also modulated by the F-actin cytoskeleton and the activation of PKC.

  14. Activation of potassium channels in erythrocytes of marine teleost Scorpaena porcus.

    PubMed

    Silkin, Y A; Silkina, E N; Sherstobitov, A O; Gusev, G P

    2001-01-01

    To assess the possibility of stimulating Ca2+-activated K+ channels, marine fish erythrocytes were incubated at 20-22 degrees C in saline containing a Ca2+-ATPase inhibitor (orthovanadate), a Ca2+ ionophore (A23187), propranolol or Pb2+. Incubation of the cells for up to 2 h under control conditions or in the presence of 5 mM NH4VO3 and 1 mM Ca2+ did not affect the intracellular K+ and Na+ concentrations. About 50% cellular K+ was lost from erythrocytes incubated in the presence of 0.01 mM A23187, 1 mM EGTA and 0.4-1.0 mM Ca2+. There was a significant loss of cellular K+ after the addition of 0.05-0.2 mM propranolol to the incubation medium. The stimulatory effect of propranolol on the K+ efflux was independent of external Ca2+. Blockers of Ca2+ transport, verapamil and Co2+, caused only a small decrease in the K+ loss induced by propranolol. The treatment of erythrocytes with 1-2 microM Pb2+ led to a minor K+ loss, but at a Pb2+ concentration of 20-50 microM, about 70% cellular K+ was lost. The K+ efflux induced by propranolol or Pb2+ was completely blocked by 1 mM quinine. The induced K+ loss from the erythrocytes was accompanied by a slight increase in the intracellular Na+ concentration. These data indicate the possibility of inducing Ca2+- and Pb2+-activated potassium channels in erythrocytes of S. porcus. A distinctive feature of the cells is a high sensitivity to propranolol, which activates K+ channels in the absence of external Ca2+.

  15. Rare Mutations of CACNB2 Found in Autism Spectrum Disease-Affected Families Alter Calcium Channel Function

    PubMed Central

    Breitenkamp, Alexandra F. S.; Matthes, Jan; Nass, Robert Daniel; Sinzig, Judith; Lehmkuhl, Gerd; Nürnberg, Peter; Herzig, Stefan

    2014-01-01

    Autism Spectrum Disorders (ASD) are complex neurodevelopmental diseases clinically defined by dysfunction of social interaction. Dysregulation of cellular calcium homeostasis might be involved in ASD pathogenesis, and genes coding for the L-type calcium channel subunits CaV1.2 (CACNA1C) and CaVβ2 (CACNB2) were recently identified as risk loci for psychiatric diseases. Here, we present three rare missense mutations of CACNB2 (G167S, S197F, and F240L) found in ASD-affected families, two of them described here for the first time (G167S and F240L). All these mutations affect highly conserved regions while being absent in a sample of ethnically matched controls. We suggest the mutations to be of physiological relevance since they modulate whole-cell Ba2+ currents through calcium channels when expressed in a recombinant system (HEK-293 cells). Two mutations displayed significantly decelerated time-dependent inactivation as well as increased sensitivity of voltage-dependent inactivation. In contrast, the third mutation (F240L) showed significantly accelerated time-dependent inactivation. By altering the kinetic parameters, the mutations are reminiscent of the CACNA1C mutation causing Timothy Syndrome, a Mendelian disease presenting with ASD. In conclusion, the results of our first-time biophysical characterization of these three rare CACNB2 missense mutations identified in ASD patients support the hypothesis that calcium channel dysfunction may contribute to autism. PMID:24752249

  16. Transcainide causes two modes of open-channel block with different voltage sensitivities in batrachotoxin-activated sodium channels.

    PubMed Central

    Zamponi, G W; French, R J

    1994-01-01

    Transcainide, a complex derivative of lidocaine, blocks the open state of BTX-activated sodium channels from bovine heart and rat skeletal muscle in two distinct ways. When applied to either side of the membrane, transcainide caused discrete blocking events a few hundred milliseconds in duration (slow block), and a concomitant reduction in apparent single-channel amplitude, presumably because of rapid block beyond the temporal resolution of our recordings (fast block). We quantitatively analyzed block from the cytoplasmic side. Both modes of block occurred via binding of the drug to the open channel, approximately followed 1:1 stoichiometry, and were similar for both channel subtypes. For slow block, the blocking rate increased, and the unblocking rate decreased with depolarization, yielding an overall enhancement of block at positive potentials, and suggesting a blocking site at an apparent electrical distance about 45% of the way from the cytoplasmic end of the channel (z delta approximately 0.45). In contrast, the fast blocking mode was only slightly enhanced by depolarization (z delta approximately 0.15). Phenomenologically, the bulky and complex transcainide molecule combines the almost voltage-insensitive blocking action of phenylhydrazine (Zamponi and French, 1994a (companion paper)) with a slow open-channel blocking action that shows a voltage dependence typical of simpler amines. Only the slower blocking mode was sensitive to the removal of external sodium ions, suggesting that the two types of block occur at distinct sites. Dose-response relations were also consistent with independent binding of transcainide to two separate sites on the channel. PMID:7811913

  17. Peptide fragments of the dihydropyridine receptor can modulate cardiac ryanodine receptor channel activity and sarcoplasmic reticulum Ca2+ release.

    PubMed Central

    Dulhunty, Angela F; Curtis, Suzanne M; Cengia, Louise; Sakowska, Magdalena; Casarotto, Marco G

    2004-01-01

    We show that peptide fragments of the dihydropyridine receptor II-III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793-Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671-Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at > or = 10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 microM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations >1 mM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II-III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers. PMID:14678014

  18. Computational modeling of anoctamin 1 calcium-activated chloride channels as pacemaker channels in interstitial cells of Cajal

    PubMed Central

    Gibbons, Simon J.; Farrugia, Gianrico; Sneyd, James; Cheng, Leo K.

    2014-01-01

    Interstitial cells of Cajal (ICC) act as pacemaker cells in the gastrointestinal tract by generating electrical slow waves to regulate rhythmic smooth muscle contractions. Intrinsic Ca2+ oscillations in ICC appear to produce the slow waves by activating pacemaker currents, currently thought to be carried by the Ca2+-activated Cl− channel anoctamin 1 (Ano1). In this article we present a novel model of small intestinal ICC pacemaker activity that incorporates store-operated Ca2+ entry and a new model of Ano1 current. A series of simulations were carried out with the ICC model to investigate current controversies about the reversal potential of the Ano1 Cl− current in ICC and to predict the characteristics of the other ion channels that are necessary to generate slow waves. The model results show that Ano1 is a plausible pacemaker channel when coupled to a store-operated Ca2+ channel but suggest that small cyclical depolarizations may still occur in ICC in Ano1 knockout mice. The results predict that voltage-dependent Ca2+ current is likely to be negligible during the slow wave plateau phase. The model shows that the Cl− equilibrium potential is an important modulator of slow wave morphology, highlighting the need for a better understanding of Cl− dynamics in ICC. PMID:24481603

  19. A cyclic model for bimodal activation of calcium activated potassium channels in radish vacuoles.

    PubMed

    Carpaneto, A

    2001-01-01

    This paper presents the mathematical framework of a cyclic model proposed for describing the transition between a fast and a slow mode (fast-slow effect) induced by the application of step membrane potentials to ion channels from radish vacuoles. A voltage stimulation pulse with frequency in the range of 2 Hz or higher increased the activation time (slow mode) of the recorded currents. When the frequency of the stimulation pattern was restored to 0.1 Hz the activation time decreased twofold (fast mode). This experimental result cannot be explained by classical kinetic theory. The model, based on a simple extension of the Hodgkin and Huxley chain, describes the whole current experimental data and provides hints on the structural conformation of ion channels.

  20. 16 CFR 801.3 - Activities in or affecting commerce.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 1 2014-01-01 2014-01-01 false Activities in or affecting commerce. 801.3 Section 801.3 Commercial Practices FEDERAL TRADE COMMISSION RULES, REGULATIONS, STATEMENTS AND INTERPRETATIONS UNDER THE HART-SCOTT-RODINO ANTITRUST IMPROVEMENTS ACT OF 1976 COVERAGE RULES § 801.3...

  1. 16 CFR 801.3 - Activities in or affecting commerce.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 16 Commercial Practices 1 2012-01-01 2012-01-01 false Activities in or affecting commerce. 801.3 Section 801.3 Commercial Practices FEDERAL TRADE COMMISSION RULES, REGULATIONS, STATEMENTS AND INTERPRETATIONS UNDER THE HART-SCOTT-RODINO ANTITRUST IMPROVEMENTS ACT OF 1976 COVERAGE RULES § 801.3...

  2. 16 CFR 801.3 - Activities in or affecting commerce.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Activities in or affecting commerce. 801.3 Section 801.3 Commercial Practices FEDERAL TRADE COMMISSION RULES, REGULATIONS, STATEMENTS AND INTERPRETATIONS UNDER THE HART-SCOTT-RODINO ANTITRUST IMPROVEMENTS ACT OF 1976 COVERAGE RULES § 801.3...

  3. 16 CFR 801.3 - Activities in or affecting commerce.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 16 Commercial Practices 1 2013-01-01 2013-01-01 false Activities in or affecting commerce. 801.3 Section 801.3 Commercial Practices FEDERAL TRADE COMMISSION RULES, REGULATIONS, STATEMENTS AND INTERPRETATIONS UNDER THE HART-SCOTT-RODINO ANTITRUST IMPROVEMENTS ACT OF 1976 COVERAGE RULES § 801.3...

  4. 16 CFR 801.3 - Activities in or affecting commerce.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Activities in or affecting commerce. 801.3 Section 801.3 Commercial Practices FEDERAL TRADE COMMISSION RULES, REGULATIONS, STATEMENTS AND INTERPRETATIONS UNDER THE HART-SCOTT-RODINO ANTITRUST IMPROVEMENTS ACT OF 1976 COVERAGE RULES § 801.3...

  5. Monitoring Affect States during Effortful Problem Solving Activities

    ERIC Educational Resources Information Center

    D'Mello, Sidney K.; Lehman, Blair; Person, Natalie

    2010-01-01

    We explored the affective states that students experienced during effortful problem solving activities. We conducted a study where 41 students solved difficult analytical reasoning problems from the Law School Admission Test. Students viewed videos of their faces and screen captures and judged their emotions from a set of 14 states (basic…

  6. Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst

    SciTech Connect

    Ahluwalia, Jatinder

    2008-10-31

    Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I{sub e}. In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 {mu}M), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.

  7. Dual effects of microwaves on single Ca(2+)-activated K+ channels in cultured kidney cells Vero.

    PubMed

    Geletyuk, V I; Kazachenko, V N; Chemeris, N K; Fesenko, E E

    1995-02-06

    Using the patch voltage-clamp method, possible effects of millimetre microwaves (42.25 GHz) on single Ca(2+)-activated K+ channels in cultured kidney cells (Vero) were investigated. It was found that exposure to the field of non-thermal power (about 100 microW/cm2) for 20-30 min greatly modifies both the Hill coefficient and an apparent affinity of the channels for Ca2+(i). The data suggest that the field alters both cooperativity and binding characteristics of the channel activation by internal Ca2+. The effects depend on initial sensitivity of the channels to Ca2+ and the Ca2+ concentration applied.

  8. A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels, an arachidonate-regulated store-independent Orai channel.

    PubMed

    Thompson, Jill L; Shuttleworth, Trevor J

    2012-01-01

    The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.

  9. Role of Calcium-activated Potassium Channels in Atrial Fibrillation Pathophysiology and Therapy

    PubMed Central

    Diness, Jonas G.; Bentzen, Bo H.; Sørensen, Ulrik S.

    2015-01-01

    Abstract: Small-conductance Ca2+-activated potassium (SK) channels are relative newcomers within the field of cardiac electrophysiology. In recent years, an increased focus has been given to these channels because they might constitute a relatively atrial-selective target. This review will give a general introduction to SK channels followed by their proposed function in the heart under normal and pathophysiological conditions. It is revealed how antiarrhythmic effects can be obtained by SK channel inhibition in a number of species in situations of atrial fibrillation. On the contrary, the beneficial effects of SK channel inhibition in situations of heart failure are questionable and still needs investigation. The understanding of cardiac SK channels is rapidly increasing these years, and it is hoped that this will clarify whether SK channel inhibition has potential as a new anti–atrial fibrillation principle. PMID:25830485

  10. Activation of single cardiac and skeletal ryanodine receptor channels by flash photolysis of caged Ca2+.

    PubMed Central

    Györke, S; Vélez, P; Suárez-Isla, B; Fill, M

    1994-01-01

    Single ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels isolated from rabbit skeletal and canine cardiac muscle were reconstituted in planar lipid bilayers. Single channel activity was measured in simple solutions (no ATP or Mg2+) with 250 mM symmetrical Cs+ as charge carrier. A laser flash was used to photolyze caged-Ca2+ (DM-nitrophen) in a small volume directly in front of the bilayer. The free [Ca2+] in this small volume and in the bulk solution was monitored with Ca2+ electrodes. This setup allowed fast, calibrated free [Ca2+] stimuli to be applied repetitively to single SR Ca2+ release channels. A standard photolytically induced free [Ca2+] step (pCa 7-->6) was applied to both the cardiac and skeletal release channels. The rate of channel activation was determined by fitting a single exponential to ensemble currents generated from at least 50 single channel sweeps. The time constants of activation were 1.43 +/- 0.65 ms (mean +/- SD; n = 5) and 1.28 +/- 0.61 ms (n = 5) for cardiac and skeletal channels, respectively. This study presents a method for defining the fast Ca2+ regulation kinetics of single SR Ca2+ release channels and shows that the activation rate of skeletal SR Ca2+ release channels is consistent with a role for CICR in skeletal muscle excitation-contraction coupling. PMID:8075325

  11. Effect of trimethyllead chloride on slowly activating (SV) channels in red beet (Beta vulgaris L.) taproots.

    PubMed

    Trela, Zenon; Burdach, Zbigniew; Przestalski, Stanisław; Karcz, Waldemar

    2012-12-01

    The patch-clamp technique was used to examine the effect of trimethyllead chloride (Met(3)PbCl) on SV channel activity in red beet (Beta vulgaris L.) taproot vacuoles. It was found that in the control bath the macroscopic currents showed the typical slow activation and a strong outward rectification of the steady-state currents. An addition of Met(3)PbCl to the bath solution blocked, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant τ increased several times in the presence of 100 μM trimethyllead chloride at all voltages tested. When single channel properties were analyzed, only little channel activity could be recorded in the presence of 100 μM Met(3)PbCl. Trimethyllead chloride decreased significantly (by about one order of magnitude) the open probability of single channels. The recordings of single channel activity obtained in the presence and absence of Met(3)PbCl showed that organolead only slightly (by ca. 10%) decreased the unitary conductance of single channels. It was also found that Met(3)PbCl diminished significantly the number of SV channel openings, whereas it did not change the opening times of the channels. Taken together, these results suggest that Met(3)PbCl binding site is located outside the channel selectivity filter.

  12. Effects of cytoplasmic Mg2+ on slowly activating channels in isolated vacuoles of Beta vulgaris.

    PubMed

    Carpaneto, A; Cantù, A M; Gambale, F

    2001-07-01

    The slow vacuolar (SV) channel can mediate a large part of the ionic current in plant tonoplasts, but its actual physiological role is still unclear. We demonstrate that in vacuoles from the taproots of sugar beet (Beta vulgaris L.), besides Ca2+, cytoplasmic Mg2+ also plays an important role in promoting the activation of the SV channel. An increase in Mg2+ concentration decreases the time constants of channel activation and deactivation, and determines a consistent shift, towards negative voltages, of the conductance characteristic; as an example, when the free concentration of Mg2+ was increased from the micromolar range up to 10 mM the activation shifted by about -60 mV. The experimental results obtained, which are based on a fast perfusion procedure allowing us to change the solution bathing the vacuole in a few milliseconds, suggest that magnesium-binding is a faster process than the voltage-activation gating of the channel, which constitutes the rate-limiting step controlling channel opening. Interestingly, the activation of the channel mediated by Mg2+ depends on the cooperative binding of at least three magnesium ions. We verified that cytoplasmic magnesium favours the activation of SV channels in the presence of nanomolar cytoplasmic calcium concentrations. A critical discussion on the Calcium Induced Calcium Release (CICR) mechanism proposed for the SV channel is presented.

  13. Brain-derived neurotrophic factor inhibits calcium channel activation, exocytosis, and endocytosis at a central nerve terminal.

    PubMed

    Baydyuk, Maryna; Wu, Xin-Sheng; He, Liming; Wu, Ling-Gang

    2015-03-18

    Brain-derived neurotrophic factor (BDNF) is a neurotrophin that regulates synaptic function and plasticity and plays important roles in neuronal development, survival, and brain disorders. Despite such diverse and important roles, how BDNF, or more generally speaking, neurotrophins affect synapses, particularly nerve terminals, remains unclear. By measuring calcium currents and membrane capacitance during depolarization at a large mammalian central nerve terminal, the rat calyx of Held, we report for the first time that BDNF slows down calcium channel activation, including P/Q-type channels, and inhibits exocytosis induced by brief depolarization or single action potentials, inhibits slow and rapid endocytosis, and inhibits vesicle mobilization to the readily releasable pool. These presynaptic mechanisms may contribute to the important roles of BDNF in regulating synapses and neuronal circuits and suggest that regulation of presynaptic calcium channels, exocytosis, and endocytosis are potential mechanisms by which neurotrophins achieve diverse neuronal functions.

  14. Ion Channel Activity of Vpu Proteins Is Conserved throughout Evolution of HIV-1 and SIV

    PubMed Central

    Greiner, Timo; Bolduan, Sebastian; Hertel, Brigitte; Groß, Christine; Hamacher, Kay; Schubert, Ulrich; Moroni, Anna; Thiel, Gerhard

    2016-01-01

    The human immunodeficiency virus type 1 (HIV-1) protein Vpu is encoded exclusively by HIV-1 and related simian immunodeficiency viruses (SIVs). The transmembrane domain of the protein has dual functions: it counteracts the human restriction factor tetherin and forms a cation channel. Since these two functions are causally unrelated it remains unclear whether the channel activity has any relevance for viral release and replication. Here we examine structure and function correlates of different Vpu homologs from HIV-1 and SIV to understand if ion channel activity is an evolutionary conserved property of Vpu proteins. An electrophysiological testing of Vpus from different HIV-1 groups (N and P) and SIVs from chimpanzees (SIVcpz), and greater spot-nosed monkeys (SIVgsn) showed that they all generate channel activity in HEK293T cells. This implies a robust and evolutionary conserved channel activity and suggests that cation conductance may also have a conserved functional significance. PMID:27916968

  15. Cross talk between activation and slow inactivation gates of Shaker potassium channels.

    PubMed

    Panyi, Gyorgy; Deutsch, Carol

    2006-11-01

    This study addresses the energetic coupling between the activation and slow inactivation gates of Shaker potassium channels. To track the status of the activation gate in inactivated channels that are nonconducting, we used two functional assays: the accessibility of a cysteine residue engineered into the protein lining the pore cavity (V474C) and the liberation by depolarization of a Cs(+) ion trapped behind the closed activation gate. We determined that the rate of activation gate movement depends on the state of the inactivation gate. A closed inactivation gate favors faster opening and slower closing of the activation gate. We also show that hyperpolarization closes the activation gate long before a channel recovers from inactivation. Because activation and slow inactivation are ubiquitous gating processes in potassium channels, the cross talk between them is likely to be a fundamental factor in controlling ion flux across membranes.

  16. Flavaglines Stimulate Transient Receptor Potential Melastatin Type 6 (TRPM6) Channel Activity

    PubMed Central

    Verkaart, Sjoerd A. J.; Lameris, Anke L.; Basmadjian, Christine; Zhao, Qian; Désaubry, Laurent; Bindels, René J. M.; Hoenderop, Joost G. J.

