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Sample records for affect channel gating

  1. 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.

  2. Peptide Neurotoxins that Affect Voltage-Gated Calcium Channels: A Close-Up on ω-Agatoxins

    PubMed Central

    Pringos, Emilie; Vignes, Michel; Martinez, Jean; Rolland, Valerie

    2011-01-01

    Peptide neurotoxins found in animal venoms have gained great interest in the field of neurotransmission. As they are high affinity ligands for calcium, potassium and sodium channels, they have become useful tools for studying channel structure and activity. Peptide neurotoxins represent the clinical potential of ion-channel modulators across several therapeutic fields, especially in developing new strategies for treatment of ion channel-related diseases. The aim of this review is to overview the latest updates in the domain of peptide neurotoxins that affect voltage-gated calcium channels, with a special focus on ω-agatoxins. PMID:22069688

  3. 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

  4. 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

    potassium at a bandwidth of 5 kHz. Filtering at lower cutoff frequencies reduces the apparent single-channel conductance, the ratio of the conductance in rubidium versus potassium is, however, independent of the cutoff frequency. Our results suggest the presence of a relatively rapid process (flicker) that can occur almost independently of the gating state. Occupancy by rubidium at negative voltages favors the flicker-open state and slows the flickering rate in homomeric channels, whereas rubidium does not affect the flickering in heteromeric channels. The effects of KCNE1 on the conduction properties are consistent with an interaction of KCNE1 in the outer vestibule of the channel. PMID:10620287

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. Fluorescence detection of plant extracts that affect neuronal voltage-gated Ca2+ channels.

    PubMed

    Rogers, K L; Fong, W F; Redburn, J; Griffiths, L R

    2002-05-01

    Structurally novel compounds able to block voltage-gated Ca2+ channels (VGCCs) are currently being sought for the development of new drugs directed at neurological disorders. Fluorescence techniques have recently been developed to facilitate the analysis of VGCC blockers in a multi-well format. By utilising the small cell lung carcinoma cell line, NCI-H146, we were able to detect changes in intracellular Ca2+ concentration ([Ca2+](i)) using a fluorescence microplate reader. NCI-H146 cells have characteristics resembling those of neuronal cells and express multiple VGCC subtypes, including those of the L-, N- and P-type. We found that K+-depolarisation of fluo-3 loaded NCI-H146 cells causes a rapid and transient increase in fluorescence, which was readily detected in a 96-well plate. Extracts of Australian plants, including those used traditionally as headache or pain treatments, were tested in this study to identify those affecting Ca2+ influx following membrane depolarisation of NCI-H146 cells. We found that E. bignoniiflora, A. symphyocarpa and E. vespertilio caused dose-dependent inhibition of K+-depolarised Ca2+ influx, with IC(50) values calculated to be 234, 548 and 209 microg/ml, respectively. This data suggests an effect of these extracts on the function of VGCCs in these cells. Furthermore, we found similar effects using a fluorescence laser imaging plate reader (FLIPR) that allows simultaneous measurement of real-time fluorescence in a multi-well plate. Our results indicate that the dichloromethane extract of E. bignoniiflora and the methanolic extract of E. vespertilio show considerable promise as antagonists of neuronal VGCCs. Further analysis is required to characterise the function of the bioactive constituents in these extracts and determine their selectivity on VGCC subtypes.

  10. Point mutations affecting antagonist affinity and agonist dependent gating of GABAA receptor channels.

    PubMed Central

    Sigel, E; Baur, R; Kellenberger, S; Malherbe, P

    1992-01-01

    Two variant amino acid sequences, which differ in a single amino acid residue, have been reported for the alpha 1-subunit of the rat brain GABAA receptor. We separately co-expressed these two variants in Xenopus oocytes, in combination with beta 2 and gamma 2. This experiment showed that substitution of alpha 1-Phe64 by Leu strongly decreases the apparent affinity for GABA dependent channel gating from 6 microM to 1260 microM. Starting from this observation, we used in vitro mutagenesis to obtain information relevant for the localization of the agonist/antagonist binding site in the GABAA receptor. Homologous mutation in alpha 5 had similar consequences for alpha 5 beta 2 gamma 2. Homologous mutation in beta 2 and gamma 2 resulted in intermediate and small shifts in EC50, respectively. The apparent affinities of the competitive antagonists bicuculline methiodide and SR95531, the latter sharing close structural similarity with the agonist GABA, were decreased 60- to 200-fold by these mutations in alpha-subunits. Interestingly, these affinities remained nearly unaffected upon introduction of the homologous mutations in beta 2 and gamma 2, or upon mutation of the neighbouring amino acid in alpha 1, Phe65 to Leu. These results suggest close functional and structural association of alpha-subunits with the agonist/antagonist binding site, and involvement of N-terminal portions of the extracellular domains of all subunits in the gating of the channel. PMID:1376242