    2015-01-01

    Magnesium (Mg2+) is essential for enzymatic activity, brain function and muscle contraction. Blood Mg2+ concentrations are tightly regulated between 0.7 and 1.1 mM by Mg2+ (re)absorption in kidney and intestine. The apical entry of Mg2+ in (re)absorbing epithelial cells is mediated by the transient receptor potential melastatin type 6 (TRPM6) ion channel. Here, flavaglines are described as a novel class of stimulatory compounds for TRPM6 activity. Flavaglines are a group of natural and synthetic compounds that target the ubiquitously expressed prohibitins and thereby affect cellular signaling. By whole-cell patch clamp analyses, it was demonstrated that nanomolar concentrations of flavaglines increases TRPM6 activity by ∼2 fold. The stimulatory effects were dependent on the presence of the alpha-kinase domain of TRPM6, but did not require its phosphotransferase activity. Interestingly, it was observed that two natural occurring TRPM6 mutants with impaired insulin-sensitivity, TRPM6-p.Val1393Ile and TRPM6-p.Lys1584Glu, are not sensitive to flavagline stimulation. In conclusion, we have identified flavaglines as potent activators of TRPM6 activity. Our results suggest that flavaglines stimulate TRPM6 via the insulin receptor signaling pathway. PMID:25774985

  17. Flavaglines Stimulate Transient Receptor Potential Melastatin Type 6 (TRPM6) Channel Activity.

    PubMed

    Blanchard, Maxime G; de Baaij, Jeroen H F; Verkaart, Sjoerd A J; Lameris, Anke L; Basmadjian, Christine; Zhao, Qian; Désaubry, Laurent; Bindels, René J M; Hoenderop, Joost G J

    2015-01-01

    Magnesium (Mg2+) is essential for enzymatic activity, brain function and muscle contraction. Blood Mg2+ concentrations are tightly regulated between 0.7 and 1.1 mM by Mg2+ (re)absorption in kidney and intestine. The apical entry of Mg2+ in (re)absorbing epithelial cells is mediated by the transient receptor potential melastatin type 6 (TRPM6) ion channel. Here, flavaglines are described as a novel class of stimulatory compounds for TRPM6 activity. Flavaglines are a group of natural and synthetic compounds that target the ubiquitously expressed prohibitins and thereby affect cellular signaling. By whole-cell patch clamp analyses, it was demonstrated that nanomolar concentrations of flavaglines increases TRPM6 activity by ∼2 fold. The stimulatory effects were dependent on the presence of the alpha-kinase domain of TRPM6, but did not require its phosphotransferase activity. Interestingly, it was observed that two natural occurring TRPM6 mutants with impaired insulin-sensitivity, TRPM6-p.Val1393Ile and TRPM6-p.Lys1584Glu, are not sensitive to flavagline stimulation. In conclusion, we have identified flavaglines as potent activators of TRPM6 activity. Our results suggest that flavaglines stimulate TRPM6 via the insulin receptor signaling pathway.

  18. Mechanosensitive channel activation by diffusio-osmotic force.

    PubMed

    Bonthuis, Douwe Jan; Golestanian, Ramin

    2014-10-03

    For ion channel gating, the appearance of two distinct conformational states and the discrete transitions between them are essential, and therefore of crucial importance to all living organisms. We show that the physical interplay between two structural elements that are commonly present in bacterial mechanosensitive channels--namely, a charged vestibule and a hydrophobic constriction--creates two distinct conformational states, open and closed, as well as the gating between them. We solve the nonequilibrium Stokes-Poisson-Nernst-Planck equations, extended to include a molecular potential of mean force, and show that a first order transition between the closed and open states arises naturally from the diffusio-osmotic stress caused by the ions and the water inside the channel and the elastic restoring force from the membrane.

  19. Three-dimensional flow dynamics of an active submarine channel

    NASA Astrophysics Data System (ADS)

    Sumner, E. J.; Dorrell, R. M.; Peakall, J.; Darby, S. E.; Parsons, D. R.; Wynn, R.

    2012-12-01

    Field scale submarine channel gravity currents are notoriously difficult to measure and thus directly investigate due to their inaccessible location and infrequent nature, which is compounded by present sea-level high-stand. An exception to this is the almost continuous density-driven current that results from the inflow of saline Mediterranean water, via the Bosporus strait, into the Black Sea. This flow has carved a sinuous channel system in water depths of 70 to 120 m. The relatively shallow depths of the channel and the continuous nature of this current provide a rare opportunity to study three-dimensional flow dynamics and the interaction of the flow with a seafloor channel network. Thus, it provides a rare analogue for channelized dilute sediment-laden turbidity currents. Sediment erosion, transport and deposition within submarine channel bends is primarily controlled by the magnitude and direction of near bed flow. Flow around channel bends is characterized by a helical or spiralling structure. In rivers this helical flow is characterized by near-surface fluid moving toward the outer bank and near-bed fluid moving toward the inner bank. Following fierce debate over the last decade, it is now accepted that helical flow in submarine channel bends can display a variety of complex structures. Most importantly for understanding sediment transport, near bed flow can be directed towards the outer bank, which is in the opposite sense to in a river. The next challenge is to understand what the exact controls on the orientation of helical flow cells within submarine flows are, and their spatial evolution around bends. We present data from the Black Sea showing how the three-dimensional velocity and density of a submarine gravity current evolves at multiple cross sections as the flow travels around a bend. We use this data to calculate the magnitude, relative importance and interaction of centrifugal, coriolis and pressure gradients in controlling the structure of

  20. Ferroelectric active models of ion channels in biomembranes.

    PubMed

    Bystrov, V S; Lakhno, V D; Molchanov, M

    1994-06-21

    Ferroactive models of ion channels in the theory of biological membranes are presented. The main equations are derived and their possible solutions are shown. The estimates of some experimentally measured parameters are given. Possible physical consequences of the suggested models are listed and the possibility of their experimental finding is discussed. The functioning of the biomembrane's ion channel is qualitatively described on the basis of the suggested ferroactive models. The main directions and prospects for development of the ferroactive approach to the theory of biological membranes and their structures are indicated.

  1. Asymmetric frontal cortical activity and negative affective responses to ostracism.

    PubMed

    Peterson, Carly K; Gravens, Laura C; Harmon-Jones, Eddie

    2011-06-01

    Ostracism arouses negative affect. However, little is known about variables that influence the intensity of these negative affective responses. Two studies fill this void by incorporating work on approach- and withdrawal-related emotional states and their associated cortical activations. Study 1 found that following ostracism anger related directly to relative left frontal cortical activation. Study 2 used unilateral hand contractions to manipulate frontal cortical activity prior to an ostracizing event. Right-hand contractions, compared to left-hand contractions, caused greater relative left frontal cortical activation during the hand contractions as well as ostracism. Also, right-hand contractions caused more self-reported anger in response to being ostracized. Within-condition correlations revealed patterns of associations between ostracism-induced frontal asymmetry and emotive responses to ostracism consistent with Study 1. Taken together, these results suggest that asymmetrical frontal cortical activity is related to angry responses to ostracism, with greater relative left frontal cortical activity being associated with increased anger.

  2. CRL4A(CRBN) E3 ubiquitin ligase restricts BK channel activity and prevents epileptogenesis.

    PubMed

    Liu, Jiye; Ye, Jia; Zou, Xiaolong; Xu, Zhenghao; Feng, Yan; Zou, Xianxian; Chen, Zhong; Li, Yuezhou; Cang, Yong

    2014-05-21

    Ion channels regulate membrane excitation, and mutations of ion channels often cause serious neurological disorders including epilepsy. Compared with extensive analyses of channel protein structure and function, much less is known about the fine tuning of channel activity by post-translational modification. Here we report that the large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels are targeted by the E3 ubiquitin ligase CRL4A(CRBN) for polyubiquitination and retained in the endoplasmic reticulum (ER). Inactivation of CRL4A(CRBN) releases deubiquitinated BK channels from the ER to the plasma membrane, leading to markedly enhanced channel activity. Mice with CRL4A(CRBN) mutation in the brain or treated with a CRL4A(CRBN) inhibitor are very sensitive to seizure induction, which can be attenuated by blocking BK channels. Finally, the mutant mice develop spontaneous epilepsy when aged. Therefore, ubiquitination of BK channels before their cell surface expression is an important step to prevent systemic neuronal excitability and epileptogenesis.

  3. Dopamine suppresses neuronal activity of Helisoma B5 neurons via a D2-like receptor, activating PLC and K channels.

    PubMed

    Zhong, L R; Artinian, L; Rehder, V

    2013-01-03

    Dopamine (DA) plays fundamental roles as a neurotransmitter and neuromodulator in the central nervous system. How DA modulates the electrical excitability of individual neurons to elicit various behaviors is of great interest in many systems. The buccal ganglion of the freshwater pond snail Helisoma trivolvis contains the neuronal circuitry for feeding and DA is known to modulate the feeding motor program in Helisoma. The buccal neuron B5 participates in the control of gut contractile activity and is surrounded by dopaminergic processes, which are expected to release DA. In order to study whether DA modulates the electrical activity of individual B5 neurons, we performed experiments on physically isolated B5 neurons in culture and on B5 neurons within the buccal ganglion in situ. We report that DA application elicited a strong hyperpolarization in both conditions and turned the electrical activity from a spontaneously firing state to an electrically silent state. Using the cell culture system, we demonstrated that the strong hyperpolarization was inhibited by the D2 receptor antagonist sulpiride and the phospholipase C (PLC) inhibitor U73122, indicating that DA affected the membrane potential of B5 neurons through the activation of a D2-like receptor and PLC. Further studies revealed that the DA-induced hyperpolarization was inhibited by the K channel blockers 4-aminopyridine and tetraethylammonium, suggesting that K channels might serve as the ultimate target of DA signaling. Through its modulatory effect on the electrical activity of B5 neurons, the release of DA in vivo may contribute to a neuronal output that results in a variable feeding motor program.

  4. The AQP-3 water channel is a pivotal modulator of glycerol-induced chloride channel activation in nasopharyngeal carcinoma cells.

    PubMed

    Zhang, Haifeng; Deng, Zhiqin; Yang, Lili; Luo, Hai; Liu, Shanwen; Li, Yuan; Wei, Yan; Peng, Shuang; Zhu, Linyan; Wang, Liwei; Chen, Lixin

    2016-03-01

    Aquaporin (AQP) and chloride channels are ubiquitous in virtually all living cells, playing pivotal roles in cell proliferation, migration and apoptosis. We previously reported that AQP-3 aquaglyceroporin and ClC-3 chloride channels could form complexes to regulate cell volume in nasopharyngeal carcinoma cells. In this study, the roles of AQP-3 in their hetero-complexes were further investigated. Glycerol entered the cells via AQP-3 and induced two different Cl(-) currents through cell swelling-dependent or -independent pathways. The swelling-dependent Cl(-) current was significantly inhibited by pretreatment with CuCl2 and AQP-3-siRNA. After siRNA-induced AQP-3 knock-down, the 140 mM glycerol isoosmotic solution swelled cells by 22% (45% in AQP-3-intact cells) and induced a smaller Cl(-) current; this current was smaller than that activated by 8% cell volume swelling, which induced by the 140 mM glycerol hyperosmotic solution in AQP-3-intact cells. This suggests that the interaction between AQP-3 and ClC-3 plays an important role in cell volume regulation and that AQP-3 may be a modulator that opens volume-regulated chloride channels. The swelling-independent Cl(-) current, which was activated by extracellular glycerol, was reduced by CuCl2 and AQP-3-siRNA pretreatment. Dialyzing glycerol into cells via the pipette directly induced the swelling-independent Cl(-) current; however this current was blocked by AQP-3 down-regulation, suggesting AQP-3 is essential for the opening of chloride channels. In conclusion, AQP-3 is the pathway for water, glycerol and other small solutes to enter cells, and it may be an essential modulator for the gating of chloride channels.

  5. Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

    PubMed Central

    Larsen, EH; Gabriei, SE; Stutts, MJ; Fullton, J; Price, EM; Boucher, RC

    1996-01-01

    The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1

  6. Membrane-pipette interactions underlie delayed voltage activation of mechanosensitive channels in Xenopus oocytes.

    PubMed Central

    Gil, Z; Magleby, K L; Silberberg, S D

    1999-01-01

    To investigate the mechanism for the delayed activation by voltage of the predominant mechanosensitive (MS) channel in Xenopus oocytes, currents were recorded from on-cell and excised patches of membrane with the patch clamp technique and from intact oocytes with the two-electrode voltage clamp technique. MS channels could be activated by stretch in inside-out, on-cell, and outside-out patch configurations, using pipettes formed of either borosilicate or soft glass. In inside-out patches formed with borosilicate glass pipettes, depolarizing voltage steps activated MS channels in a cooperative manner after delays of seconds. This voltage-dependent activation was not observed for outside-out patches. Voltage-dependent activation was also not observed when the borosilicate pipettes were either replaced with soft glass pipettes or coated with soft glass. When depolarizing voltage steps were applied to the whole oocyte with a two-electrode voltage clamp, currents that could be attributed to MS channels were not observed. Yet the same depolarizing steps activated MS channels in on-cell patches formed with borosilicate pipettes on the same oocyte. These observations suggest that the delayed cooperative activation of MS channels by depolarization is not an intrinsic property of the channels, but requires interaction between the membrane and patch pipette. PMID:10354436

  7. 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

  8. How does the anthropogenic activity affect the spring discharge?

    NASA Astrophysics Data System (ADS)

    Hao, Yonghong; Zhang, Juan; Wang, Jiaojiao; Li, Ruifang; Hao, Pengmei; Zhan, Hongbin

    2016-09-01

    Karst hydrological process has largely been altered by climate change and human activity. In many places throughout the world, human activity (e.g. groundwater pumping and dewatering from mining) has intensified and surpassed climate change, where human activity becomes the primary factor that affects groundwater system. But it is still largely unclear how the human activity affects spring discharge in magnitude and periodicity. This study investigates the effects of anthropogenic activity on spring discharge, using the Xin'an Springs of China as an example. The Xin'an Spring discharge were divided into two time periods: the pre-development period from 1956 to 1971 and the post-development period from 1972 to 2013. We confirm the dividing time (i.e. 1971) of these two periods using the Wilcoxon rank-sum test. Then the wavelet transform and wavelet coherence were used to analyze the karst hydrological processes for the two periods respectively. We analyze the correlations of precipitation and the Xin'an spring discharge with the monsoons including the Indian Summer Monsoon (ISM) and the West North Pacific Monsoon (WNPM) and the climate teleconnections including El Niño Southern Oscillation (ENSO) and Pacific Decadal Oscillation (PDO), respectively. The results indicated that the spring discharge was attenuated about 19.63% under the influence of human activity in the Xin'an Springs basin. However, human activity did not alter the size of the resonance frequencies between the spring discharge and the monsoons. In contrast, it reinforced the periodicities of the monsoons-driven spring discharge. It suggested that human has adapted to the major climate periodicities, and human activity had the same rhyme with the primary climate periodicity. In return, human activity enhances the correlation between the monsoons and the spring discharge.

  9. Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel

    PubMed Central

    Wu, Shengjun; Gao, Weihua; Xie, Changan; Xu, Xinping; Vorvis, Christina; Marni, Farzana; Hackett, Amber R.; Liu, Qinglian

    2012-01-01

    Recently, applications of the patch-clamp fluorometry (PCF) technique in studies of cyclic nucleotide–gated (CNG) and hyperpolarization-activated, cyclic nucleotide–regulated (HCN) channels have provided direct evidence for the long-held notion that ligands preferably bind to and stabilize these channels in an open state. This state-dependent ligand–channel interaction involves contributions from not only the ligand-binding domain but also other discrete structural elements within the channel protein. This insight led us to investigate whether the pore of the HCN channel plays a role in the ligand–whole channel interaction. We used three well-characterized HCN channel blockers to probe the ion-conducting passage. The PCF technique was used to simultaneously monitor channel activity and cAMP binding. Two ionic blockers, Cs+ and Mg2+, effectively block channel conductance but have no obvious effect on cAMP binding. Surprisingly, ZD7288, an open channel blocker specific for HCN channels, significantly reduces the activity-dependent increase in cAMP binding. Independent biochemical assays exclude any nonspecific interaction between ZD7288 and isolated cAMP-binding domain. Because ZD7228 interacts with the inner pore region, where the activation gate is presumably located, we did an alanine scanning of the intracellular end of S6, from T426 to A435. Mutations of three residues, T426, M430, and H434, which are located at regular intervals on the S6 α-helix, enhance cAMP binding. In contrast, mutations of two residues in close proximity, F431A and I432A, dampen the response. Our results demonstrate that movements of the structural elements near the activation gate directly affect ligand binding affinity, which is a simple mechanistic explanation that could be applied to the interpretation of ligand gating in general. PMID:22689828

  10. A pervasive mechanism for analgesia: activation of GIRK2 channels.

    PubMed

    Blednov, Y A; Stoffel, M; Alva, H; Harris, R A

    2003-01-07

    G protein-coupled inwardly rectifying potassium channels (GIRKs) provide a common link between numerous neurotransmitter receptors and the regulation of synaptic transmission. We asked whether GIRKs specify a single behavioral action that is produced by drugs acting on the diverse receptors coupled with GIRKs. By using GIRK2-null mutant mice, we found marked reduction or complete elimination of the antinociceptive (hot plate test) effects of ethanol, oxotremorine, nicotine, baclofen, clonidine, and the cannabinoid receptor agonist WIN 55,212. However, ketamine analgesia remained intact. For most drugs, there was a sex difference in antinociceptive action, and the impact of deletion of the GIRK2 channel was less in female mice. The deletion of the GIRK2 channel blocks the opioid-dependent component of stress-induced analgesia (SIA), whereas nonopioid SIA was not changed. We propose that opioid, alpha adrenergic, muscarinic cholinergic, gamma-aminobutyric acid-B, and cannabinoid receptors are coupled with postsynaptic GIRK2 channels in vivo. Furthermore, this pathway accounts for essentially all of the antinociceptive effects in males, although females appear to recruit additional signal transduction mechanisms for some analgesic drugs.

  11. BK channel activity determines the extent of cell degeneration after oxygen and glucose deprivation: a study in organotypical hippocampal slice cultures.

    PubMed

    Rundén-Pran, E; Haug, F M; Storm, J F; Ottersen, O P

    2002-01-01

    BK channels are voltage- and calcium-dependent potassium channels whose activation tends to reduce cellular excitability. In hippocampal pyramidal cells, BK channels repolarize somatic action potentials, and recent immunogold and electrophysiological analyses have revealed a presynaptic pool of BK channels that can regulate glutamate release. Agents that modulate BK channel activity would therefore be expected to affect cell excitability and neurotransmitter release also under pathological conditions. We have investigated the role of BK potassium channels in a model of ischemia-induced nerve cell degeneration. Organotypical slice cultures of rat hippocampus were exposed to oxygen and glucose deprivation (OGD), and cell death was assessed by the fluorescent dye propidium iodide. OGD induced cell death in the CA1 region and to a lesser extent in CA3. Treatment with the BK channel blockers, paxilline and iberiotoxin, during and after OGD induced increased cell death in CA1 and CA3. Both BK channel blockers also sensitized the relatively resistant granule cells in fascia dentata to OGD. The effect of paxilline and iberiotoxin was evident from 3 h after OGD, indicating a role of BK channels early in the post-ischemic phase or during OGD itself. The BK channel opener, NS1619, turned out to be gliotoxic, and this effect was not counteracted by paxilline and iberiotoxin. Our data show that blockade of BK channels aggravates OGD-induced cell damage and suggest that BK channels act as a kind of 'emergency brake' during and/or after ischemia. Accordingly, the BK channel is a potential molecular target for neuroprotective therapy in stroke.

  12. Fragile X mental retardation protein controls gating of the sodium-activated potassium channel Slack

    PubMed Central

    Brown, Maile R.; Kronengold, Jack; Gazula, Valeswara-Rao; Chen, Yi; Strumbos, John G.; Sigworth, Fred J.; Navaratnam, Dhasakumar; Kaczmarek, Leonard K.

    2010-01-01

    In humans, absence of Fragile X mental retardation protein (FMRP), an RNA-binding protein, results in Fragile X syndrome (FXS), the most common inherited form of intellectual disability. Here we report through biochemical and electrophysiological studies that FMRP binds the C-terminus of the Slack sodium-activated potassium channel to activate the channel. The findings suggest that Slack activity may provide a link between patterns of neuronal firing and changes in protein translation. PMID:20512134

  13. Luminal acetylcholine does not affect the activity of the CFTR in tracheal epithelia of pigs.

    PubMed

    Dittrich, Nikolaus P; Kummer, Wolfgang; Clauss, Wolfgang G; Fronius, Martin

    2015-11-01

    Fluid homeostasis mediated by the airway epithelium is required for proper lung function, and the CFTR (cystic fibrosis transmembrane conductance regulator) Cl(-) channel is crucial for these processes. Luminal acetylcholine (ACh) acts as an auto-/paracrine mediator to activate Cl(-) channels in airway epithelia and evidence exists showing that nicotinic ACh receptors activate CFTR in murine airway epithelia. The present study investigated whether or not luminal ACh regulates CFTR activity in airway epithelia of pigs, an emerging model for investigations of human airway disease and cystic fibrosis (CF) in particular. Transepithelial ion currents of freshly dissected pig tracheal preparations were measured with Ussing chambers. Application of luminal ACh (100 μM) induced an increase of the short-circuit current (I(SC)). The ACh effect was mimicked by muscarine and pilocarpine (100 μM each) and was sensitive to muscarinic receptor antagonists (atropine, 4-DAMP, pirenzepine). No changes of the I(SC) were observed by nicotine (100 μM) and ACh responses were not affected by nicotine or mecamylamine (25 μM). Luminal application of IBMX (I, 100 μM) and forskolin (F, 10 μM), increase the I(SC) and the I/F-induced current were decreased by the CFTR inhibitor GlyH-101 (GlyH, 50 μM) indicating increased CFTR activity by I/F. In contrast, GlyH did not affect the ACh-induced current, indicating that the ACh response does not involve the activation of the CFTR. Results from this study suggest that luminal ACh does not regulate the activity of the CFTR in tracheal epithelia of pigs which opposes observation from studies using mice airway epithelium.