  11. TRP channel gating physiology.

    PubMed

    Nieto-Posadas, Andrés; Jara-Oseguera, Andrés; Rosenbaum, Tamara

    2011-01-01

    Transient Receptor Potential (TRP) cation channels participate in several processes of vital importance in cell and organism physiology, and have been demonstrated to participate in the detection of sensory stimuli. The thermo TRP's reviewed: TRPV1 (vanilloid 1), TRPM8 (melastatin 8) and TRPA1 (ankyrin-like 1) are known to integrate different chemical and physical stimuli such as changes in temperature and sensing different irritant or pungent compounds. However, despite the physiological importance of these channels the mechanisms by which they detect incoming stimuli, how the sensing domains are coupled to channel gating and how these processes are connected to specific structural regions in the channel are not fully understood, but valuable information is available. Many sites involved in agonist detection have been characterized and gating models that describe many features of the channel's behavior have been put forward. In this review we will survey some of the key findings concerning the structural and molecular mechanisms of TRPV1, TRPA1 and TRPM8 activation.

  12. 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.

  13. Voltage-Gated Calcium Channels

    NASA Astrophysics Data System (ADS)

    Zamponi, Gerald Werner

    Voltage Gated Calcium Channels is the first comprehensive book in the calcium channel field, encompassing over thirty years of progress towards our understanding of calcium channel structure, function, regulation, physiology, pharmacology, and genetics. This book balances contributions from many of the leading authorities in the calcium channel field with fresh perspectives from risings stars in the area, taking into account the most recent literature and concepts. This is the only all-encompassing calcium channel book currently available, and is an essential resource for academic researchers at all levels in the areas neuroscience, biophysics, and cardiovascular sciences, as well as to researchers in the drug discovery area.

  14. Voltage-gated Proton Channels

    PubMed Central

    DeCoursey, Thomas E.

    2014-01-01

    Voltage-gated proton channels, HV1, have vaulted from the realm of the esoteric into the forefront of a central question facing ion channel biophysicists, namely the mechanism by which voltage-dependent gating occurs. This transformation is the result of several factors. Identification of the gene in 2006 revealed that proton channels are homologues of the voltage-sensing domain of most other voltage-gated ion channels. Unique, or at least eccentric, properties of proton channels include dimeric architecture with dual conduction pathways, perfect proton selectivity, a single-channel conductance ~103 smaller than most ion channels, voltage-dependent gating that is strongly modulated by the pH gradient, ΔpH, and potent inhibition by Zn2+ (in many species) but an absence of other potent inhibitors. The recent identification of HV1 in three unicellular marine plankton species has dramatically expanded the phylogenetic family tree. Interest in proton channels in their own right has increased as important physiological roles have been identified in many cells. Proton channels trigger the bioluminescent flash of dinoflagellates, facilitate calcification by coccolithophores, regulate pH-dependent processes in eggs and sperm during fertilization, secrete acid to control the pH of airway fluids, facilitate histamine secretion by basophils, and play a signaling role in facilitating B-cell receptor mediated responses in B lymphocytes. The most elaborate and best-established functions occur in phagocytes, where proton channels optimize the activity of NADPH oxidase, an important producer of reactive oxygen species. Proton efflux mediated by HV1 balances the charge translocated across the membrane by electrons through NADPH oxidase, minimizes changes in cytoplasmic and phagosomal pH, limits osmotic swelling of the phagosome, and provides substrate H+ for the production of H2O2 and HOCl, reactive oxygen species crucial to killing pathogens. PMID:23798303

  15. 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.

  16. Gating of two pore domain potassium channels

    PubMed Central

    Mathie, Alistair; Al-Moubarak, Ehab; Veale, Emma L

    2010-01-01

    Two-pore-domain potassium (K2P) channels are responsible for background leak currents which regulate the membrane potential and excitability of many cell types. Their activity is modulated by a variety of chemical and physical stimuli which act to increase or decrease the open probability of individual K2P channels. Crystallographic data and homology modelling suggest that all K+ channels possess a highly conserved structure for ion selectivity and gating mechanisms. Like other K+ channels, K2P channels are thought to have two primary conserved gating mechanisms: an inactivation (or C-type) gate at the selectivity filter close to the extracellular side of the channel and an activation gate at the intracellular entrance to the channel involving key, identified, hinge glycine residues. Zinc and hydrogen ions regulate Drosophila KCNK0 and mammalian TASK channels, respectively, by interacting with the inactivation gate of these channels. In contrast, the voltage dependence of TASK3 channels is mediated through its activation gate. For KCNK0 it has been shown that the gates display positive cooperativity. It is of much interest to determine whether other K2P regulatory compounds interact with either the activation gate or the inactivation gate to alter channel activity or, indeed, whether additional regulatory gating pathways exist. PMID:20566661