  14. Factors affecting farm noise during common agricultural activities.

    PubMed

    Franklin, R C; Depczynski, J; Challinor, K; Williams, W; Fragar, L J

    2006-05-01

    Hearing injury due to exposure to excessive noise during common farming activities is a significant problem for farmers. The aim of this study was to investigate factors that affect the level of risk to hearing caused by common farming activities. Noise levels on farms were measured across a range of activities and producer groups, and situational factors that effect noise levels were also investigated. Older tractors were found to be 6 dB louder than newer tractors. Cabs reduced noise to the operator by 16 dB, which was halved to 8 dB if a door was open. Radios added between 3 and 5 dB to the noise in the cab. These variables significantly affect the noise level at the ear of operators and others in the workplace, and affect the subsequent exposure limits that are considered safe. Situational factors need to be considered in assessing the level of risk to farmers' hearing and in choosing noise management strategies on the farm. This information has been incorporated into material about hearing and discussions with farmers who participated in field day hearing screening programs in Australia.

  15. Substituted N-(Biphenyl-4′-yl)methyl (R)-2-Acetamido-3-methoxypropionamides: Potent Anticonvulsants That Affect Frequency (Use) Dependence and Slow Inactivation of Sodium Channels

    PubMed Central

    2015-01-01

    We prepared 13 derivatives of N-(biphenyl-4′-yl)methyl (R)-2-acetamido-3-methoxypropionamide that differed in type and placement of a R-substituent in the terminal aryl unit. We demonstrated that the R-substituent impacted the compound’s whole animal and cellular pharmacological activities. In rodents, select compounds exhibited excellent anticonvulsant activities and protective indices (PI = TD50/ED50) that compared favorably with clinical antiseizure drugs. Compounds with a polar, aprotic R-substituent potently promoted Na+ channel slow inactivation and displayed frequency (use) inhibition of Na+ currents at low micromolar concentrations. The possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed. PMID:25004277

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

    PubMed

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

    2013-10-01

    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 Ca(2+)/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 Ca(2+)/calmodulin, one at submicromolar Ca(2+) concentrations and one in the micromolar Ca(2+) 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 Ca(2+)/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca(2+) 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 Ca(2+) regulation in anoctamin Cl(-) channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.

  17. 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

  18. 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.

  19. 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 (Kv 3.1 and Kv 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, Cav 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.

  20. 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.

  1. Small-conductance Ca2+ -activated K+ channels and cardiac arrhythmias.

    PubMed

    Zhang, Xiao-Dong; Lieu, Deborah K; Chiamvimonvat, Nipavan

    2015-08-01

    Small-conductance Ca2+ -activated K+ (SK, KCa2) channels are unique in that they are gated solely by changes in intracellular Ca2+ and, hence, function to integrate intracellular Ca2+ and membrane potentials on a beat-to-beat basis. Recent studies have provided evidence for the existence and functional significance of SK channels in the heart. Indeed, our knowledge of cardiac SK channels has been greatly expanded over the past decade. Interests in cardiac SK channels are further driven by recent studies suggesting the critical roles of SK channels in human atrial fibrillation, the SK channel as a possible novel therapeutic target in atrial arrhythmias, and upregulation of SK channels in heart failure in animal models and in human heart failure. However, there remain critical gaps in our knowledge. Specifically, blockade of SK channels in cardiac arrhythmias has been shown to be both antiarrhythmic and proarrhythmic. This contemporary review provides an overview of the literature on the role of cardiac SK channels in cardiac arrhythmias and serves as a discussion platform for the current clinical perspectives. At the translational level, development of SK channel blockers as a new therapeutic strategy in the treatment of atrial fibrillation and the possible proarrhythmic effects merit further considerations and investigations.

  2. ZD7288 inhibits low-threshold Ca(2+) channel activity and regulates sperm function.

    PubMed

    Felix, Ricardo; Sandoval, Alejandro; Sánchez, Daniel; Gómora, Juan Carlos; De la Vega-Beltrán, José L; Treviño, Claudia L; Darszon, Alberto

    2003-11-07

    In this study, ZD7288, a blocker of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, has been found to inhibit the mouse sperm acrosome reaction (AR). HCN channels have not yet been either recorded or implicated in mouse sperm AR, but low-threshold (T-type) Ca(2+) channels have. Interestingly, ZD7288 blocked native T-type Ca(2+) currents in mouse spermatogenic cells with an IC(50) of about 100 microM. This blockade was more effective at voltages producing low levels of inactivation, suggesting a differential affinity of ZD7288 for different channel conformations. Furthermore, ZD7288 inhibited all cloned T-type but not high-threshold N-type channels heterologously expressed in HEK-293 cells. Our results further support the role of T-type Ca(2+) channels in the mouse sperm AR.

  3. Transient potassium channels augment degeneracy in hippocampal active dendritic spectral tuning

    PubMed Central

    Rathour, Rahul Kumar; Malik, Ruchi; Narayanan, Rishikesh

    2016-01-01

    Hippocampal pyramidal neurons express an intraneuronal map of spectral tuning mediated by hyperpolarization-activated cyclic-nucleotide-gated nonspecific-cation channels. Modeling studies have predicted a critical regulatory role for A-type potassium (KA) channels towards augmenting functional robustness of this map. To test this, we performed patch-clamp recordings from soma and dendrites of rat hippocampal pyramidal neurons, and measured spectral tuning before and after blocking KA channels using two structurally distinct pharmacological agents. Consistent with computational predictions, we found that blocking KA channels resulted in a significant reduction in resonance frequency and significant increases in input resistance, impedance amplitude and action-potential firing frequency across the somato-apical trunk. Furthermore, across all measured locations, blocking KA channels enhanced temporal summation of postsynaptic potentials and critically altered the impedance phase profile, resulting in a significant reduction in total inductive phase. Finally, pair-wise correlations between intraneuronal percentage changes (after blocking KA channels) in different measurements were mostly weak, suggesting differential regulation of different physiological properties by KA channels. Our results unveil a pivotal role for fast transient channels in regulating theta-frequency spectral tuning and intrinsic phase response, and suggest that degeneracy with reference to several coexisting functional maps is mediated by cross-channel interactions across the active dendritic arbor. PMID:27094086

  4. The expression and regulation of depolarization-activated K+ channels in the insulin-secreting cell line INS-1.

    PubMed

    Su, J; Yu, H; Lenka, N; Hescheler, J; Ullrich, S

    2001-04-01

    The aim of the present study was to characterize depolarization-activated outward currents in insulin-secreting INS-1 cells and to investigate the role of K+ channels other than the KATP channels in the regulation of insulin release. Outward currents were inhibited by 4-aminopyridine (4-AP, 10 mmol/l), tetraethylammonium (TEA, 10 mmol/l) and tetrapentylammonium (TPeA, 100 mumol/l) by 55.1 +/- 3.8% (n = 3), 78.1 +/- 3.2% (n = 6) and 98.7 +/- 0.8% (n = 5), respectively. Margatoxin (5 nmol/l) and charybdotoxin (3 mumol/l) had no effect. 4-AP inhibited mainly a fast-activating, slowly inactivating current, whereas the TEA- and TPeA-sensitive current components were slowly activating and non-inactivating. Forskolin and the forskolin analogue 1,9-dideoxyforskolin, which does not stimulate adenylyl cyclase, also inhibited the outward current, suggesting a direct effect on the channels. Using reverse transcriptase polymerase chain reaction (RT/PCR). Kv channel mRNAs of Kv1.4, Kv1.5, Kv2.1, Kv2.2, Kv3.1 and Kv3.2 were detected whereas other Kv channels, Kv1.1, Kv1.2, Kv1.3, Kv1.6 and Kv3.4 were not detected. Insulin secretion in the presence of tolbutamide (100 mumol/l) was increased by 4-AP, TEA and TPeA by 65%, 41% and 150%, respectively. Basal secretion was not affected by these blockers. Our study reveals that the opening of voltage-dependent K+ channels negatively controls insulin secretion in depolarized cells, probably by shortening the action potential thus reducing Ca2+ influx.

  5. Activation of inositol trisphosphate-sensitive Ca2+ channels of sarcoplasmic reticulum from frog skeletal muscle.

    PubMed Central

    Suárez-Isla, B A; Alcayaga, C; Marengo, J J; Bull, R

    1991-01-01

    1. The modulation by Ca2+ of the activation by inositol 1,4,5-trisphosphate (IP3) of Ca2+ channels present in native sarcoplasmic reticulum membranes from frog skeletal muscle was studied after channel incorporation into planar phospholipid bilayers in the presence of Ca2+ or Ba2+ as current carrier species. 2. Channel activity expressed as fractional open time (Po) was low (less than or equal to 0.15) in the presence of varying free Ca2+ concentrations bathing the myoplasmic face of the channel (cis side), and did not increase significantly between 0.01 and 30 microM-Ca2+. 3. Channel activation mediated by IP3 could be elicited from free Ca2+ levels similar to those of resting skeletal muscle (about 0.1 microM) and was found to be strongly regulated by the free Ca2+ concentration present at the myoplasmic moiety of the channel. 4. Channel activation by 10 microM-IP3 depended on the Ca2+ concentration on the cis side. Po reached a maximum between pCa 7.0 and 6.0, but decreased at higher concentrations of free Ca2+. Thus, Ca2+ exerted a modulatory influence on IP3-mediated activation in a concentration range where the channel was insensitive to Ca2+. 5. The results indicate that Ca2+ ions act as modulators of IP3 efficacy to open the channel. This could arise from an interaction of Ca2+ with the channel gating mechanism or with the agonist binding site. PMID:1667801

  6. Characterization of ryanodine receptor type 1 single channel activity using "on-nucleus" patch clamp.

    PubMed

    Wagner, Larry E; Groom, Linda A; Dirksen, Robert T; Yule, David I

    2014-08-01

    In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca(2+) release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca(2+)] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ∼750pS or 450pS in symmetrical 250mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ∼40% of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca(2+), and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation.

  7. Inverse coupling in leak and voltage-activated K+ channel gates underlies distinct roles in electrical signaling.

    PubMed

    Ben-Abu, Yuval; Zhou, Yufeng; Zilberberg, Noam; Yifrach, Ofer

    2009-01-01

    Voltage-activated (Kv) and leak (K(2P)) K(+) channels have key, yet distinct, roles in electrical signaling in the nervous system. Here we examine how differences in the operation of the activation and slow inactivation pore gates of Kv and K(2P) channels underlie their unique roles in electrical signaling. We report that (i) leak K(+) channels possess a lower activation gate, (ii) the activation gate is an important determinant controlling the conformational stability of the K(+) channel pore, (iii) the lower activation and upper slow inactivation gates of leak channels cross-talk and (iv) unlike Kv channels, where the two gates are negatively coupled, these two gates are positively coupled in K(2P) channels. Our results demonstrate how basic thermodynamic properties of the K(+) channel pore, particularly conformational stability and coupling between gates, underlie the specialized roles of Kv and K(2P) channel families in electrical signaling.

  8. Mechanosensitive Channel Activation by Diffusio-Osmotic Force

    NASA Astrophysics Data System (ADS)

    Bonthuis, Douwe Jan; Golestanian, Ramin

    2014-10-01

    For ion channel gating, the appearance of two distinct conformational states and the discrete transitions between them are essential, and therefore of crucial importance to all living organisms. We show that the physical interplay between two structural elements that are commonly present in bacterial mechanosensitive channels—namely, a charged vestibule and a hydrophobic constriction—creates two distinct conformational states, open and closed, as well as the gating between them. We solve the nonequilibrium Stokes-Poisson-Nernst-Planck equations, extended to include a molecular potential of mean force, and show that a first order transition between the closed and open states arises naturally from the diffusio-osmotic stress caused by the ions and the water inside the channel and the elastic restoring force from the membrane.

  9. Schizophrenia-Associated MIR204 Regulates Noncoding RNAs and Affects Neurotransmitter and Ion Channel Gene Sets

    PubMed Central

    Cammaerts, Sophia; Strazisar, Mojca; Smets, Bart; Weckhuysen, Sarah; Nordin, Annelie; De Jonghe, Peter; Adolfsson, Rolf; De Rijk, Peter; Del Favero, Jurgen

    2015-01-01

    As regulators of gene expression, microRNAs (miRNAs) are likely to play an important role in the development of disease. In this study we present a large-scale strategy to identify miRNAs with a role in the regulation of neuronal processes. Thereby we found variant rs7861254 located near the MIR204 gene to be significantly associated with schizophrenia. This variant resulted in reduced expression of miR-204 in neuronal-like SH-SY5Y cells. Analysis of the consequences of the altered miR-204 expression on the transcriptome of these cells uncovered a new mode of action for miR-204, being the regulation of noncoding RNAs (ncRNAs), including several miRNAs, such as MIR296. Furthermore, pathway analysis showed downstream effects of miR-204 on neurotransmitter and ion channel related gene sets, potentially mediated by miRNAs regulated through miR-204. PMID:26714269

  10. Overexpression of the rice AKT1 potassium channel affects potassium nutrition and rice drought tolerance

    PubMed Central

    Ahmad, Izhar; Mian, Afaq; Maathuis, Frans J. M.

    2016-01-01

    Potassium (K+) is the most important cationic nutrient for all living organisms and has roles in most aspects of plant physiology. To assess the impact of one of the main K+ uptake components, the K+ inward rectifying channel AKT1, we characterized both loss of function and overexpression of OsAKT1 in rice. In many conditions, AKT1 expression correlated with K+ uptake and tissue K+ levels. No salinity-related growth phenotype was observed for either loss or gain of function mutants. However, a correlation between AKT1 expression and root Na+ when the external Na/K ratio was high suggests that there may be a role for AKT1 in Na+ uptake in such conditions. In contrast to findings with Arabidopsis thaliana, we did not detect any change in growth of AKT1 loss of function mutants in the presence of NH4 +. Nevertheless, NH4 +-dependent inhibition was detected during K+ uptake assays in loss of function and wild type plants, depending on pre-growth conditions. The most prominent result of OsAKT1 overexpression was a reduction in sensitivity to osmotic/drought stress in transgenic plants: the data suggest that AKT1 overexpression improved rice osmotic and drought stress tolerance by increasing tissue levels of K+, especially in the root. PMID:26969743

  11. Myotonia congenita-associated mutations in chloride channel-1 affect zebrafish body wave swimming kinematics.

    PubMed

    Cheng, Wei; Tian, Jing; Burgunder, Jean-Marc; Hunziker, Walter; Eng, How-Lung

    2014-01-01

    Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.

  12. Structure-activity studies on 1,4-dihydropyridine calcium channel antagonists and activators

    SciTech Connect

    Joslyn, A.F.

    1986-01-01

    Four series of 1,4-dihydropyridine Ca{sup 2+} channel antagonists related to mifedipine were synthesized by a modified Hantzsch procedure to determine the effects of ester (C{sub 3} = CO{sub 2}Me, C{sub 5} = CO{sub 2}R) and phenyl (C{sub 4}) substituents on pharmacological and radioligand binding ((H)nitrendipine) activities in guinea pig ileal longitudinal smooth muscle. Two series of Ca{sup 2+} channel activator 1,4-dihydropyridines, BAY K 8644 (C{sub 3} = NO{sub 2}, C{sub 5} = CO{sub 2}Me) and CGP 28392 (C{sub 2,3} = lactone, C{sub 5} = CO{sub 2}Me) were biochemically evaluated by inhibition of ({sup 3}H)nitrendipine binding in guinea pig ileal longitudinal smooth muscle membranes to establish fundamental structure-activity requirements. A homologous series of bis-1,4-dihydropyridines were synthesized, pharmacologically and biochemically evaluated in an attempt to explore the distribution of the 1,4-dihydropyridine receptor in guinea pig ileal longitudinal smooth muscle membranes. Several potential affinity labels including ester substituted 3- and 4-fluorosulfonyl benzoyl and isothiocyanate derivatives were synthesized and evaluated by inhibition of ({sup 3}H)nitrendipine binding.

  13. HCN channels contribute to serotonergic modulation of ventral surface chemosensitive neurons and respiratory activity

    PubMed Central

    Hawkins, Virginia E.; Hawryluk, Joanna M.; Takakura, Ana C.; Tzingounis, Anastasios V.; Moreira, Thiago S.

    2014-01-01

    Chemosensitive neurons in the retrotrapezoid nucleus (RTN) provide a CO2/H+-dependent drive to breathe and function as an integration center for the respiratory network, including serotonergic raphe neurons. We recently showed that serotonergic modulation of RTN chemoreceptors involved inhibition of KCNQ channels and activation of an unknown inward current. Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are the molecular correlate of the hyperpolarization-activated inward current (Ih) and have a high propensity for modulation by serotonin. To investigate whether HCN channels contribute to basal activity and serotonergic modulation of RTN chemoreceptors, we characterize resting activity and the effects of serotonin on RTN chemoreceptors in vitro and on respiratory activity of anesthetized rats in the presence or absence of blockers of KCNQ (XE991) and/or HCN (ZD7288, Cs+) channels. We found in vivo that bilateral RTN injections of ZD7288 increased respiratory activity and in vitro HCN channel blockade increased activity of RTN chemoreceptors under control conditions, but this was blunted by KCNQ channel inhibition. Furthermore, in vivo unilateral RTN injection of XE991 plus ZD7288 eliminated the serotonin response, and in vitro serotonin sensitivity was eliminated by application of XE991 and ZD7288 or SQ22536 (adenylate cyclase blocker). Serotonin-mediated activation of RTN chemoreceptors was blocked by a 5-HT7-receptor blocker and mimicked by a 5-HT7-receptor agonist. In addition, serotonin caused a depolarizing shift in the voltage-dependent activation of Ih. These results suggest that HCN channels contribute to resting chemoreceptor activity and that serotonin activates RTN chemoreceptors and breathing in part by a 5-HT7 receptor-dependent mechanism and downstream activation of Ih. PMID:25429115

  14. Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle.

    PubMed

    Hristov, Kiril L; Smith, Amy C; Parajuli, Shankar P; Malysz, John; Petkov, Georgi V

    2014-03-01

    Large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca(2+) imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca(2+) sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca(2+) levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca(2+)-dependent mechanism, thus increasing DSM contractility.

  15. Crizotinib Inhibits Hyperpolarization-activated Cyclic Nucleotide-Gated Channel 4 Activity

    PubMed Central

    Zhang, Zhushan; Huang, Tai-Qin; Nepliouev, Igor; Zhang, Hengtao; Barnett, Adam S.; Rosenberg, Paul B.; Ou, Sai-Hong I.; Stiber, Jonathan A.

    2017-01-01

    Background Sinus bradycardia is frequently observed in patients treated with crizotinib, a receptor tyrosine kinase inhibitor used for the treatment of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). We investigated whether crizotinib could influence heart rate (HR) through direct cardiac effects. Methods The direct effect of crizotinib on HR was studied using ECG analysis of Langendorff-perfused mouse hearts. The whole-cell patch clamp technique was used to measure the effects of crizotinib on the hyperpolarization-activated funny current, If, in mouse sinoatrial node cells (SANCs) and hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) activity in HEK-293 cells stably expressing human HCN4. Results Crizotinib resulted in a dose-dependent reduction in HR in isolated intact mouse hearts with a half maximal inhibitory concentration (IC50) of 1.7 ± 0.4 μmol/L. Because ECG analysis revealed that crizotinib (0–5 μmol/L) resulted in significant reductions in HR in isolated mouse hearts without changes in PR, QRS, or QT intervals, we performed whole-cell patch clamp recordings of SANCs which showed that crizotinib inhibited If which regulates cardiac pacemaker activity. Crizotinib resulted in diminished current density of HCN4, the major molecular determinant of If, with an IC50 of 1.4 ± 0.3 μmol/L. Crizotinib also slowed HCN4 activation and shifted the activation curve to the left towards more hyperpolarized potentials. Conclusions Our results suggest that crizotinib’s effects on HCN4 channels play a significant role in mediating its observed effects on HR. PMID:28217366

  16. Bothriurus bonariensis scorpion venom activates voltage-dependent sodium channels in insect and mammalian nervous systems.

    PubMed

    Dos Santos, Douglas Silva; Carvalho, Evelise Leis; de Lima, Jeferson Camargo; Breda, Ricardo Vaz; Oliveira, Raquel Soares; de Freitas, Thiago Carrazoni; Salamoni, Simone Denise; Domingues, Michelle Flores; Piovesan, Angela Regina; Boldo, Juliano Tomazzoni; de Assis, Dênis Reis; da Costa, Jaderson Costa; Dal Belo, Cháriston André; Pinto, Paulo Marcos

    2016-10-25

    Animal venoms have been widely recognized as a major source of biologically active molecules. Bothriurus bonariensis, popularly known as black scorpion, is the arthropod responsible for the highest number of accidents involving scorpion sting in Southern Brazil. Here we reported the first attempt to investigate the neurobiology of B. bonariensis venom (BBV) in the insect and mammalian nervous system. BBV (32 μg/g) induced a slow neuromuscular blockade in the in vivo cockroach nerve-muscle preparations (70 ± 4%, n = 6, p < 0.001), provoking repetitive twitches and significantly decreasing the frequency of spontaneous leg action potentials (SNCAPs) from 82 ± 3 min(-1) to 36 ± 1.3 min(-1) (n = 6, p < 0.05), without affecting the amplitude. When tested in primary cultures of rat hippocampal cells, BBV induced a massive increase of Ca(2+) influx (250 ± 1% peak increase, n = 3, p < 0.0001). The disturbance of calcium homeostasis induced by BBV on the mammalian central nervous system was not accompanied by cellular death and was prevented by the co-treatment of the hippocampal cells with tetrodotoxin, a selective sodium channel blocker. The results suggest that the biological activity of BBV is mostly related to a modulation of sodium channels function. Our biological activity survey suggests that BBV may have a promising insecticidal and therapeutic potential.