  17. Gating of two pore domain potassium channels.

    PubMed

    Mathie, Alistair; Al-Moubarak, Ehab; Veale, Emma L

    2010-09-01

    Two-pore-domain potassium (K2P) channels are responsible for background leak currents which regulate the membrane potential and excitability of many cell types. Their activity is modulated by a variety of chemical and physical stimuli which act to increase or decrease the open probability of individual K2P channels. Crystallographic data and homology modelling suggest that all K(+) channels possess a highly conserved structure for ion selectivity and gating mechanisms. Like other K(+) channels, K2P channels are thought to have two primary conserved gating mechanisms: an inactivation (or C-type) gate at the selectivity filter close to the extracellular side of the channel and an activation gate at the intracellular entrance to the channel involving key, identified, hinge glycine residues. Zinc and hydrogen ions regulate Drosophila KCNK0 and mammalian TASK channels, respectively, by interacting with the inactivation gate of these channels. In contrast, the voltage dependence of TASK3 channels is mediated through its activation gate. For KCNK0 it has been shown that the gates display positive cooperativity. It is of much interest to determine whether other K2P regulatory compounds interact with either the activation gate or the inactivation gate to alter channel activity or, indeed, whether additional regulatory gating pathways exist.

  18. Hydrophobic Gating in Ion Channels

    PubMed Central

    Aryal, Prafulla; Sansom, Mark S.P.; Tucker, Stephen J.

    2016-01-01

    Biological ion channels are nanoscale transmembrane pores. When water and ions are enclosed within the narrow confines of a sub-nanometer hydrophobic pore, they exhibit behavior not evident from macroscopic descriptions. At this nanoscopic level, the unfavorable interaction between the lining of a hydrophobic pore and water may lead to liquid-vapor oscillations. The resultant transient vapor state is ‘dewetted’ i.e. effectively devoid of water molecules within all, or part of the pore, thus leading to an energetic barrier to ion conduction. This process, termed ‘hydrophobic gating’, was first observed in molecular dynamics simulations of model nanopores, where the principles underlying hydrophobic gating (i.e. changes in diameter, polarity, or transmembrane voltage) have now been extensively validated. Computational, structural and functional studies now indicate that biological ion channels may also exploit hydrophobic gating to regulate ion flow within their pores. Here we review the evidence for this process, and propose that this unusual behavior of water represents an increasingly important element in understanding the relationship between ion channel structure and function. PMID:25106689

  19. Mechanosensitive gating of Kv channels.

    PubMed

    Morris, Catherine E; Prikryl, Emil A; Joós, Béla

    2015-01-01

    K-selective voltage-gated channels (Kv) are multi-conformation bilayer-embedded proteins whose mechanosensitive (MS) Popen(V) implies that at least one conformational transition requires the restructuring of the channel-bilayer interface. Unlike Morris and colleagues, who attributed MS-Kv responses to a cooperative V-dependent closed-closed expansion↔compaction transition near the open state, Mackinnon and colleagues invoke expansion during a V-independent closed↔open transition. With increasing membrane tension, they suggest, the closed↔open equilibrium constant, L, can increase >100-fold, thereby taking steady-state Popen from 0→1; "exquisite sensitivity to small…mechanical perturbations", they state, makes a Kv "as much a mechanosensitive…as…a voltage-dependent channel". Devised to explain successive gK(V) curves in excised patches where tension spontaneously increased until lysis, their L-based model falters in part because of an overlooked IK feature; with recovery from slow inactivation factored in, their g(V) datasets are fully explained by the earlier model (a MS V-dependent closed-closed transition, invariant L≥4). An L-based MS-Kv predicts neither known Kv time courses nor the distinctive MS responses of Kv-ILT. It predicts Kv densities (hence gating charge per V-sensor) several-fold different from established values. If opening depended on elevated tension (L-based model), standard gK(V) operation would be compromised by animal cells' membrane flaccidity. A MS V-dependent transition is, by contrast, unproblematic on all counts. Since these issues bear directly on recent findings that mechanically-modulated Kv channels subtly tune pain-related excitability in peripheral mechanoreceptor neurons we undertook excitability modeling (evoked action potentials). Kvs with MS V-dependent closed-closed transitions produce nuanced mechanically-modulated excitability whereas an L-based MS-Kv yields extreme, possibly excessive (physiologically

  20. Channel gating pore: a new therapeutic target.

    PubMed

    Kornilov, Polina; Peretz, Asher; Attali, Bernard

    2013-09-01

    Each subunit of voltage-gated cation channels comprises a voltage-sensing domain and a pore region. In a paper recently published in Cell Research, Li et al. showed that the gating charge pathway of the voltage sensor of the KCNQ2 K+ channel can accommodate small opener molecules and offer a new target to treat hyperexcitability disorders.