  17. STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels.

    PubMed

    Huang, Guo N; Zeng, Weizhong; Kim, Joo Young; Yuan, Joseph P; Han, Linhuang; Muallem, Shmuel; Worley, Paul F

    2006-09-01

    Receptor-evoked Ca2+ signalling involves Ca2+ release from the endoplasmic reticulum, followed by Ca2+ influx across the plasma membrane. Ca2+ influx is essential for many cellular functions, from secretion to transcription, and is mediated by Ca2+-release activated Ca2+ (I(crac)) channels and store-operated calcium entry (SOC) channels. Although the molecular identity and regulation of I(crac) and SOC channels have not been precisely determined, notable recent findings are the identification of STIM1, which has been indicated to regulate SOC and I(crac) channels by functioning as an endoplasmic reticulum Ca2+ sensor, and ORAI1 (ref. 7) or CRACM1 (ref. 8)--both of which may function as I(crac) channels or as an I(crac) subunit. How STIM1 activates the Ca2+ influx channels and whether STIM1 contributes to the channel pore remains unknown. Here, we identify the structural features that are essential for STIM1-dependent activation of SOC and I(crac) channels, and demonstrate that they are identical to those involved in the binding and activation of TRPC1. Notably, the cytosolic carboxyl terminus of STIM1 is sufficient to activate SOC, I(crac) and TRPC1 channels even when native STIM1 is depleted by small interfering RNA. Activity of STIM1 requires an ERM domain, which mediates the selective binding of STIM1 to TRPC1, 2 and 4, but not to TRPC3, 6 or 7, and a cationic lysine-rich region, which is essential for gating of TRPC1. Deletion of either region in the constitutively active STIM1(D76A) yields dominant-negative mutants that block native SOC channels, expressed TRPC1 in HEK293 cells and I(crac) in Jurkat cells. These observations implicate STIM1 as a key regulator of activity rather than a channel component, and reveal similar regulation of SOC, I(crac) and TRPC channel activation by STIM1.

  18. Fast and slow activation of voltage-dependent ion channels in radish vacuoles.

    PubMed Central

    Gambale, F; Cantu, A M; Carpaneto, A; Keller, B U

    1993-01-01

    The molecular processes associated with voltage-dependent opening and closing (gating) of ion channels were investigated using a new preparation from plant cells, i.e., voltage and calcium-activated ion channels in radish root vacuoles. These channels display a main single channel conductance of approximately 90 pS and are characterized by long activation times lasting several hundreds of milliseconds. Here, we demonstrate that these channels have a second kinetically distinct activation mode which is characterized by even longer activation times. Different membrane potential protocols allowed to switch between the fast and the slow mode in a controlled and reversible manner. At transmembrane potentials of -100 mV, the ratio between the fast and slow activation time constant was around 1:5. Correspondingly, activation times lasting several seconds were observed in the slow mode. The molecular process controlling fast and slow activation may represent an effective modulator of voltage-dependent gating of ion channels in other plant and animal systems. PMID:7507716

  19. Serotonin and Dopamine: Unifying Affective, Activational, and Decision Functions

    PubMed Central

    Cools, Roshan; Nakamura, Kae; Daw, Nathaniel D

    2011-01-01

    Serotonin, like dopamine (DA), has long been implicated in adaptive behavior, including decision making and reinforcement learning. However, although the two neuromodulators are tightly related and have a similar degree of functional importance, compared with DA, we have a much less specific understanding about the mechanisms by which serotonin affects behavior. Here, we draw on recent work on computational models of dopaminergic function to suggest a framework by which many of the seemingly diverse functions associated with both DA and serotonin—comprising both affective and activational ones, as well as a number of other functions not overtly related to either—can be seen as consequences of a single root mechanism. PMID:20736991

  20. Pulsatile atheroprone shear stress affects the expression of transient receptor potential channels in human endothelial cells.

    PubMed

    Thilo, Florian; Vorderwülbecke, Bernd J; Marki, Alex; Krueger, Katharina; Liu, Ying; Baumunk, Daniel; Zakrzewicz, Andreas; Tepel, Martin

    2012-06-01

    The goal of the study was to assess whether pulsatile atheroprone shear stress modulates the expression of transient receptor potential (TRP) channels, TRPC3, TRPC6, TRPM7, and TRPV1 mRNA, in human umbilical vascular endothelial cells. Exposure of cultured vascular endothelial cells to defined shear stress, producing a constant laminar flow (generating a shear stress of 6 dyne/cm(2)), laminar pulsatile atheroprotective flow (with a mean shear stress of 20 dyne/cm(2)), or laminar atheroprone bidirectional flow (with a mean shear stress of 0 dyne/cm(2)) differentially induced TRPC6 and TRPV1 mRNA as measured by quantitative real-time RT-PCR and normalized to GAPDH expression. Thereby, TRPC6 and TRPV1 mRNA expressions were significantly increased after 24 hours of exposure to an atheroprone flow profile compared with an atheroprotective flow profile. Furthermore, the expression of transcription factors GATA1 and GATA4 was significantly correlated with the expression of TRPC6 mRNA. In contrast, after 24 hours of constant laminar flow, the expression of TRPC6 and TRPV1 mRNA was unchanged, whereas the expression of TRPC3 and TRPM7 was significantly higher in endothelial cells exposed to shear stress in comparison with endothelial cells grown under static conditions. There was a significant association between the expression of TRPC6 and tumor necrosis factor-α mRNA in human vascular tissue. No-flow and atheroprone flow conditions are equally characterized by an increase in the expression of tumor necrosis factor-α; however, inflammation-associated endothelial cell reactions may be further aggravated at atheroprone flow conditions by the increase of TRPV1 and TRPC6, as observed in our study.

  1. Upregulation of the large conductance voltage- and Ca2+-activated K+ channels by Janus kinase 2.

    PubMed

    Hosseinzadeh, Zohreh; Almilaji, Ahmad; Honisch, Sabina; Pakladok, Tatsiana; Liu, GuoXing; Bhavsar, Shefalee K; Ruth, Peter; Shumilina, Ekaterina; Lang, Florian

    2014-06-01

    The iberiotoxin-sensitive large conductance voltage- and Ca(2+)-activated potassium (BK) channels (maxi-K(+)-channels) hyperpolarize the cell membrane thus supporting Ca(2+) entry through Ca(2+)-release activated Ca(2+) channels. Janus kinase-2 (JAK2) has been identified as novel regulator of ion transport. To explore whether JAK2 participates in the regulation of BK channels, cRNA encoding Ca(2+)-insensitive BK channels (BK(M513I+Δ899-903)) was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, gain-of-function (V617F)JAK2, or inactive (K882E)JAK2. K(+) conductance was determined by dual electrode voltage clamp and BK-channel protein abundance by confocal microscopy. In A204 alveolar rhabdomyosarcoma cells, iberiotoxin-sensitive K(+) current was determined utilizing whole cell patch clamp. A204 cells were further transfected with JAK2 and BK-channel transcript, and protein abundance was quantified by RT-PCR and Western blotting, respectively. As a result, the K(+) current in BK(M513I+Δ899-903)-expressing oocytes was significantly increased following coexpression of JAK2 or (V617F)JAK2 but not (K882E)JAK2. Coexpression of the BK channel with (V617F)JAK2 but not (K882E)JAK2 enhanced BK-channel protein abundance in the oocyte cell membrane. Exposure of BK-channel and (V617F)JAK2-expressing oocytes to the JAK2 inhibitor AG490 (40 μM) significantly decreased K(+) current. Inhibition of channel insertion by brefeldin A (5 μM) decreased the K(+) current to a similar extent in oocytes expressing the BK channel alone and in oocytes expressing the BK channel and (V617F)JAK2. The iberiotoxin (50 nM)-sensitive K(+) current in rhabdomyosarcoma cells was significantly decreased by AG490 pretreatment (40 μM, 12 h). Moreover, overexpression of JAK2 in A204 cells significantly enhanced BK channel mRNA and protein abundance. In conclusion, JAK2 upregulates BK channels by increasing channel protein abundance in the cell membrane.

  2. BK and Kv3.1 potassium channels control different aspects of deep cerebellar nuclear neurons action potentials and spiking activity.

    PubMed

    Pedroarena, Christine M

    2011-12-01

    Deep cerebellar nuclear neurons (DCNs) display characteristic electrical properties, including spontaneous spiking and the ability to discharge narrow spikes at high frequency. These properties are thought to be relevant to processing inhibitory Purkinje cell input and transferring well-timed signals to cerebellar targets. Yet, the underlying ionic mechanisms are not completely understood. BK and Kv3.1 potassium channels subserve similar functions in spike repolarization and fast firing in many neurons and are both highly expressed in DCNs. Here, their role in the abovementioned spiking characteristics was addressed using whole-cell recordings of large and small putative-glutamatergic DCNs. Selective BK channel block depolarized DCNs of both groups and increased spontaneous firing rate but scarcely affected evoked activity. After adjusting the membrane potential to control levels, the spike waveforms under BK channel block were indistinguishable from control ones, indicating no significant BK channel involvement in spike repolarization. The increased firing rate suggests that lack of DCN-BK channels may have contributed to the ataxic phenotype previously found in BK channel-deficient mice. On the other hand, block of Kv3.1 channels with low doses of 4-aminopyridine (20 μM) hindered spike repolarization and severely depressed evoked fast firing. Therefore, I propose that despite similar characteristics of BK and Kv3.1 channels, they play different roles in DCNs: BK channels control almost exclusively spontaneous firing rate, whereas DCN-Kv3.1 channels dominate the spike repolarization and enable fast firing. Interestingly, after Kv3.1 channel block, BK channels gained a role in spike repolarization, demonstrating how the different function of each of the two channels is determined in part by their co-expression and interplay.

  3. Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

    PubMed

    Al Khamici, Heba; Brown, Louise J; Hossain, Khondker R; Hudson, Amanda L; Sinclair-Burton, Alxcia A; Ng, Jane Phui Mun; Daniel, Elizabeth L; Hare, Joanna E; Cornell, Bruce A; Curmi, Paul M G; Davey, Mary W; Valenzuela, Stella M

    2015-01-01

    The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

  4. Large-conductance Ca2+-activated potassium channels in secretory neurons.

    PubMed

    Lara, J; Acevedo, J J; Onetti, C G

    1999-09-01

    Large-conductance Ca2+-activated K+ channels (BK) are believed to underlie interburst intervals and contribute to the control of hormone release in several secretory cells. In crustacean neurosecretory cells, Ca2+ entry associated with electrical activity could act as a modulator of membrane K+ conductance. Therefore we studied the contribution of BK channels to the macroscopic outward current in the X-organ of crayfish, and their participation in electrophysiological activity, as well as their sensitivity toward intracellular Ca2+, ATP, and voltage, by using the patch-clamp technique. The BK channels had a conductance of 223 pS and rectified inwardly in symmetrical K+. These channels were highly selective to K+ ions; potassium permeability (PK) value was 2.3 x 10(-13) cm(3) s(-1). The BK channels were sensitive to internal Ca2+ concentration, voltage dependent, and activated by intracellular MgATP. Voltage sensitivity (k) was approximately 13 mV, and the half-activation membrane potentials depended on the internal Ca2+ concentration. Calcium ions (0.3-3 microM) applied to the internal membrane surface caused an enhancement of the channel activity. This activation of BK channels by internal calcium had a KD(0) of 0.22 microM and was probably due to the binding of only one or two Ca2+ ions to the channel. Addition of MgATP (0.01-3 mM) to the internal solution increased steady state-open probability. The dissociation constant for MgATP (KD) was 119 microM, and the Hill coefficient (h) was 0.6, according to the Hill analysis. Ca2+-activated K+ currents recorded from whole cells were suppressed by either adding Cd2+ (0.4 mM) or removing Ca2+ ions from the external solution. TEA (1 mM) or charybdotoxin (100 nM) blocked these currents. Our results showed that both BK and K(ATP) channels are present in the same cell. Even when BK and K(ATP) channels were voltage dependent and modulated by internal Ca2+ and ATP, the profile of sensitivity was quite different for each kind

  5. The membrane protein Pannexin1 forms two open channel conformations depending on the mode of activation

    PubMed Central

    Wang, Junjie; Ambrosi, Cinzia; Qiu, Feng; Jackson, David G.; Sosinsky, Gina; Dahl, Gerhard

    2014-01-01

    Pannexin1 (Panx1) participates in several signaling events that involve ATP release, including the innate immune response, ciliary beat in airway epithelia and oxygen supply in the vasculature. The view that Panx1 forms a large ATP-release channel has been challenged by the association of a low conductance, small anion-selective channel with the presence of Panx1. We showed that Panx1 membrane channels can function in two distinct modes with different conductances and permeabilities when heterologously expressed in Xenopus oocytes. When stimulated by potassium ions (K+), Panx1 formed a high conductance channel of ~500 pS that was permeable to ATP. Various physiological stimuli can induce this ATP-permeable conformation of the channel in several cell types. In contrast, the channel had a low conductance (~50 pS) with no detectable ATP permeability when activated by voltage in the absence of K+. The two channel states were associated with different reactivities of the terminal cysteine of Panx1 to thiol reagents, suggesting different conformations. Single particle electron microscopic analysis revealed that K+ stimulated the formation of channels with a larger pore diameter than those formed in the absence of K+. These data suggest that different stimuli lead to distinct channel structures with distinct biophysical properties. PMID:25056878

  6. Differential distribution of the sodium-activated potassium channels slick and slack in mouse brain.

    PubMed

    Rizzi, Sandra; Knaus, Hans-Günther; Schwarzer, Christoph

    2016-07-01

    The sodium-activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are high-conductance potassium channels of the Slo family. In neurons, Slick and Slack channels are involved in the generation of slow afterhyperpolarization, in the regulation of firing patterns, and in setting and stabilizing the resting membrane potential. The distribution and subcellular localization of Slick and Slack channels in the mouse brain have not yet been established in detail. The present study addresses this issue through in situ hybridization and immunohistochemistry. Both channels were widely distributed and exhibited distinct distribution patterns. However, in some brain regions, their expression overlapped. Intense Slick channel immunoreactivity was observed in processes, varicosities, and neuronal cell bodies of the olfactory bulb, granular zones of cortical regions, hippocampus, amygdala, lateral septal nuclei, certain hypothalamic and midbrain nuclei, and several regions of the brainstem. The Slack channel showed primarily a diffuse immunostaining pattern, and labeling of cell somata and processes was observed only occasionally. The highest Slack channel expression was detected in the olfactory bulb, lateral septal nuclei, basal ganglia, and distinct areas of the midbrain, brainstem, and cerebellar cortex. In addition, comparing our data obtained from mouse brain with a previously published study on rat brain revealed some differences in the expression and distribution of Slick and Slack channels in these species. J. Comp. Neurol. 524:2093-2116, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

  7. Differential distribution of the sodium‐activated potassium channels slick and slack in mouse brain

    PubMed Central

    Knaus, Hans‐Günther; Schwarzer, Christoph

    2015-01-01

    ABSTRACT The sodium‐activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are high‐conductance potassium channels of the Slo family. In neurons, Slick and Slack channels are involved in the generation of slow afterhyperpolarization, in the regulation of firing patterns, and in setting and stabilizing the resting membrane potential. The distribution and subcellular localization of Slick and Slack channels in the mouse brain have not yet been established in detail. The present study addresses this issue through in situ hybridization and immunohistochemistry. Both channels were widely distributed and exhibited distinct distribution patterns. However, in some brain regions, their expression overlapped. Intense Slick channel immunoreactivity was observed in processes, varicosities, and neuronal cell bodies of the olfactory bulb, granular zones of cortical regions, hippocampus, amygdala, lateral septal nuclei, certain hypothalamic and midbrain nuclei, and several regions of the brainstem. The Slack channel showed primarily a diffuse immunostaining pattern, and labeling of cell somata and processes was observed only occasionally. The highest Slack channel expression was detected in the olfactory bulb, lateral septal nuclei, basal ganglia, and distinct areas of the midbrain, brainstem, and cerebellar cortex. In addition, comparing our data obtained from mouse brain with a previously published study on rat brain revealed some differences in the expression and distribution of Slick and Slack channels in these species. J. Comp. Neurol. 524:2093–2116, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:26587966

  8. Mutations affecting export and activity of cytolysin A from Escherichia coli.

    PubMed

    Ludwig, Albrecht; Völkerink, Guido; von Rhein, Christine; Bauer, Susanne; Maier, Elke; Bergmann, Birgit; Goebel, Werner; Benz, Roland

    2010-08-01

    Cytolysin A (known as ClyA, HlyE, and SheA) is a cytolytic pore-forming protein toxin found in several Escherichia coli and Salmonella enterica strains. The structure of its water-soluble monomeric form and that of dodecameric ClyA pores is known, but the mechanisms of ClyA export from bacterial cells and of pore assembly are only partially understood. Here we used site-directed mutagenesis to study the importance of different regions of the E. coli ClyA protein for export and activity. The data indicate that ClyA translocation to the periplasm requires several protein segments located closely adjacent to each other in the "tail" domain of the ClyA monomer, namely, the N- and C-terminal regions and the hydrophobic sequence ranging from residues 89 to 101. Deletion of most of the "head" domain of the monomer (residues 181 to 203), on the other hand, did not strongly affect ClyA secretion, suggesting that the tail domain plays a particular role in export. Furthermore, we found that the N-terminal amphipathic helix alphaA1 of ClyA is crucial for the formation and the properties of the transmembrane channel, and hence for hemolytic activity. Several mutations affecting the C-terminal helix alphaG, the "beta-tongue" region in the head domain, or the hydrophobic region in the tail domain of the ClyA monomer strongly impaired the hemolytic activity and reduced the activity toward planar lipid bilayer membranes but did not totally prevent formation of wild-type-like channels in these artificial membranes. The latter regions thus apparently promote membrane interaction without being directly required for pore formation in a lipid bilayer.

  9. Oxalomalate affects the inducible nitric oxide synthase expression and activity.

    PubMed

    Irace, Carlo; Esposito, Giuseppe; Maffettone, Carmen; Rossi, Antonietta; Festa, Michela; Iuvone, Teresa; Santamaria, Rita; Sautebin, Lidia; Carnuccio, Rosa; Colonna, Alfredo

    2007-03-13

    Inducible nitric oxide synthase (iNOS) is an homodimeric enzyme which produces large amounts of nitric oxide (NO) in response to inflammatory stimuli. Several factors affect the synthesis and catalytic activity of iNOS. Particularly, dimerization of NOS monomers is promoted by heme, whereas an intracellular depletion of heme and/or L-arginine considerably decreases NOS resistance to proteolysis. In this study, we found that oxalomalate (OMA, oxalomalic acid, alpha-hydroxy-beta-oxalosuccinic acid), an inhibitor of both aconitase and NADP-dependent isocitrate dehydrogenase, inhibited nitrite production and iNOS protein expression in lipopolysaccharide (LPS)-activated J774 macrophages, without affecting iNOS mRNA content. Furthermore, injection of OMA precursors to LPS-stimulated rats also decreased nitrite production and iNOS expression in isolated peritoneal macrophages. Interestingly, alpha-ketoglutarate or succinyl-CoA administration reversed OMA effect on NO production, thus correlating NO biosynthesis with the anabolic capacity of Krebs cycle. When protein synthesis was blocked by cycloheximide in LPS-activated J774 cells treated with OMA, iNOS protein levels, evaluated by Western blot analysis and (35)S-metabolic labelling, were decreased, suggesting that OMA reduces iNOS biosynthesis and induces an increase in the degradation rate of iNOS protein. Moreover, we showed that OMA inhibits the activity of the iNOS from lung of LPS-treated rats by enzymatic assay. Our results, demonstrating that OMA acts regulating synthesis, catalytic activity and degradation of iNOS, suggest that this compound might have a potential role in reducing the NO overproduction occurring in some pathological conditions.

  10. 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.

  11. Dendrotoxins: structure-activity relationships and effects on potassium ion channels.

    PubMed

    Harvey, A L; Robertson, B

    2004-12-01

    Dendrotoxins are small proteins isolated from mamba (Dendroaspis) snakes. The original dendrotoxin was found in venom of the Eastern green mamba, Dendroaspis angusticeps, and related proteins were subsequently found in other mamba venoms. The dendrotoxins contain 57-60 amino acid residues cross-linked by three disulphide bridges, and they are homologous to Kunitz-type serine protease inhibitors, such as aprotinin (BPTI). The dendrotoxins have little or no anti-protease activity, but they block particular subtypes of voltage-dependent potassium channels of the Kv1 subfamily in neurones. Alpha-dendrotoxin from green mamba Dendroaspis angusticeps and toxin I from the black mamba Dendroaspis polylepis block cloned Kv1.1, Kv1.2 and Kv1.6 channels in the low nanomolar range; toxin K, also from the black mamba Dendroaspis polylepis, preferentially blocks Kv1.1 channels and is active at picomolar concentrations. Structural modifications and mutations to dendrotoxins have helped to define the molecular recognition properties of different types of K+ channels, although more work is needed to characterise the chemical features of the toxins that underlie their selectivity and potency at particular subtypes of channels. Dendrotoxins have been useful markers of subtypes of K+ channels in vivo, and dendrotoxins have become widely used as probes for studying the function of K+ channels in physiology and pathophysiology. With some pathological conditions being associated with voltage-gated K+ channels, analogues of dendrotoxins might have therapeutic potential.

  12. Physical basis of apparent pore-dilation of ATP-activated P2X receptor channels

    PubMed Central

    Li, Mufeng; Toombes, Gilman E S; Silberberg, Shai D; Swartz, Kenton J

    2016-01-01

    The selectivity of ion channels is fundamental for their roles in electrical and chemical signaling, and ion homeostasis. Although most ion channels exhibit stable ion selectivity, the prevailing view for purinergic P2X receptor channels, transient receptor potential V1 (TRPV1) channels and acid sensing ion channels (ASICs) is that their ion conduction pores dilate upon prolonged activation. We investigated this mechanism in P2X receptors and found that the hallmark shift in equilibrium potential observed with prolonged channel activation does not result from pore dilation, but from time-dependent alterations in the concentration of intracellular ions. We derived a physical model to calculate ion concentration changes during patch-clamp recordings, which validates our experimental findings and provides a quantitative guideline for effectively controlling ion concentration. Our results have fundamental implications for understanding ion permeation and gating in P2X receptor channels, and more broadly for using patch-clamp techniques to study ion channels and neuronal excitability. PMID:26389841

  13. Cryo-EM structure of the Slo2.2 Na+-activated K+ channel

    PubMed Central

    Hite, Richard; Yuan, Peng; Li, Zongli; Hsuing, Yichun; Walz, Thomas; MacKinnon, Roderick

    2015-01-01

    Na+-activated K+ channels are members of the Slo family of large conductance K+ channels that are widely expressed in the brain, where their opening regulates neuronal excitability. These channels are fascinating for the biological roles they fulfill as well as for their intriguing biophysical properties, including conductance levels ten times most other K+ channels and gating sensitivity to intracellular Na+. Here we present the structure a complete Na+-activated K+ channel, Slo2.2, in the Na+-free state, determined by cryo-electron microscopy at a nominal resolution of 4.5 Å. The channel is composed of a large cytoplasmic gating ring within which resides the Na+-binding site and a transmembrane domain that closely resembles voltage-gated K+ channels. In the structure, the cytoplasmic domain adopts a closed conformation and the ion conduction pore is also closed. The structure provides a first view of a member of the Slo K+ channel family, which reveals features explaining their high conductance and gating mechanism. PMID:26436452

  14. Relaxin Affects Smooth Muscle Biophysical Properties and Mechanical Activity of the Female Mouse Colon.

    PubMed

    Squecco, Roberta; Garella, Rachele; Idrizaj, Eglantina; Nistri, Silvia; Francini, Fabio; Baccari, Maria Caterina

    2015-12-01

    The hormone relaxin (RLX) has been reported to influence gastrointestinal motility in mice. However, at present, nothing is known about the effects of RLX on the biophysical properties of the gastrointestinal smooth muscle cells (SMCs). Other than extending previous knowledge of RLX on colonic motility, the purpose of this study was to investigate the ability of the hormone to induce changes in resting membrane potential (RMP) and on sarcolemmal ion channels of colonic SMCs of mice that are related to its mechanical activity. To this aim, we used a combined mechanical and electrophysiological approach. In the mechanical experiments, we observed that RLX caused a decay of the basal tone coupled to an increase of the spontaneous contractions, completely abolished by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ). The electrophysiological results indicate for the first time that RLX directly affects the SMC biophysical properties inducing hyperpolarization of RMP and cycles of slow hyperpolarization/depolarization oscillations. The effects of RLX on RMP were abolished by ODQ as well as by a specific inhibitor of the cGMP-dependent protein kinase, KT5823. RLX reduced Ca(2+) entry through the voltage-dependent L-type channels and modulated either voltage- or ATP-dependent K(+) channels. These effects were abolished by ODQ, suggesting the involvement of the nitric oxide/guanylate cyclase pathway in the effects of RLX on RMP and ion channel modulation. These actions of RLX on membrane properties may contribute to the regulation of the proximal colon motility by the nitric oxide/cGMP/cGMP-dependent protein kinase pathway.

  15. Sequential acquisition of cacophony calcium currents, sodium channels and voltage-dependent potassium currents affects spike shape and dendrite growth during postembryonic maturation of an identified Drosophila motoneuron.

    PubMed

    Ryglewski, Stefanie; Kilo, Lukas; Duch, Carsten

    2014-05-01

    During metamorphosis the CNS undergoes profound changes to accommodate the switch from larval to adult behaviors. In Drosophila and other holometabolous insects, adult neurons differentiate either from respecified larval neurons, newly born neurons, or are born embryonically but remain developmentally arrested until differentiation during pupal life. This study addresses the latter in the identified Drosophila flight motoneuron 5. In situ patch-clamp recordings, intracellular dye fills and immunocytochemistry address the interplay between dendritic shape, excitability and ionic current development. During pupal life, changes in excitability and spike shape correspond to a stereotyped, progressive appearance of voltage-gated ion channels. High-voltage-activated calcium current is the first current to appear at pupal stage P4, prior to the onset of dendrite growth. This is followed by voltage-gated sodium as well as transient potassium channel expression, when first dendrites grow, and sodium-dependent action potentials can be evoked by somatic current injection. Sustained potassium current appears later than transient potassium current. During the early stages of rapid dendritic growth, sodium-dependent action potentials are broadened by a calcium component. Narrowing of spike shape coincides with sequential increases in transient and sustained potassium currents during stages when dendritic growth ceases. Targeted RNAi knockdown of pupal calcium current significantly reduces dendritic growth. These data indicate that the stereotyped sequential acquisition of different voltage-gated ion channels affects spike shape and excitability such that activity-dependent calcium influx serves as a partner of genetic programs during critical stages of motoneuron dendrite growth.

  16. Investigation of Channel-Forming Activity of Polyene Macrolide Antibiotics in Planar Lipid Bilayers in the Presence of Dipole Modifiers

    PubMed Central

    Efimova, S. S.; Schagina, L. V.; Ostroumova, O. S.

    2014-01-01

    The role of membrane components, sterols, phospholipids and sphingolipids in the formation and functioning of ion-permeable nanopores formed by antifungal macrolide antibiotics, amphotericin B, nystatin and filipin in planar lipid bilayers was studied. Dipole modifiers, flavonoids and styryl dyes, were used as a tool to study the molecular mechanisms of polyene channel-forming activity. The introduction of dipole modifiers into the membrane bathing solutions was shown to change the conductance of single channels and the steadystate transmembrane current induced by polyene antibiotics in the sterol-containing phospholipid-bilayers. The conductance of single amphotericin B channels was found to depend on the dipole potential of the membrane. The experiments with various phospholipids, sterols, and polyenes led to the assumption that the shape of a phospholipid molecule, the presence of double bonds at the positions 7 and 22 of a sterol molecule, the number of conjugated double bonds, and the presence of an amino sugar in the polyene antibiotic molecule are important factors impacting the stability of polyene-lipid complexes forming ion-permeable pores. Experimental and literature data presented in the paper suggest that the channel-forming activity of polyene antibiotics is also affected by the physicochemical properties of polyene-enriched ordered membrane domains. PMID:25558397

  17. Direct Activation of β-Cell KATP Channels with a Novel Xanthine Derivative

    PubMed Central

    Raphemot, Rene; Swale, Daniel R.; Dadi, Prasanna K.; Jacobson, David A.; Cooper, Paige; Wojtovich, Andrew P.; Banerjee, Sreedatta; Nichols, Colin G.

    2014-01-01

    ATP-regulated potassium (KATP) channel complexes of inward rectifier potassium channel (Kir) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet β-cell membrane potential, calcium influx, and insulin secretion, and consequently, represent important drug targets for metabolic disorders of glucose homeostasis. The KATP channel opener diazoxide is used clinically to treat intractable hypoglycemia caused by excessive insulin secretion, but its use is limited by off-target effects due to lack of potency and selectivity. Some progress has been made in developing improved Kir6.2/SUR1 agonists from existing chemical scaffolds and compound screening, but there are surprisingly few distinct chemotypes that are specific for SUR1-containing KATP channels. Here we report the serendipitous discovery in a high-throughput screen of a novel activator of Kir6.2/SUR1: VU0071063 [7-(4-(tert-butyl)benzyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione]. The xanthine derivative rapidly and dose-dependently activates Kir6.2/SUR1 with a half-effective concentration (EC50) of approximately 7 μM, is more efficacious than diazoxide at low micromolar concentrations, directly activates the channel in excised membrane patches, and is selective for SUR1- over SUR2A-containing Kir6.1 or Kir6.2 channels, as well as Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and voltage-gated potassium channel 2.1. Finally, we show that VU0071063 activates native Kir6.2/SUR1 channels, thereby inhibiting glucose-stimulated calcium entry in isolated mouse pancreatic β cells. VU0071063 represents a novel tool/compound for investigating β-cell physiology, KATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry. PMID:24646456

  18. Activities affecting surface water resources: A general overview

    SciTech Connect

    Not Available

    1990-01-01

    In November 1987, P.E.I. signed a federal/provincial work-sharing arrangement on water resource management focusing on groundwater pollution, surface water degradation and estuarine eutrophication. The surface water program was designed to identify current surface water uses and users within 12 major watersheds across the Island containing 26 individual rivers, as well as problems arising due to practices that degrade the quality of surface water and restricts its value to other user groups. This report presents a general overview of the program, covering the general characteristics of the Island; operations in agriculture, fish and wildlife, forestry, recreation, fisheries, and industry; alterations of natural features of waterways; wetlands; additional watershed activities such as hydrometric stations and subdivision development; and activities affecting surface water resources such as sedimentation sources, pollution point sources and instream obstructions.

  19. Intolerance of uncertainty correlates with insula activation during affective ambiguity

    PubMed Central

    Simmons, Alan; Matthews, Scott C.; Paulus, Martin P.; Stein, Murray B.

    2009-01-01

    Intolerance of uncertainty (IU), or the increased affective response to situations with uncertain outcomes, is an important component process of anxiety disorders. Increased IU is observed in panic disorder (PD), obsessive compulsive disorder (OCD) and generalized anxiety disorder (GAD), and is thought to relate to dysfunctional behaviors and thought patterns in these disorders. Identifying what brain systems are associated with IU would contribute to a comprehensive model of anxiety processing, and increase our understanding of the neurobiology of anxiety disorders. Here, we used a behavioral task, Wall of Faces (WOF), during functional magnetic resonance imaging (fMRI), which probes both affect and ambiguity, to examine the neural circuitry of IU in fourteen (10 females) college age (18.8 yrs) subjects. All subjects completed the Intolerance of Uncertainty Scale (IUS), Anxiety Sensitivity Index (ASI), and a measure of neuroticism (i.e. the NEO-N). IUS scores but neither ASI nor NEO-N scores, correlated positively with activation in bilateral insula during affective ambiguity. Thus, the experience of IU during certain types of emotion processing may relate to the degree to which bilateral insula processes uncertainty. Previously observed insula hyperactivity in anxiety disorder individuals may therefore be directly linked to altered processes of uncertainty. PMID:18079060

  20. Intolerance of uncertainty correlates with insula activation during affective ambiguity.

    PubMed

    Simmons, Alan; Matthews, Scott C; Paulus, Martin P; Stein, Murray B

    2008-01-10

    Intolerance of uncertainty (IU), or the increased affective response to situations with uncertain outcomes, is an important component process of anxiety disorders. Increased IU is observed in panic disorder (PD), obsessive compulsive disorder (OCD) and generalized anxiety disorder (GAD), and is thought to relate to dysfunctional behaviors and thought patterns in these disorders. Identifying what brain systems are associated with IU would contribute to a comprehensive model of anxiety processing, and increase our understanding of the neurobiology of anxiety disorders. Here, we used a behavioral task, Wall of Faces (WOFs), during functional magnetic resonance imaging (fMRI), which probes both affect and ambiguity, to examine the neural circuitry of IU in 14 (10 females) college age (18.8 years) subjects. All subjects completed the Intolerance of Uncertainty Scale (IUS), Anxiety Sensitivity Index (ASI), and a measure of neuroticism (i.e. the NEO-N). IUS scores but neither ASI nor NEO-N scores, correlated positively with activation in bilateral insula during affective ambiguity. Thus, the experience of IU during certain types of emotion processing may relate to the degree to which bilateral insula processes uncertainty. Previously observed insula hyperactivity in anxiety disorder individuals may therefore be directly linked to altered processes of uncertainty.

  1. Mg2+ mediates interaction between the voltage sensor and cytosolic domain to activate BK channels.

    PubMed

    Yang, Huanghe; Hu, Lei; Shi, Jingyi; Delaloye, Kelli; Horrigan, Frank T; Cui, Jianmin

    2007-11-13

    The voltage-sensor domain (VSD) of voltage-dependent ion channels and enzymes is critical for cellular responses to membrane potential. The VSD can also be regulated by interaction with intracellular proteins and ligands, but how this occurs is poorly understood. Here, we show that the VSD of the BK-type K(+) channel is regulated by a state-dependent interaction with its own tethered cytosolic domain that depends on both intracellular Mg(2+) and the open state of the channel pore. Mg(2+) bound to the cytosolic RCK1 domain enhances VSD activation by electrostatic interaction with Arg-213 in transmembrane segment S4. Our results demonstrate that a cytosolic domain can come close enough to the VSD to regulate its activity electrostatically, thereby elucidating a mechanism of Mg(2+)-dependent activation in BK channels and suggesting a general pathway by which intracellular factors can modulate the function of voltage-dependent proteins.

  2. The Fungal Sexual Pheromone Sirenin Activates the Human CatSper Channel Complex.

    PubMed

    Syeda, Shameem Sultana; Carlson, Erick J; Miller, Melissa R; Francis, Rawle; Clapham, David E; Lishko, Polina V; Hawkinson, Jon E; Hook, Derek; Georg, Gunda I

    2016-02-19

    The basal fungus Allomyces macrogynus (A. macrogynus) produces motile male gametes displaying well-studied chemotaxis toward their female counterparts. This chemotaxis is driven by sirenin, a sexual pheromone released by the female gametes. The pheromone evokes a large calcium influx in the motile gametes, which could proceed through the cation channel of sperm (CatSper) complex. Herein, we report the total synthesis of sirenin in 10 steps and 8% overall yield and show that the synthetic pheromone activates the CatSper channel complex, indicated by a concentration-dependent increase in intracellular calcium in human sperm. Sirenin activation of the CatSper channel was confirmed using whole-cell patch clamp electrophysiology with human sperm. Based on this proficient synthetic route and confirmed activation of CatSper, analogues of sirenin can be designed as blockers of the CatSper channel that could provide male contraceptive agents.

  3. Activity-dependent regulation of T-type calcium channels by submembrane calcium ions

    PubMed Central

    Cazade, Magali; Bidaud, Isabelle; Lory, Philippe; Chemin, Jean

    2017-01-01

    Voltage-gated Ca2+ channels are involved in numerous physiological functions and various mechanisms finely tune their activity, including the Ca2+ ion itself. This is well exemplified by the Ca2+-dependent inactivation of L-type Ca2+ channels, whose alteration contributes to the dramatic disease Timothy Syndrome. For T-type Ca2+ channels, a long-held view is that they are not regulated by intracellular Ca2+. Here we challenge this notion by using dedicated electrophysiological protocols on both native and expressed T-type Ca2+ channels. We demonstrate that a rise in submembrane Ca2+ induces a large decrease in T-type current amplitude due to a hyperpolarizing shift in the steady-state inactivation. Activation of most representative Ca2+-permeable ionotropic receptors similarly regulate T-type current properties. Altogether, our data clearly establish that Ca2+ entry exerts a feedback control on T-type channel activity, by modulating the channel availability, a mechanism that critically links cellular properties of T-type Ca2+ channels to their physiological roles. DOI: http://dx.doi.org/10.7554/eLife.22331.001 PMID:28109159

  4. 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

  5. Calmodulin regulation of TMEM16A and 16B Ca2+-activated chloride channels

    PubMed Central

    Yang, Tingting; Colecraft, Henry M

    2016-01-01

    Ca2+-activated chloride channels encoded by TMEM16A and 16B are important for regulating epithelial mucus secretion, cardiac and neuronal excitability, smooth muscle contraction, olfactory transduction, and cell proliferation. Whether and how the ubiquitous Ca2+ sensor calmodulin (CaM) regulates the activity of TMEM16A and 16B channels has been controversial and the subject of an ongoing debate. Recently, using a bioengineering approach termed ChIMP (Channel Inactivation induced by Membrane-tethering of an associated Protein) we argued that Ca2+-free CaM (apoCaM) is pre-associated with functioning TMEM16A and 16B channel complexes in live cells. Further, the pre-associated apoCaM mediates Ca2+-dependent sensitization of activation (CDSA) and Ca2+-dependent inactivation (CDI) of some TMEM16A splice variants. In this review, we discuss these findings in the context of previous and recent results relating to Ca2+-dependent regulation of TMEM16A/16B channels and the putative role of CaM. We further discuss potential future directions for these nascent ideas on apoCaM regulation of TMEM16A/16B channels, noting that such future efforts will benefit greatly from the pioneering work of Dr. David T. Yue and colleagues on CaM regulation of voltage-dependent calcium channels. PMID:26083059

  6. Permeation Mechanisms in the TMEM16B Calcium-Activated Chloride Channels.

    PubMed

    Pifferi, Simone

    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.

  7. Activity-dependent regulation of T-type calcium channels by submembrane calcium ions.

    PubMed

    Cazade, Magali; Bidaud, Isabelle; Lory, Philippe; Chemin, Jean

    2017-01-21

    Voltage-gated Ca(2+) channels are involved in numerous physiological functions and various mechanisms finely tune their activity, including the Ca(2+) ion itself. This is well exemplified by the Ca(2+)-dependent inactivation of L-type Ca(2+) channels, whose alteration contributes to the dramatic disease Timothy Syndrome. For T-type Ca(2+) channels, a long-held view is that they are not regulated by intracellular Ca(2+). Here we challenge this notion by using dedicated electrophysiological protocols on both native and expressed T-type Ca(2+) channels. We demonstrate that a rise in submembrane Ca(2+) induces a large decrease in T-type current amplitude due to a hyperpolarizing shift in the steady-state inactivation. Activation of most representative Ca(2+)-permeable ionotropic receptors similarly regulate T-type current properties. Altogether, our data clearly establish that Ca(2+) entry exerts a feedback control on T-type channel activity, by modulating the channel availability, a mechanism that critically links cellular properties of T-type Ca(2+) channels to their physiological roles.

  8. Attracting Views and Going Viral: How Message Features and News-Sharing Channels Affect Health News Diffusion

    PubMed Central

    Kim, Hyun Suk

    2015-01-01

    This study examined how intrinsic as well as perceived message features affect the extent to which online health news stories prompt audience selections and social retransmissions, and how news-sharing channels (e-mail vs. social media) shape what goes viral. The study analyzed actual behavioral data on audience viewing and sharing of New York Times health news articles, and associated article content and context data. News articles with high informational utility and positive sentiment invited more frequent selections and retransmissions. Articles were also more frequently selected when they presented controversial, emotionally evocative, and familiar content. Informational utility and novelty had stronger positive associations with e-mail-specific virality, while emotional evocativeness, content familiarity, and exemplification played a larger role in triggering social media-based retransmissions. PMID:26441472

  9. Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators

    PubMed Central

    Wang, Wei; Hong, Jeong S.; Rab, Andras; Sorscher, Eric J.; Kirk, Kevin L.

    2016-01-01

    W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3–5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein. PMID:27007499

  10. Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators.

    PubMed

    Wang, Wei; Hong, Jeong S; Rab, Andras; Sorscher, Eric J; Kirk, Kevin L

    2016-01-01

    W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3-5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein.

  11. Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites.

    PubMed

    Gu, Yuanzheng; Barry, Joshua; Gu, Chen

    2013-05-15

    Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.

  12. Modulation of Ca2+ oscillation and melatonin secretion by BKCa channel activity in rat pinealocytes.

    PubMed

    Mizutani, Hiroya; Yamamura, Hisao; Muramatsu, Makoto; Hagihara, Yumiko; Suzuki, Yoshiaki; Imaizumi, Yuji

    2016-05-01

    The pineal glands regulate circadian rhythm through the synthesis and secretion of melatonin. The stimulation of nicotinic acetylcholine receptor due to parasympathetic nerve activity causes an increase in intracellular Ca(2+) concentration and eventually downregulates melatonin production. Our previous report shows that rat pinealocytes have spontaneous and nicotine-induced Ca(2+) oscillations that are evoked by membrane depolarization followed by Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs). These Ca(2+) oscillations are supposed to contribute to the inhibitory mechanism of melatonin secretion. Here we examined the involvement of large-conductance Ca(2+)-activated K(+) (BKCa) channel conductance on the regulation of Ca(2+) oscillation and melatonin production in rat pinealocytes. Spontaneous Ca(2+) oscillations were markedly enhanced by BKCa channel blockers (1 μM paxilline or 100 nM iberiotoxin). Nicotine (100 μM)-induced Ca(2+) oscillations were also augmented by paxilline. In contrast, spontaneous Ca(2+) oscillations were abolished by BKCa channel opener [3 μM 12,14-dichlorodehydroabietic acid (diCl-DHAA)]. Under whole cell voltage-clamp configurations, depolarization-elicited outward currents were significantly activated by diCl-DHAA and blocked by paxilline. Expression analyses revealed that the α and β3 subunits of BKCa channel were highly expressed in rat pinealocytes. Importantly, the activity of BKCa channels modulated melatonin secretion from whole pineal gland of the rat. Taken together, BKCa channel activation attenuates these Ca(2+) oscillations due to depolarization-synchronized Ca(2+) influx through VDCCs and results in a recovery of reduced melatonin secretion during parasympathetic nerve activity. BKCa channels may play a physiological role for melatonin production via a negative-feedback mechanism.

  13. Gating of Recombinant Small-Conductance Ca-activated K+ Channels by Calcium

    PubMed Central

    Hirschberg, Birgit; Maylie, James; Adelman, John P.; Marrion, Neil V.

    1998-01-01

    Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 μM Ca2+ and was maximal above 2 μM Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was ∼0.6 in 1 μM free Ca2+. A lower open probability of ∼0.05 in 1 μM Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches. PMID:9524139

  14. 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

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

    PubMed

    Liedtke, W; Choe, Y; Martí-Renom, M A; Bell, A M; Denis, C S; Sali, A; Hudspeth, A J; Friedman, J M; Heller, S

    2000-10-27

    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 nervous system, the channel is expressed in 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.

  16. PKA and phosphatases attached to the Ca(V)1.2 channel regulate channel activity in cell-free patches.

    PubMed

    Xu, Jianjun; Yu, Lifeng; Minobe, Etsuko; Lu, Liting; Lei, Ming; Kameyama, Masaki

    2016-01-15

    Calmodulin (CaM) + ATP can reprime voltage-gated L-type Ca(2+) channels (Ca(V)1.2) in inside-out patches for activation, but this effect decreases time dependently. This suggests that the Ca(V)1.2 channel activity is regulated by additional cytoplasmic factors. To test this hypothesis, we examined the role of cAMP-dependent protein kinase A (PKA) and protein phosphatases in the regulation of Ca(V)1.2 channel activity in the inside-out mode in guinea pig ventricular myocytes. Ca(V)1.2 channel activity quickly disappeared after the patch was excised from the cell and recovered to only 9% of that in the cell-attached mode on application of CaM + ATP at 10 min after the inside out. However, immediate exposure of the excised patch to the catalytic subunit of PKA + ATP or the nonspecific phosphatase inhibitor okadaic acid significantly increased the Ca(V)1.2 channel activity recovery by CaM + ATP (114 and 96%, respectively) at 10 min. Interestingly, incubation of the excised patches with cAMP + ATP also increased CaM/ATP-induced Ca(V)1.2 channel activity recovery (108%), and this effect was blocked by the nonspecific protein kinase inhibitor K252a. The channel activity in the inside-out mode was not maintained by either catalytic subunit of PKA or cAMP + ATP in the absence of CaM, but was stably maintained in the presence of CaM for more than 40 min. These results suggest that PKA and phosphatase(s) attached on or near the Ca(V)1.2 channel regulate the basal channel activity, presumably through modulation of the dynamic CaM interaction with the channel.

  17. Voltage dependence and pH regulation of human polycystin-2-mediated cation channel activity.

    PubMed

    Gonzalez-Perrett, Silvia; Batelli, Marisa; Kim, Keetae; Essafi, Makram; Timpanaro, Gustavo; Moltabetti, Nicolas; Reisin, Ignacio L; Arnaout, M Amin; Cantiello, Horacio F

    2002-07-12

    Polycystin-2, the product of the human PKD2 gene, whose mutations cause autosomal dominant polycystic kidney disease, is a large conductance, Ca(2+)-permeable non-selective cation channel. Polycystin-2 is functionally expressed in the apical membrane of the human syncytiotrophoblast, where it may play a role in the control of fetal electrolyte homeostasis. Little is known, however, about the mechanisms that regulate polycystin-2 channel function. In this study, the role of pH in the regulation of polycystin-2 was assessed by ion channel reconstitution of both apical membranes of human syncytiotrophoblast and the purified FLAG-tagged protein from in vitro transcribed/translated material. A kinetic analysis of single channel currents, including dwell time histograms, confirmed two open and two close states for spontaneous channel behavior and a strong voltage dependence of the open probability of the channel (P(o)). A reduction of cis pH (pH(cis)) decreased P(o) and shifted the voltage dependence of channel function but had no effect on the single channel conductance. An increase in pH(cis), in contrast, increased NP(o) (channel number times P(o)). Elimination of the H(+) chemical gradient did not reverse the low pH(cis) inhibition of polycystin-2. Similar findings confirmed the pH effect on the in vitro translated, FLAG-tagged purified polycystin-2. The data indicate the presence of an H(+) ion regulatory site in the channel protein, which is accessible from the cytoplasmic side of the protein. This protonation site controls polycystin-2 cation-selective channel activity.

  18. Ba2+ release from soda glass modifies single maxi K+ channel activity in patch clamp experiments.

    PubMed Central

    Copello, J; Simon, B; Segal, Y; Wehner, F; Ramanujam, V M; Alcock, N; Reuss, L

    1991-01-01

    Glasses used to fabricate patch pipettes may release components which affect ion channels (Cota, G., and C.M. Armstrong. 1988. Biophys. J. 53:107-109; Furman, R.E., and J.C. Tanaka. 1988. Biophys. J. 53:287-292; Rojas, L., and C. Zuazaga. 1988. Neurosci. Lett. 88:39-44). The gating properties of maxi K+ channels from Necturus gallbladder epithelium depend on whether borosilicate glass (BG) or blue tip hematocrit glass (SG) is used to construct the patch pipettes. The data are consistent with solubilization from SG of a component which exerts voltage-dependent, cytosolic-side specific block, closely resembling "slow block" by Ba2+ ions. Ringer's solution preincubated with SG, but not with BG, blocked inside-out maxi K+ channels when used as bathing solution. Mass spectrometry revealed that Ba2+ is released by the glass from fast and slow-release compartments (SG contains 3% wt/wt BaO), and is the only ion found in the solution at concentrations consistent with the observed channel block. Additionally, SG released O2-, Na+, Ca2+, and Mg2+, all to micromolar concentrations. These elements do not interfere with maxi K+ channels but they could in principle alter the properties of other ion channels. Thus, screening for channel-modifying substances released by the glass may be necessary for the adequate interpretation of patch-clamp results. PMID:1742460

  19. Calcium entry via TRPC1 channels activates chloride currents in human glioma cells

    PubMed Central

    Cuddapah, Vishnu Anand; Turner, Kathryn L.; Sontheimer, Harald

    2012-01-01

    Malignant gliomas are highly-invasive brain cancers that carry a dismal prognosis. Recent studies indicate that Cl− channels facilitate glioma cell invasion by promoting hydrodynamic cell shape and volume changes. Here we asked how Cl− channels are regulated in the context of migration. Using patch-clamp recordings we show Cl− currents are activated by physiological increases of [Ca2+]i to 65 and 180 nM. Cl− currents appear to be mediated by ClC-3, a voltage-gated, CaMKII-regulated Cl− channel highly expressed by glioma cells. ClC-3 channels colocalized with TRPC1 on caveolar lipid rafts on glioma cell processes. Using perforated-patch electrophysiological recordings, we demonstrate that inducible knockdown of TRPC1 expression with shRNA significantly inhibited glioma Cl− currents in a Ca2+-dependent fashion, placing Cl− channels under the regulation of Ca2+ entry via TRPC1. In chemotaxis assays epidermal growth factor (EGF)-induced invasion was inhibition by TRPC1 knockdown to the same extent as pharmacological block of Cl− channels. Thus endogenous glioma Cl− channels are regulated by TRPC1. Cl− channels could be an important downstream target of TRPC1 in many other cells types, coupling elevations in [Ca2+]i to the shape and volume changes associated with migrating cells. PMID:23261316

  20. Negative-shift activation, current reduction and resurgent currents induced by β-toxins from Centruroides scorpions in sodium channels.

    PubMed

    Schiavon, Emanuele; Pedraza-Escalona, Martha; Gurrola, Georgina B; Olamendi-Portugal, Timoteo; Corzo, Gerardo; Wanke, Enzo; Possani, Lourival D

    2012-02-01

    The β-toxins purified from the New World scorpion venoms of the Centruroides species affect several voltage-gated sodium channels (VGSCs) and thus are essential tools not only for the discrimination of different channel sub-types but also for studying the structure-function relationship between channels and toxins. This communication reports the results obtained with four different peptides purified from three species of Centruroides scorpions and assayed on seven distinct isoforms of VGSC (Na(v)1.1-Na(v)1.7) by specific functional analysis conducted through single cell electrophysiology. The toxins studied were CssII from Centruroides suffusus suffusus, Cll1 and Cll2 from Centruroides limpidus limpidus and a novel toxin from Centruroides noxius, which was characterized for the first time here. It has 67 amino acid residues and four disulfide bridges with a molecular mass of 7626 Da. Three different functional features were identified: current reduction of macroscopic conductance, left shift of the voltage-dependent activation and induction of resurgent currents at negative voltages following brief, strong depolarizations. The isoforms which revealed to be more affected resulted to be Na(v)1.6 > 1.1 > 1.2 and, for the first time, a β-toxin is here shown to induce resurgent current also in isoforms different from Na(v)1.6. Additionally, these results were analyzed with molecular modelling. In conclusion, although the four toxins have a high degree of identity, they display tri-modal function, each of which shows selectivity among the different sub-types of Na+ -channels. Thus, they are invaluable as tools for structure-function studies of β-toxins and offer a basis for the design of novel ion channel-specific drugs.

  1. Regulation of ion channels and transporters by AMP-activated kinase (AMPK)

    PubMed Central

    Lang, Florian; Föller, Michael

    2014-01-01

    The energy-sensing AMP-activated kinase AMPK ensures survival of energy-depleted cells by stimulating ATP production and limiting ATP utilization. Both energy production and energy consumption are profoundly influenced by transport processes across the cell membane including channels, carriers and pumps. Accordingly, AMPK is a powerful regulator of transport across the cell membrane. AMPK regulates diverse K+ channels, Na+ channels, Ca2+ release activated Ca2+ channels, Cl- channels, gap junctional channels, glucose carriers, Na+/H+-exchanger, monocarboxylate-, phosphate-, creatine-, amino acid-, peptide- and osmolyte-transporters, Na+/Ca2+-exchanger, H+-ATPase and Na+/K+-ATPase. AMPK activates ubiquitin ligase Nedd4–2, which labels several plasma membrane proteins for degradation. AMPK further regulates transport proteins by inhibition of Rab GTPase activating protein (GAP) TBC1D1. It stimulates phosphatidylinositol 3-phosphate 5-kinase PIKfyve and inhibits phosphatase and tensin homolog (PTEN) via glycogen synthase kinase 3β (GSK3β). Moreover, it stabilizes F-actin as well as downregulates transcription factor NF-κB. All those cellular effects serve to regulate transport proteins. PMID:24366036

  2. Coupling between Voltage Sensors and Activation Gate in Voltage-gated K+ Channels

    PubMed Central

    Lu, Zhe; Klem, Angela M.; Ramu, Yajamana

    2002-01-01

    Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1–S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5–S6, equivalent to M1–M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4–S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues. PMID:12407078

  3. Satellite glial cells in situ within mammalian prevertebral ganglia express K+ channels active at rest potential.

    PubMed

    Gola, M; Niel, J P; Delmas, P; Jacquet, G

    1993-10-01

    Patch-clamp experiments were performed on satellite glial cells wrapped around sympathetic neurons in the rabbit coeliac ganglion. With the cleaning method used, the glial cells could be kept in place and were directly accessible to the patch-clamp pipettes. Whole-cell recordings showed that glial cells had almost ohmic properties. Their resting potential (-79.1 +/- 1.2 mV) was found to be very nearly the same as the K+ reversal potential and approximately 20 mV more negative than that of the neurons they encapsulated. Unitary currents from ionic channels present in the glial membrane were recorded in the cell-attached configuration with pipettes filled with various amounts of K+, Na+ and gluconate. Only K(+)-selective channels with slight inwardly rectifying properties (in the presence of 150 mM [K+]o) were detected. These channels were active (Po = 0.7-0.8) at the cell resting potential. The channel conductance, but not its opening probability, was dependent on the [K+] in the pipette. Cl(-)-selective channels (outwardly rectifying and large conductance channels) were detected in excised patches. The properties of the K+ channels (increased inward current with [K+] and detectable outward current at low [K+]) are well suited for siphoning the K+ released by active neurons.

  4. Modulation of channel activity and gadolinium block of MscL by static magnetic fields.

    PubMed

    Petrov, Evgeny; Martinac, Boris

    2007-02-01

    The magnetic field of the Earth has for long been known to influence the behaviour and orientation of a variety of living organisms. Experimental studies of the magnetic sense have, however, been impaired by the lack of a plausible cellular and/or molecular mechanism providing meaningful explanation for detection of magnetic fields by these organisms. Recently, mechanosensitive (MS) ion channels have been implied to play a role in magnetoreception. In this study we have investigated the effect of static magnetic fields (SMFs) of moderate intensity on the activity and gadolinium block of MscL, the bacterial MS channel of large conductance, which has served as a model channel to study the basic physical principles of mechanosensory transduction in living cells. In addition to showing that direct application of the magnetic field decreased the activity of the MscL channel, our study demonstrates for the first time that SMFs can reverse the effect of gadolinium, a well-known blocker of MS channels. The results of our study are consistent with a notion that (1) the effects of SMFs on the MscL channels may result from changes in physical properties of the lipid bilayer due to diamagnetic anisotropy of phospholipid molecules and consequently (2) cooperative superdiamagnetism of phospholipid molecules under influence of SMFs could cause displacement of Gd(3+) ions from the membrane bilayer and thus remove the MscL channel block.

  5. 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.

  6. Upregulation of swelling-activated Cl− channel sensitivity to cell volume by activation of EGF receptors in murine mammary cells

    PubMed Central

    Abdullaev, Iskandar F; Sabirov, Ravshan Z; Okada, Yasunobu

    2003-01-01

    Whole-cell recordings showed that, in mouse mammary C127 cells transfected with the full genome of the bovine papilloma virus (BPV), a hypotonic challenge induced the activation of outwardly rectifying Cl− currents with a peak amplitude 2.7 times greater than that in control C127 cells. Cell-attached single-channel recordings showed that BPV-induced augmentation of the peak amplitude of the whole-cell current could not chiefly be explained by a small increase (1.2 times) in unitary conductance. There was no difference between control and BPV-transfected cells in the osmotic cell swelling rate, and hence, osmotic water permeability. However, a plot of the whole-cell current density as a function of cell volume, which was measured simultaneously, showed that the BPV-transfected cells had a strikingly greater volume sensitivity than control cells. Since the E5 protein of BPV has been reported to induce constitutive activation of the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor in a variety of cell lines including C127 cells, effects of the growth factors on volume-sensitive outwardly rectifying (VSOR) Cl− currents were examined in C127 cells. Application of PDGF peptides failed to affect the Cl− currents in control and BPV-transfected cells, although C127 cells are known to endogenously express PDGF receptors. In contrast, EGF peptides significantly increased the VSOR Cl− current in control cells. However, they failed to induce further augmentation of the current in BPV-transfected cells. VSOR Cl− currents were inhibited by tyrphostin B46, an inhibitor of the EGF receptor tyrosine kinase, in both control and BPV-transfected cells. The IC50 value in BPV-transfected cells (12 μm) was lower than that in control cells (31 μm). However, the VSOR Cl− currents in both cell types were insensitive to tyrphostin AG1296, an inhibitor of the PDGF receptor tyrosine kinase. The rate of regulatory volume decrease (RVD) was

  7. Faster voltage-dependent activation of Na+ channels in growth cones versus somata of neuroblastoma N1E-115 cells.

    PubMed Central

    Zhang, J; Loew, L M; Davidson, R M

    1996-01-01

    Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells. PMID:8913589

  8. Glial potassium channels activated by neuronal firing or intracellular cyclic AMP in Helix.

    PubMed

    Gommerat, I; Gola, M

    1996-09-15

    1. Cell-attached and whole cell patch clamp experiments were performed on satellite glial cells adhering to the cell body of neurones in situ within the nervous system of the snail Helix pomatia. The underlying neurone was under current or voltage-clamp control. 2. Neuronal firing induced a delayed (20-30 s) persistent (3-4 min) increase in the opening probability of glial K+ channels. The channels were also activated by perfusing the ganglion with a depolarizing high-K+ saline, except when the underlying neurone was prevented from depolarizing under voltage-clamp conditions. 3. Two K(+)-selective channels were detected in the glial membrane. The channel responding to neuronal firing was present in 95% of the patches (n = 393). It had a unitary conductance of 56 pS, a Na+ :K+ permeability ratio < 0.02 and displayed slight inward rectification in symmetrical [K+] conditions. It was sensitive to TEA, Ba2+ and Cs+. The following results refer to this channel as studied in the cell-attached configuration. 4. The glial K+ channel was activated by bath application of the membrane-permeant cyclic AMP derivatives 8-bromo-cAMP and dibutyryl-cAMP, the adenylyl cyclase activator forskolin and the diesterase inhibitors IBMX, theophylline and caffeine. It was insensitive to cyclic GMP activators and to conditions that might alter the intracellular [Ca2+] (ionomycin, low-Ca2+ saline and Ca2+ channel blockers). 5. The forskolin-induced changes in channel behaviour (open and closed time distributions, burst duration, short and long gaps within bursts) could be accounted for by a four-state model (3 closed states, 1 open state) by simply changing one of the six rate parameters. 6. The present results suggest that the signal sent by an active neurone to satellite glial cells is confined to the glial cells round that neurone. The effect of this signal on the class of glial K+ channels studied can be mimicked by an increase in glial cAMP concentration. The subsequent delayed opening

  9. Glial potassium channels activated by neuronal firing or intracellular cyclic AMP in Helix.

    PubMed Central

    Gommerat, I; Gola, M

    1996-01-01

    1. Cell-attached and whole cell patch clamp experiments were performed on satellite glial cells adhering to the cell body of neurones in situ within the nervous system of the snail Helix pomatia. The underlying neurone was under current or voltage-clamp control. 2. Neuronal firing induced a delayed (20-30 s) persistent (3-4 min) increase in the opening probability of glial K+ channels. The channels were also activated by perfusing the ganglion with a depolarizing high-K+ saline, except when the underlying neurone was prevented from depolarizing under voltage-clamp conditions. 3. Two K(+)-selective channels were detected in the glial membrane. The channel responding to neuronal firing was present in 95% of the patches (n = 393). It had a unitary conductance of 56 pS, a Na+ :K+ permeability ratio < 0.02 and displayed slight inward rectification in symmetrical [K+] conditions. It was sensitive to TEA, Ba2+ and Cs+. The following results refer to this channel as studied in the cell-attached configuration. 4. The glial K+ channel was activated by bath application of the membrane-permeant cyclic AMP derivatives 8-bromo-cAMP and dibutyryl-cAMP, the adenylyl cyclase activator forskolin and the diesterase inhibitors IBMX, theophylline and caffeine. It was insensitive to cyclic GMP activators and to conditions that might alter the intracellular [Ca2+] (ionomycin, low-Ca2+ saline and Ca2+ channel blockers). 5. The forskolin-induced changes in channel behaviour (open and closed time distributions, burst duration, short and long gaps within bursts) could be accounted for by a four-state model (3 closed states, 1 open state) by simply changing one of the six rate parameters. 6. The present results suggest that the signal sent by an active neurone to satellite glial cells is confined to the glial cells round that neurone. The effect of this signal on the class of glial K+ channels studied can be mimicked by an increase in glial cAMP concentration. The subsequent delayed opening

  10. Bisphenol A activates BK channels through effects on α and β1 subunits

    PubMed Central

    Rottgen, Trey S; Fancher, Ibra S; Asano, Shinichi; Widlanski, Theodore S; Dick, Gregory M

    2014-01-01

    We demonstrated previously that BK (KCa1.1) channel activity (NPo) increases in response to bisphenol A (BPA). Moreover, BK channels containing regulatory β1 subunits were more sensitive to the stimulatory effect of BPA. How BPA increases BK channel NPo remains mostly unknown. Estradiol activates BK channels by binding to an extracellular site, but neither the existence nor location of a BPA binding site has been demonstrated. We tested the hypothesis that an extracellular binding site is responsible for activation of BK channels by BPA. We synthesized membrane-impermeant BPA-monosulfate (BPA-MS) and used patch clamp electrophysiology to study channels composed of α or α + β1 subunits in cell-attached (C-A), whole-cell (W-C), and inside-out (I-O) patches. In C-A patches, bath application of BPA-MS (100 μM) had no effect on the NPo of BK channels, regardless of their subunit composition. Importantly, however, subsequent addition of membrane-permeant BPA (100 μM) increased the NPo of both α and α + β1 channels in C-A patches. The C-A data indicate that in order to alter BK channel NPo, BPA must interact with the channel itself (or some closely associated partner) and diffusible messengers are not involved. In W-C patches, 100 μM BPA-MS activated current in cells expressing α subunits, whereas cells expressing α + β1 subunits responded similarly to a log-order lower concentration (10 μM). The W-C data suggest that an extracellular activation site exists, but do not eliminate the possibility that an intracellular site may also be present. In I-O patches, where the cytoplasmic face was exposed to the bath, BPA-MS had no effect on the NPo of BK α subunits, but BPA increased it. BPA-MS increased the NPo of α + β1 channels in I-O patches, but not as much as BPA. We conclude that BPA activates BK α via an extracellular site and that BPA-sensitivity is increased by the β1 subunit, which may also constitute part of an intracellular binding site. PMID

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

    PubMed

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

    2015-06-22

    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.

  12. 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.

  13. Zinc supplementation affects the activity patterns of rural Guatemalan infants.

    PubMed

    Bentley, M E; Caulfield, L E; Ram, M; Santizo, M C; Hurtado, E; Rivera, J A; Ruel, M T; Brown, K H

    1997-07-01

    Zinc deficiency has been associated with growth deficits, reduced dietary intake and appetite, and has been hypothesized to result in reduced activity. This randomized, double-blind, placebo-controlled study examined whether 10 mg of oral zinc as zinc sulfate, given daily for up to 7 mo, affected activity patterns of 85 Guatemalan infants recruited at 6-9 mo of age. Infant activity was assessed by time sampling-observation method at 10-min intervals during a 12-h data collection period, at base line, 3 and 7 mo follow-up. Motor development and the percentage of time infants were observed in various positions (being carried, lying down, sitting, crawling, standing or walking) and engaged in various activities (eating, sleeping, resting, crying/whining or playing) were compared by treatment group. No differences in motor development were observed by treatment group. However, at follow-up 2 (after 7 mo of supplementation), zinc-supplemented infants were significantly more frequently observed sitting up compared with lying down, and were playing during 4.18 +/- 1.95% (P < 0.05) more observations than unsupplemented infants. They were also somewhat less likely to be observed crying or whining (P < 0.10) compared with those receiving the placebo. These effects are independent of other factors including infant age, motor development, sex, maternal education, family socioeconomic status and nutritional status at base line. Further research must be conducted to determine the long-term developmental importance of these differences in activity patterns associated with zinc supplementation in this setting.

  14. 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.

  15. Activity-induced internalization and rapid degradation of sodium channels in cultured fetal neurons

    PubMed Central

    1996-01-01

    A regulatory mechanism for neuronal excitability consists in controlling sodium channel density at the plasma membrane. In cultured fetal neurons, activation of sodium channels by neurotoxins, e.g., veratridine and alpha-scorpion toxin (alpha-ScTx) that enhance the channel open state probability induced a rapid down-regulation of surface channels. Evidence that the initial step of activity-induced sodium channel down-regulation is mediated by internalization was provided by using 125I-alpha-ScTx as both a channel probe and activator. After its binding to surface channels, the distribution of 125I-alpha-ScTx into five subcellular compartments was quantitatively analyzed by EM autoradiography. 125I-alpha-ScTx was found to accumulate in tubulovesicular endosomes and disappear from the cell surface in a time-dependent manner. This specific distribution was prevented by addition of tetrodotoxin (TTX), a channel blocker. By using a photoreactive derivative to covalently label sodium channels at the surface of cultured neurons, we further demonstrated that they are degraded after veratridine-induced internalization. A time-dependent decrease in the amount of labeled sodium channel alpha subunit was observed after veratridine treatment. After 120 min of incubation, half of the alpha subunits were cleaved. This degradation was prevented totally by TTX addition and was accompanied by the appearance of an increasing amount of a 90-kD major proteolytic fragment that was already detected after 45-60 min of veratridine treatment. Exposure of the photoaffinity-labeled cells to amphotericin B, a sodium ionophore, gave similar results. In this case, degradation was prevented when Na+ ions were substituted by choline ions and not blocked by TTX. After veratridine- or amphotericin B-induced internalization of sodium channels, breakdown of the labeled alpha subunit was inhibited by leupeptin, while internalization was almost unaffected. Thus, cultured fetal neurons are capable of

  16. Non-selective voltage-activated cation channel in the human red blood cell membrane.

    PubMed

    Kaestner, L; Bollensdorff, C; Bernhardt, I

    1999-02-04

    Using the patch-clamp technique, a non-selective voltage-activated Na+ and K+ channel in the human red blood cell membrane was found. The channel operates only at positive membrane potentials from about +30 mV (inside positive) onwards. For sodium and potassium ions, similar conductances of about 21 pS were determined. Together with the recently described K+(Na+)/H+ exchanger, this channel is responsible for the increase of residual K+ and Na+ fluxes across the human red blood cell membrane when the cells are suspended in low ionic strength medium.

  17. Preliminary microwave irradiation of water solutions changes their channel-modifying activity.

    PubMed

    Fesenko, E E; Geletyuk, V I; Kazachenko, V N; Chemeris, N K

    1995-06-05

    Earlier we have shown that millimetre microwaves (42.25 GHz) of non-thermal power, upon direct admittance into an experiment bath, greatly influence activation characteristics of single Ca(2+)-dependent K+ channels (in particular, the channel open state probability, Po). Here we present new data showing that similar changes in Po arise due to the substitution of a control bath solution for a preliminary microwave irradiated one of the same composition (100 mmol/l KCl with Ca2+ added), with irradiation time being 20-30 min. Therefore, due to the exposure to the field the solution acquires some new properties that are important for the channel activity. The irradiation terminated, the solution retains a new state for at least 10-20 min (solution memory). The data suggest that the effects of the field on the channels are mediated, at least partially, by changes in the solution properties.

  18. Molecular heterogeneity of large-conductance calcium-activated potassium channels in canine intracardiac ganglia.

    PubMed

    Selga, Elisabet; Pérez-Serra, Alexandra; Moreno-Asso, Alba; Anderson, Seth; Thomas, Kristen; Desai, Mayurika; Brugada, Ramon; Pérez, Guillermo J; Scornik, Fabiana S

    2013-01-01

    Large conductance calcium-activated potassium (BK) channels are widely expressed in the nervous system. We have recently shown that principal neurons from canine intracardiac ganglia (ICG) express a paxilline- and TEA-sensitive BK current, which increases neuronal excitability. In the present work, we further explore the molecular constituents of the BK current in canine ICG. We found that the β1 and β4 regulatory subunits are expressed in ICG. Single channel voltage-dependence at different calcium concentrations suggested that association of the BKα with a particular β subunit was not enough to explain the channel activity in this tissue. Indeed, we detected the presence of several splice variants of the BKα subunit. In conclusion, BK channels in canine ICG may result from the arrangement of different BKα splice variants, plus accessory β subunits. The particular combinations expressed in canine IC neurons likely rule the excitatory role of BK current in this tissue.

  19. Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation.

    PubMed

    Gründemann, Jan; Clark, Beverley A

    2015-09-22

    Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, K(Ca)3.1) by local, activity-dependent calcium (Ca(2+)) influx at nodes of Ranvier via a T-type voltage-gated Ca(2+) current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells.

  20. Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation

    PubMed Central

    Gründemann, Jan; Clark, Beverley A.

    2015-01-01

    Summary Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, KCa3.1) by local, activity-dependent calcium (Ca2+) influx at nodes of Ranvier via a T-type voltage-gated Ca2+ current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells. PMID:26344775

  1. β1-Subunit of the Ca2+-activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle

    PubMed Central

    Petkov, Georgi V; Bonev, Adrian D; Heppner, Thomas J; Brenner, Robert; Aldrich, Richard W; Nelson, Mark T

    2001-01-01

    The large-conductance calcium-activated potassium (BK) channel plays an important role in controlling membrane potential and contractility of urinary bladder smooth muscle (UBSM). These channels are composed of a pore-forming α-subunit and an accessory, smooth muscle-specific, β1-subunit. Our aim was to determine the functional role of the β1-subunit of the BK channel in controlling the contractions of UBSM by using BK channel β1-subunit ‘knock-out’ (KO) mice. The β-galactosidase reporter (lacZ gene) was targeted to the β1 locus, which provided the opportunity to examine the expression of the β1-subunit in UBSM. Based on this approach, the β1-subunit is highly expressed in UBSM. BK channels lacking β1-subunits have reduced activity, consistent with a shift in BK channel voltage/Ca2+ sensitivity. Iberiotoxin, an inhibitor of BK channels, increased the amplitude and decreased the frequency of phasic contractions of UBSM strips from control mice. The effects of the β1-subunit deletion on contractions were similar to the effect of iberiotoxin on control mice. The UBSM strips from β1-subunit KO mice had elevated phasic contraction amplitude and decreased frequency when compared to control UBSM strips. Iberiotoxin increased the amplitude and frequency of phasic contractions, and UBSM tone of UBSM strips from β1-subunit KO mice, suggesting that BK channels still regulate contractions in the absence of the β1-subunit. The results indicate that the β1-subunit, by modulating BK channel activity, plays a significant role in the regulation of phasic contractions of the urinary bladder. PMID:11731577

  2. Downregulation of Purkinje Cell Activity by Modulators of Small Conductance Calcium-Activated Potassium Channels In Rat Cerebellum

    PubMed Central

    Karelina, T. V.; Stepanenko, Yu. D.; Abushik, P. A.; Sibarov, D. A.; Antonov, S. M.

    2016-01-01

    Small-conductance calcium-activated potassium channels (SK channels) are widely expressed in CNS tissues. Their functions, however, have not been well studied. Participation of SK channels in Purkinje cell (PC) pacemaker activity has been studied predominantly in vitro. Here we studied for the first time the effects of SK channel activation by NS309 or CyPPA on the PC simple spike frequency in vivo in adult (3 – 6 months) and aged (22 – 28 months) rats using extracellular microelectrode recordings. Both pharmacological agents caused a statistically significant decrease in the PC simple spike frequency. The maximum value of the decrease in the simple spike frequency did not depend on age, whereas a statistically significant inhibition of the spike frequency was achieved faster in aged animals than in adult ones. In experiments on cultured neurons PCs were identified by the expression of calbindin as the PC-specific marker. Registration of transmembrane currents in cerebellar neurons revealed the direct action of NS309 and CyPPA on the SK channels of PC consisted in the enhancement of outward potassium currents and action potential after-hyperpolarization. Thus, SK channel activators can compensate for age-related changes of the autorhythmic functions of the cerebellum. PMID:28050270

  3. Urinary prostasin: a candidate marker of epithelial sodium channel activation in humans.

    PubMed

    Olivieri, Oliviero; Castagna, Annalisa; Guarini, Patrizia; Chiecchi, Laura; Sabaini, Gherardo; Pizzolo, Francesca; Corrocher, Roberto; Righetti, Pier Giorgio

    2005-10-01

    Prostasin is a serine peptidase hypothesized to regulate epithelial sodium channel (ENaC) activity in animals or on in vitro cultured cells. We investigated whether urinary prostasin may be a candidate marker of ENaC activation in humans. We studied 10 healthy volunteers and 8 hypertensive patients with raised aldosterone-to-renin ratio before and after spironolactone or saline/Florinef suppression test, respectively. Four healthy subjects were also studied before and after saline. Urinary prostasin was evaluated by SDS-PAGE, 2D maps, and Western blotting. Every sample of normotensive individuals was compared with the corresponding sample of urine collected after spironolactone or saline; every sample of hypertensive patients was compared with the corresponding sample of urine collected after saline or Florinef. Prostasin was detectable in all subjects regardless of gender, dietary sodium intake, and spironolactone treatment. Spironolactone (100 mg) increased urinary Na+/K+ ratio and decreased urinary prostasin in normotensives in whom the renin/aldosterone axis was activated by a low Na+ intake, but it was ineffective in individuals with high Na+ intake. Saline infusion also reduced prostasin in normotensive subjects. In contrast, prostasin paradoxically increased in urine of patients affected by primary aldosteronism after volume expansion. By 2D immunoblotting, several protein isoforms were observed, some of them being overexpressed after inhibition tests in patients with primary aldosteronism. In addition to a "basal" aliquot of prostasin, constitutively released in human urine regardless of sodium balance and aldosterone activation, there exists a second "aldosterone-responsive" aliquot modulated by Na+ intake and potentially suitable as candidate marker of ENaC activation.

  4. How Lipid Membranes Affect Pore Forming Toxin Activity.

    PubMed

    Rojko, Nejc; Anderluh, Gregor

    2015-12-15

    Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure-function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally

  5. Kaempferol enhances endothelium-dependent relaxation in the porcine coronary artery through activation of large-conductance a2+-activated K+ channels

    PubMed Central

    Xu, Y C; Leung, S W S; Leung, G P H; Man, R Y K

    2015-01-01

    Background and Purpose Kaempferol, a plant flavonoid present in normal human diet, can modulate vasomotor tone. The present study aimed to elucidate the signalling pathway through which this flavonoid enhanced relaxation of vascular smooth muscle. Experimental Approach The effect of kaempferol on the relaxation of porcine coronary arteries to endothelium-dependent (bradykinin) and -independent (sodium nitroprusside) relaxing agents was studied in an in vitro organ chamber setup. The whole-cell patch-clamp technique was used to determine the effect of kaempferol on potassium channels in porcine coronary artery smooth muscle cells (PCASMCs). Key Results At a concentration without direct effect on vascular tone, kaempferol (3 × 10−6 M) enhanced relaxations produced by bradykinin and sodium nitroprusside. The potentiation by kaempferol of the bradykinin-induced relaxation was not affected by Nω-nitro-L-arginine methyl ester, an inhibitor of NO synthase (10−4 M) or TRAM-34 plus UCL 1684, inhibitors of intermediate- and small-conductance calcium-activated potassium channels, respectively (10−6 M each), but was abolished by tetraethylammonium chloride, a non-selective inhibitor of calcium-activated potassium channels (10−3 M), and iberiotoxin, a selective inhibitor of large-conductance calcium-activated potassium channel (KCa1.1; 10−7 M). Iberiotoxin also inhibited the potentiation by kaempferol of sodium nitroprusside-induced relaxations. Kaempferol stimulated an outward-rectifying current in PCASMCs, which was abolished by iberiotoxin. Conclusions and Implications The present results suggest that, in smooth muscle cells of the porcine coronary artery, kaempferol enhanced relaxations caused by endothelium-derived and exogenous NO as well as those due to endothelium-dependent hyperpolarization. This vascular effect of kaempferol involved the activation of KCa1.1 channels. PMID:25652142

  6. Calcium regulation of HCN channels supports persistent activity in a multiscale model of neocortex

    PubMed Central

    McDougal, Robert A.; Bulanova, Anna S.; Zeki, Mustafa; Lakatos, Peter; Terman, David; Hines, Michael L.; Lytton, William W.

    2016-01-01

    Neuronal persistent activity has been primarily assessed in terms of electrical mechanisms, without attention to the complex array of molecular events that also control cell excitability. We developed a multiscale neocortical model proceeding from the molecular to the network level to assess the contributions of calcium regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in providing additional and complementary support of continuing activation in the network. The network contained 776 compartmental neurons arranged in the cortical layers, connected using synapses containing AMPA/NMDA/GABAA/GABAB receptors. Metabotropic glutamate receptors (mGluR) produced inositol triphosphate (IP3) which caused release of Ca2+ from endoplasmic reticulum (ER) stores, with reuptake by sarco/ER Ca2+-ATP-ase pumps (SERCA), and influence on HCN channels. Stimulus-induced depolarization led to Ca2+ influx via NMDA and voltage-gated Ca2+ channels (VGCCs). After a delay, mGluR activation led to ER Ca2+ release via IP3 receptors. These factors increased HCN channel conductance and produced firing lasting for ~1 minute. The model displayed inter-scale synergies among synaptic weights, excitation/inhibition balance, firing rates, membrane depolarization, calcium levels, regulation of HCN channels, and induction of persistent activity. The interaction between inhibition and Ca2+ at the HCN channel nexus determined a limited range of inhibition strengths for which intracellular Ca2+ could prepare population-specific persistent activity. Interactions between metabotropic and ionotropic inputs to the neuron demonstrated how multiple pathways could contribute in a complementary manner to persistent activity. Such redundancy and complementarity via multiple pathways is a critical feature of biological systems. Mediation of activation at different time scales, and through different pathways, would be expected to protect against disruption, in this case providing

  7. Calcium regulation of HCN channels supports persistent activity in a multiscale model of neocortex.

    PubMed

    Neymotin, S A; McDougal, R A; Bulanova, A S; Zeki, M; Lakatos, P; Terman, D; Hines, M L; Lytton, W W

    2016-03-01

    Neuronal persistent activity has been primarily assessed in terms of electrical mechanisms, without attention to the complex array of molecular events that also control cell excitability. We developed a multiscale neocortical model proceeding from the molecular to the network level to assess the contributions of calcium (Ca(2+)) regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in providing additional and complementary support of continuing activation in the network. The network contained 776 compartmental neurons arranged in the cortical layers, connected using synapses containing AMPA/NMDA/GABAA/GABAB receptors. Metabotropic glutamate receptors (mGluR) produced inositol triphosphate (IP3) which caused the release of Ca(2+) from endoplasmic reticulum (ER) stores, with reuptake by sarco/ER Ca(2+)-ATP-ase pumps (SERCA), and influence on HCN channels. Stimulus-induced depolarization led to Ca(2+) influx via NMDA and voltage-gated Ca(2+) channels (VGCCs). After a delay, mGluR activation led to ER Ca(2+) release via IP3 receptors. These factors increased HCN channel conductance and produced firing lasting for ∼1min. The model displayed inter-scale synergies among synaptic weights, excitation/inhibition balance, firing rates, membrane depolarization, Ca(2+) levels, regulation of HCN channels, and induction of persistent activity. The interaction between inhibition and Ca(2+) at the HCN channel nexus determined a limited range of inhibition strengths for which intracellular Ca(2+) could prepare population-specific persistent activity. Interactions between metabotropic and ionotropic inputs to the neuron demonstrated how multiple pathways could contribute in a complementary manner to persistent activity. Such redundancy and complementarity via multiple pathways is a critical feature of biological systems. Mediation of activation at different time scales, and through different pathways, would be expected to protect against disruption, in

  8. sigma Receptor activation blocks potassium channels and depresses neuroexcitability in rat intracardiac neurons.

    PubMed

    Zhang, Hongling; Cuevas, Javier

    2005-06-01

    The sigma receptors have been implicated in the regulation of the cardiovascular system, and sigma-1 receptor transcripts have been found in parasympathetic intracardiac neurons. However, the cellular function of sigma-1 receptors in these cells remains to be determined. Effects of sigma receptor activation on voltage-activated K(+) channels and action potential firing were studied in isolated intracardiac neurons using whole-cell patch-clamp recording techniques. Activation of sigma receptors reversibly blocked delayed outwardly rectifying potassium channels, large conductance Ca(2+)-sensitive K(+) channels, and the M-current with maximal inhibition >80%. The inhibition of K(+) channels by sigma ligands was dose-dependent, and the rank order potency of (+)-pentazocine > ibogaine > 1,3-di-O-tolyguanidin (DTG) suggests that the effect is mediated by sigma-1 receptor activation. Preincubation of neurons with the irreversible sigma receptor antagonist metaphit blocked DTG-induced inhibition of K(+) channels, confirming that the effect is mediated by sigma receptor activation. Although bath application of sigma ligands depolarized intracardiac neurons, the number of action potentials fired by the cells in response to depolarizing current pulses was decreased in the presence of these drugs. Neither dialysis of the neurons nor application of intracellular 5'-O-(2-thiodiphosphate) trilithium salt inhibited the effect of sigma receptors on K(+) channels, which suggests that the signal transduction pathway does not involve a diffusible cytosolic second messenger or a G protein. Together, these data suggest that sigma-1 receptors are directly coupled to K(+) channels in intracardiac neurons. Furthermore, activation of sigma-1 receptors depresses the excitability of intracardiac neurons and is thus likely to block parasympathetic input to the heart.

  9. Endoxifen, the active metabolite of tamoxifen, inhibits cloned hERG potassium channels.

    PubMed

    Chae, Yun Ju; Lee, Keon Jin; Lee, Hong Joon; Sung, Ki-Wug; Choi, Jin-Sung; Lee, Eun Hui; Hahn, Sang June

    2015-04-05

    The effects of tamoxifen, and its active metabolite endoxifen (4-hydroxy-N-desmethyl-tamoxifen), on hERG currents stably expressed in HEK cells were investigated using the whole-cell patch-clamp technique and an immunoblot assay. Tamoxifen and endoxifen inhibited hERG tail currents at -50mV in a concentration-dependent manner with IC50 values of 1.2 and 1.6μM, respectively. The steady-state activation curve of the hERG currents was shifted to the hyperpolarizing direction in the presence of endoxifen. The voltage-dependent inhibition of hERG currents by endoxifen increased steeply in the voltage range of channel activation. The inhibition by endoxifen displayed a shallow voltage dependence (δ=0.18) in the full activation voltage range. A fast application of endoxifen induced a reversible block of hERG tail currents during repolarization in a concentration-dependent manner, which suggested an interaction with the open state of the channel. Endoxifen also decreased the hERG current elicited by a 5s depolarizing pulse to +60mV to inactivate the hERG currents, suggesting an interaction with the activated (open and/or inactivated) states of the channels. Tamoxifen and endoxifen inhibited the hERG channel protein trafficking to the plasma membrane in a concentration-dependent manner with endoxifen being more potent than tamoxifen. These results indicated that tamoxifen and endoxifen inhibited the hERG current by direct channel blockage and by the disruption of channel trafficking to the plasma membrane in a concentration-dependent manner. A therapeutic concentration of endoxifen inhibited the hERG current by preferentially interacting with the activated (open and/or inactivated) states of the channel.

  10. Glucose deprivation activates diversity of potassium channels in cultured rat hippocampal neurons.

    PubMed

    Velasco, Myrian; García, Esperanza; Onetti, Carlos G

    2006-05-01

    1. Glucose is one of the most important substrates for generating metabolic energy required for the maintenance of cellular functions. Glucose-mediated changes in neuronal firing pattern have been observed in the central nervous system of mammals. K(+) channels directly regulated by intracellular ATP have been postulated as a linkage between cellular energetic metabolism and excitability; the functional roles ascribed to these channels include glucose-sensing to regulate energy homeostasis and neuroprotection under energy depletion conditions. The hippocampus is highly sensitive to metabolic insults and is the brain region most sensitive to ischemic damage. Because the identity of metabolically regulated potassium channels present in hippocampal neurons is obscure, we decided to study the biophysical properties of glucose-sensitive potassium channels in hippocampal neurons. 2. The dependence of membrane potential and the sensitivity of potassium channels to glucose and ATP in rat hippocampal neurons were studied in cell-attached and excised inside-out membrane patches. 3. We found that under hypoglycemic conditions, at least three types of potassium channels were activated; their unitary conductance values were 37, 147, and 241 pS in symmetrical K(+), and they were sensitive to ATP. For K(+) channels with unitary conductance of 37 and 241, when the membrane potential was depolarized the longer closed time constant diminished and this produced an increase in the open-state probability; nevertheless, the 147-pS channels were not voltage-dependent. 4. We propose that neuronal glucose-sensitive K(+) channels in rat hippocampus include subtypes of ATP-sensitive channels with a potential role in neuroprotection during short-term or prolonged metabolic stress.

  11. Allosteric Gating of a Large Conductance Ca-activated K+ Channel

    PubMed Central

    Cox, D.H.; Cui, J.; Aldrich, R.W.

    1997-01-01

    Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca2+. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca2+ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca2+ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca2+ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca2+ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations. PMID:9276753

  12. Pituitary adenylate cyclase activating polypeptide (PACAP) dilates cerebellar arteries through activation of large-conductance Ca(2+)-activated (BK) and ATP-sensitive (K ATP) K (+) channels.

    PubMed

    Koide, Masayo; Syed, Arsalan U; Braas, Karen M; May, Victor; Wellman, George C

    2014-11-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K(+) channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of ATP-sensitive K(+) (KATP) and large-conductance Ca(2+)-activated K(+) (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell KATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca(2+) release events termed Ca(2+) sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50 % in the presence of glibenclamide (a KATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: (1) KATP channel activation and (2) enhanced BK channel activity, likely through increased Ca(2+) spark frequency.

  13. The mechano-activated K+ channels TRAAK and TREK-1 control both warm and cold perception.

    PubMed

    Noël, Jacques; Zimmermann, Katharina; Busserolles, Jérome; Deval, Emanuel; Alloui, Abdelkrim; Diochot, Sylvie; Guy, Nicolas; Borsotto, Marc; Reeh, Peter; Eschalier, Alain; Lazdunski, Michel

    2009-05-06

    The sensation of cold or heat depends on the activation of specific nerve endings in the skin. This involves heat- and cold-sensitive excitatory transient receptor potential (TRP) channels. However, we show here that the mechano-gated and highly temperature-sensitive potassium channels of the TREK/TRAAK family, which normally work as silencers of the excitatory channels, are also implicated. They are important for the definition of temperature thresholds and temperature ranges in which excitation of nociceptor takes place and for the intensity of excitation when it occurs. They are expressed with thermo-TRP channels in sensory neurons. TRAAK and TREK-1 channels control pain produced by mechanical stimulation and both heat and cold pain perception in mice. Expression of TRAAK alone or in association with TREK-1 controls heat responses of both capsaicin-sensitive and capsaicin-insensitive sensory neurons. Together TREK-1 and TRAAK channels are important regulators of nociceptor activation by cold, particularly in the nociceptor population that is not activated by menthol.

  14. RIM determines Ca2+ channel density and vesicle docking at the presynaptic active zone

    PubMed Central

    Han, Yunyun; Kaeser, Pascal S.; Südhof, Thomas C.; Schneggenburger, Ralf

    2012-01-01

    At presynaptic active zones, neurotransmitter release is initiated by the opening of voltage-gated Ca2+ channels close to docked vesicles. The mechanisms that enrich Ca2+ channels at active zones are, however, largely unknown, possibly because of the limited presynaptic accessibility of most synapses. Here, we have established a Cre-lox based conditional knock-out approach at a presynaptically accessible CNS synapse, the calyx of Held, to directly study the functions of RIM proteins. Removal of all RIM1/2 isoforms strongly reduced the presynaptic Ca2+ channel density, revealing a new role of RIM proteins in Ca2+ channel targeting. Removal of RIMs also reduced the readily-releasable pool, paralleled by a similar reduction of the number of docked vesicles, and the Ca2+ channel - vesicle coupling was decreased. Thus, RIM proteins co-ordinately regulate key functions for fast transmitter release: enabling a high presynaptic Ca2+ channel density, and vesicle docking at the active zone. PMID:21262468

  15. STIM1 activates CRAC channels through rotation of the pore helix to open a hydrophobic gate

    PubMed Central

    Yamashita, Megumi; Yeung, Priscilla S.-W.; Ing, Christopher E.; McNally, Beth A.; Pomès, Régis; Prakriya, Murali

    2017-01-01

    Store-operated Ca2+ release-activated Ca2+ (CRAC) channels constitute a major pathway for Ca2+ influx and mediate many essential signalling functions in animal cells, yet how they open remains elusive. Here, we investigate the gating mechanism of the human CRAC channel Orai1 by its activator, stromal interacting molecule 1 (STIM1). We find that two rings of pore-lining residues, V102 and F99, work together to form a hydrophobic gate. Mutations of these residues to polar amino acids produce channels with leaky gates that conduct ions in the resting state. STIM1-mediated channel activation occurs through rotation of the pore helix, which displaces the F99 residues away from the pore axis to increase pore hydration, allowing ions to flow through the V102-F99 hydrophobic band. Pore helix rotation by STIM1 also explains the dynamic coupling between CRAC channel gating and ion selectivity. This hydrophobic gating mechanism has implications for CRAC channel function, pharmacology and disease-causing mutations. PMID:28220789

  16. STIM1 activates CRAC channels through rotation of the pore helix to open a hydrophobic gate

    NASA Astrophysics Data System (ADS)

    Yamashita, Megumi; Yeung, Priscilla S.-W.; Ing, Christopher E.; McNally, Beth A.; Pomès, Régis; Prakriya, Murali

    2017-02-01

    Store-operated Ca2+ release-activated Ca2+ (CRAC) channels constitute a major pathway for Ca2+ influx and mediate many essential signalling functions in animal cells, yet how they open remains elusive. Here, we investigate the gating mechanism of the human CRAC channel Orai1 by its activator, stromal interacting molecule 1 (STIM1). We find that two rings of pore-lining residues, V102 and F99, work together to form a hydrophobic gate. Mutations of these residues to polar amino acids produce channels with leaky gates that conduct ions in the resting state. STIM1-mediated channel activation occurs through rotation of the pore helix, which displaces the F99 residues away from the pore axis to increase pore hydration, allowing ions to flow through the V102-F99 hydrophobic band. Pore helix rotation by STIM1 also explains the dynamic coupling between CRAC channel gating and ion selectivity. This hydrophobic gating mechanism has implications for CRAC channel function, pharmacology and disease-causing mutations.

  17. Gating charges per channel of Ca(V)2.2 channels are modified by G protein activation in rat sympathetic neurons.

    PubMed

    Rebolledo-Antúnez, Santiago; Farías, José M; Arenas, Isabel; García, David E

    2009-06-01

    It has been suggested that voltage-dependent G protein modulation of Ca(V)2.2 channels is carried out at closed states of the channel. Our purpose was to estimate the number of gating charges of Ca(V)2.2 channel in control and G protein-modulated conditions. By using a Cole-Moore protocol we observed a significant delay in Ca(V)2.2 channel activation according to a transit of the channel through a series of closed states before channel opening. If G protein voltage-dependent modulation were carried out at these closed states, then we would have expected a greater Cole-Moore lag in the presence of a neurotransmitter. This prediction was confirmed for noradrenaline, while no change was observed in the presence of angiotensin II, a voltage-insensitive G protein modulator. We used the limiting slope method for calculation of the gating charge per channel. Effective charge z was 6.32+/-0.65 for Ca(V)2.2 channels in unregulated conditions, while GTPgammaS reduced elementary charge by approximately 4 e(0). Accordingly, increased concentration of noradrenaline induced a gradual decrease on z, indicating that this decrement was due to a G protein voltage-sensitive modulation. This paper shows for the first time a significant and reversible decrease in charge transfer of Ca(V)2.2 channels under G protein modulation, which might depend on the activated G protein inhibitory pathway.

  18. Neuronal Ca2+-Activated K+ Channels Limit Brain Infarction and Promote Survival

    PubMed Central

    Liao, Yiliu; Gu, Ning; Rundén-Pran, Elise; Ruth, Peter; Sausbier, Matthias; Storm, Johan F.

    2010-01-01

    Neuronal calcium-activated potassium channels of the BK type are activated by membrane depolarization and intracellular Ca2+ ions. It has been suggested that these channels may play a key neuroprotective role during and after brain ischemia, but this hypothesis has so far not been tested by selective BK-channel manipulations in vivo. To elucidate the in vivo contribution of neuronal BK channels in acute focal cerebral ischemia, we performed middle cerebral artery occlusion (MCAO) in mice lacking BK channels (homozygous mice lacking the BK channel alpha subunit, BK−/−). MCAO was performed in BK−/− and WT mice for 90 minutes followed by a 7-hour-reperfusion period. Coronal 1 mm thick sections were stained with 2,3,5-triphenyltetrazolium chloride to reveal the infarction area. We found that transient focal cerebral ischemia by MCAO produced larger infarct volume, more severe neurological deficits, and higher post-ischemic mortality in BK−/− mice compared to WT littermates. However, the regional cerebral blood flow was not significantly different between genotypes as measured by Laser Doppler (LD) flowmetry pre-ischemically, intra-ischemically, and post-ischemically, suggesting that the different impact of MCAO in BK−/− vs. WT was not due to vascular BK channels. Furthermore, when NMDA was injected intracerebrally in non-ischemic mice, NMDA-induced neurotoxicity was found to be larger in BK−/− mice compared to WT. Whole-cell patch clamp recordings from CA1 pyramidal cells in organotypic hippocampal slice cultures revealed that BK channels contribute to rapid action potential repolarization, as previously found in acute slices. When these cultures were exposed to ischemia-like conditions this induced significantly more neuronal death in BK−/− than in WT cultures. These results indicate that neuronal BK channels are important for protection against ischemic brain damage. PMID:21209897

  19. KCNQ channel openers reverse depressive symptoms via an active resilience mechanism

    PubMed Central

    Friedman, Allyson K.; Juarez, Barbara; Ku, Stacy M.; Zhang, Hongxing; Calizo, Rhodora C.; Walsh, Jessica J.; Chaudhury, Dipesh; Zhang, Song; Hawkins, Angel; Dietz, David M.; Murrough, James W.; Ribadeneira, Maria; Wong, Erik H.; Neve, Rachael L.; Han, Ming-Hu

    2016-01-01

    Less than half of patients suffering from major depressive disorder, a leading cause of disability worldwide, achieve remission with current antidepressants, making it imperative to develop more effective treatment. A new therapeutic direction is emerging from the increased understanding of natural resilience as an active stress-coping process. It is known that potassium (K+) channels in the ventral tegmental area (VTA) are an active mediator of resilience. However, no druggable targets have been identified to potentiate active resilience mechanisms. In the chronic social defeat stress model of depression, we report that KCNQ-type K+ channel openers, including FDA-approved drug retigabine (ezogabine), show antidepressant efficacy. We demonstrate that overexpression of KCNQ channels in the VTA dopaminergic neurons and either local infusion or systemic administration of retigabine normalized neuronal hyperactivity and depressive behaviours. These findings identify KCNQ as a target for conceptually novel antidepressants that function through the potentiation of active resilience mechanisms. PMID:27216573

  20. 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

  1. Proapoptotic N-truncated BCL-xL protein activates endogenous mitochondrial channels in living synaptic terminals

    PubMed Central

    Jonas, Elizabeth A.; Hickman, John A.; Chachar, Mushtaque; Polster, Brian M.; Brandt, Teresa A.; Fannjiang, Yihru; Ivanovska, Iva; Basañez, Gorka; Kinnally, Kathleen W.; Zimmerberg, Joshua; Hardwick, J. Marie; Kaczmarek, Leonard K.

    2004-01-01

    Neuronal death is often preceded by functional alterations at nerve terminals. Anti- and proapoptotic BCL-2 family proteins not only regulate the neuronal death pathway but also affect excitability of healthy neurons. We found that exposure of squid stellate ganglia to hypoxia, a death stimulus for neurons, causes a cysteine protease-dependent loss of full-length antiapoptotic BCL-xL, similar to previous findings in mammalian cells. Therefore, to determine the direct effect of the naturally occurring proapoptotic cleavage product of BCL-xL on mitochondria, recombinant N-truncated BCL-xL was applied to mitochondria inside the squid presynaptic terminal and to purified mitochondria isolated from yeast. N-truncated BCL-xL rapidly induced large multi-conductance channels with a maximal conductance significantly larger than those produced by full-length BCL-xL. This activity required the hydrophobic C terminus and the BH3 domain of BCL-xL. Moreover, N-truncated BCL-xL failed to produce any channel activity when applied to plasma membranes, suggesting that a component of the mitochondrial membrane is necessary for its actions. Consistent with this idea, the large channels induced by N-truncated BCL-xL are inhibited by NADH and require the presence of VDAC, a voltage-dependent anion channel present in the outer mitochondrial membrane. These observations suggest that the mitochondrial channels specific to full-length and N-truncated BCL-xL contribute to their opposite effects on synaptic transmission, and are consistent with their opposite effects on the cell death pathway. PMID:15342906

  2. MscS-Like10 is a stretch-activated ion channel from Arabidopsis thaliana with a preference for anions

    PubMed Central

    Maksaev, Grigory; Haswell, Elizabeth S.

    2012-01-01

    Like many other organisms, plants are capable of sensing and responding to mechanical stimuli such as touch, osmotic pressure, and gravity. One mechanism for the perception of force is the activation of mechanosensitive (or stretch-activated) ion channels, and a number of mechanosensitive channel activities have been described in plant membranes. Based on their homology to the bacterial mechanosensitive channel MscS, the 10 MscS-Like (MSL) proteins of Arabidopsis thaliana have been hypothesized to form mechanosensitive channels in plant cell and organelle membranes. However, definitive proof that MSLs form mechanosensitive channels has been lacking. Here we used single-channel patch clamp electrophysiology to show that MSL10 is capable of providing a MS channel activity when heterologously expressed in Xenopus laevis oocytes. This channel had a conductance of ∼100 pS, consistent with the hypothesis that it underlies an activity previously observed in the plasma membrane of plant root cells. We found that MSL10 formed a channel with a moderate preference for anions, which was modulated by strongly positive and negative membrane potentials, and was reversibly inhibited by gadolinium, a known inhibitor of mechanosensitive channels. MSL10 demonstrated asymmetric activation/inactivation kinetics, with the channel closing at substantially lower tensions than channel opening. The electrophysiological characterization of MSL10 reported here provides insight into the evolution of structure and function of this important family of proteins. PMID:23112188

  3. Discovery, structure-activity relationship study, and oral analgesic efficacy of cyproheptadine derivatives possessing N-type calcium channel inhibitory activity.

    PubMed

    Yamamoto, Takashi; Niwa, Seiji; Iwayama, Satoshi; Koganei, Hajime; Fujita, Shin-ichi; Takeda, Tomoko; Kito, Morikazu; Ono, Yukitsugu; Saitou, Yuki; Takahara, Akira; Iwata, Seinosuke; Yamamoto, Hiroshi; Shoji, Masataka

    2006-08-01

    Antiallergic drug cyproheptadine (Cyp) is known to have inhibitory activities for L-type calcium channels in addition to histamine and serotonin receptors. Since we found that Cyp had an inhibitory activity against N-type calcium channel, Cyp was optimized to obtain more selective N-type calcium channel blocker with analgesic action. As a consequence of the optimization, we found 13 with potent N-type calcium channel inhibitory activity which had lower inhibitory activities against L-type calcium channel, histamine (H1), and serotonin (5-HT2A) receptors than those of Cyp. 13 showed an oral analgesic activity in rat formalin-induced pain model.

  4. Constitutively active G-protein-gated inwardly rectifying K+ channels in dendrites of hippocampal CA1 pyramidal neurons.

    PubMed

    Chen, Xixi; Johnston, Daniel

    2005-04-13

    A diversity of ion channels contributes to the active properties of neuronal dendrites. From the apical dendrites of hippocampal CA1 pyramidal neurons, we recorded inwardly rectifying K+ channels with a single-channel conductance of 33 pS. The inwardly rectifying K+ channels were constitutively active at the resting membrane potential. The amount of constitutive channel activity was significantly larger in the apical dendrites than in the soma. Activities of these inwardly rectifying K+ channels were inhibited by Ba2+ (200 microM) and tertiapin (10 nM), both of which are believed to block G-protein-coupled inwardly rectifying K+ (GIRK) channels. Intracellularly applied GTPgammaS (20 microM) during dual dendritic recordings significantly increased constitutive channel activity. Baclofen (20 microM), an agonist for the G-protein-coupled GABA(B) receptor, also significantly increased the level of channel activity. Therefore, these channels are GIRK channels, which are constitutively active at rest in the apical dendrites of CA1 pyramidal neurons and can be further activated via G-protein-coupled neurotransmitter receptors.

  5. Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation.

    PubMed

    Ordaz, Benito; Tang, Jisen; Xiao, Rui; Salgado, Alfonso; Sampieri, Alicia; Zhu, Michael X; Vaca, Luis

    2005-09-02

    TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.

  6. The Activation Effect of Hainantoxin-I, a Peptide Toxin from the Chinese Spider, Ornithoctonus hainana, on Intermediate-Conductance Ca2+-Activated K+ Channels

    PubMed Central

    Huang, Pengfei; Zhang, Yiya; Chen, Xinyi; Zhu, Li; Yin, Dazhong; Zeng, Xiongzhi; Liang, Songping

    2014-01-01

    Intermediate-conductance Ca2+-activated K+ (IK) channels are calcium/calmodulin-regulated voltage-independent K+ channels. Activation of IK currents is important in vessel and respiratory tissues, rendering the channels potential drug targets. A variety of small organic molecules have been synthesized and found to be potent activators of IK channels. However, the poor selectivity of these molecules limits their therapeutic value. Venom-derived peptides usually block their targets with high specificity. Therefore, we searched for novel peptide activators of IK channels by testing a series of toxins from spiders. Using electrophysiological experiments, we identified hainantoxin-I (HNTX-I) as an IK-channel activator. HNTX-I has little effect on voltage-gated Na+ and Ca2+ channels from rat dorsal root ganglion neurons and on the heterologous expression of voltage-gated rapidly activating delayed rectifier K+ channels (human ether-à-go-go-related gene; human ERG) in HEK293T cells. Only 35.2% ± 0.4% of the currents were activated in SK channels, and there was no effect on BK channels. We demonstrated that HNTX-I was not a phrenic nerve conduction blocker or acutely toxic. This is believed to be the first report of a peptide activator effect on IK channels. Our study suggests that the activity and selectivity of HNTX-I on IK channels make HNTX-I a promising template for designing new drugs for cardiovascular diseases. PMID:25153257

  7. Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes.

    PubMed

    Chraïbi, A; Vallet, V; Firsov, D; Hess, S K; Horisberger, J D

    1998-01-01

    We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits alpha, beta, and gamma of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 microg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (approximately 20-fold) with the channel obtained by coexpression of the alpha subunit of Xenopus ENaC with the beta and gamma subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel pre