  1. Voltage-gated sodium channels

    PubMed Central

    Abdelsayed, Mena; Sokolov, Stanislav

    2013-01-01

    Epilepsy is a brain disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels, Nav1.1 and Nav1.2. The effects of antiepileptic drugs (AEDs) as effective therapies for epilepsy have been characterized by extensive research. Most of the classic AEDs targeting Nav share a common mechanism of action by stabilizing the channel’s fast-inactivated state. In contrast, novel AEDs, such as lacosamide, stabilize the slow-inactivated state in neuronal Nav1.1 and Nav1.7 isoforms. This paper reviews the different mechanisms by which this stabilization occurs to determine new methods for treatment. PMID:23531742

  2. Mutations in the extracellular domains of glutamate-gated chloride channel alpha3 and beta subunits from ivermectin-resistant Cooperia oncophora affect agonist sensitivity.

    PubMed

    Njue, Annete I; Hayashi, Jon; Kinne, Lyle; Feng, Xiao-Peng; Prichard, Roger K

    2004-06-01

    Two full-length glutamate-gated chloride channel (GluCl) cDNAs, encoding GluClalpha3 and GluClbeta subunits, were cloned from ivermectin-susceptible (IVS) and -resistant (IVR) Cooperia oncophora adult worms. The IVS and IVR GluClalpha3 subunits differ at three amino acid positions, while the IVS and IVR GluClbeta subunits differ at two amino acid positions. The aim of this study was to determine whether mutations in the IVR subunits affect agonist sensitivity. The subunits were expressed singly and in combination in Xenopus laevis oocytes. Electrophysiological whole-cell voltage-clamp recordings showed that mutations in the IVR GluClalpha3 caused a modest but significant threefold loss of sensitivity to glutamate, the natural ligand for GluCl receptors. As well, a significant decrease in sensitivity to the anthelmintics ivermectin and moxidectin was observed in the IVR GluClalpha3 receptor. Mutations in the IVR GluClbeta subunit abolished glutamate sensitivity. Co-expressing the IVS GluClalpha3 and GluClbeta subunits resulted in heteromeric channels that were more sensitive to glutamate than the respective homomeric channels, demonstrating co-assembly of the subunits. In contrast, the heteromeric IVR channels were less sensitive to glutamate than the homomeric IVR GluClalpha3 channels. The heteromeric IVS channels were significantly more sensitive to glutamate than the heteromeric IVR channels. Of the three amino acids distinguishing the IVS and IVR GluClalpha3 subunits, only one of them, L256F, accounted for the differences in response between the IVS and IVR GluClalpha3 homomeric channels.

  3. Mechanosensitive Gating of Kv Channels

    PubMed Central

    Morris, Catherine E.; Prikryl, Emil A.; Joós, Béla

    2015-01-01

    K-selective voltage-gated channels (Kv) are multi-conformation bilayer-embedded proteins whose mechanosensitive (MS) Popen(V) implies that at least one conformational transition requires the restructuring of the channel-bilayer interface. Unlike Morris and colleagues, who attributed MS-Kv responses to a cooperative V-dependent closed-closed expansion↔compaction transition near the open state, Mackinnon and colleagues invoke expansion during a V-independent closed↔open transition. With increasing membrane tension, they suggest, the closed↔open equilibrium constant, L, can increase >100-fold, thereby taking steady-state Popen from 0→1; “exquisite sensitivity to small…mechanical perturbations”, they state, makes a Kv “as much a mechanosensitive…as…a voltage-dependent channel”. Devised to explain successive gK(V) curves in excised patches where tension spontaneously increased until lysis, their L-based model falters in part because of an overlooked IK feature; with recovery from slow inactivation factored in, their g(V) datasets are fully explained by the earlier model (a MS V-dependent closed-closed transition, invariant L≥4). An L-based MS-Kv predicts neither known Kv time courses nor the distinctive MS responses of Kv-ILT. It predicts Kv densities (hence gating charge per V-sensor) several-fold different from established values. If opening depended on elevated tension (L-based model), standard gK(V) operation would be compromised by animal cells’ membrane flaccidity. A MS V-dependent transition is, by contrast, unproblematic on all counts. Since these issues bear directly on recent findings that mechanically-modulated Kv channels subtly tune pain-related excitability in peripheral mechanoreceptor neurons we undertook excitability modeling (evoked action potentials). Kvs with MS V-dependent closed-closed transitions produce nuanced mechanically-modulated excitability whereas an L-based MS-Kv yields extreme, possibly excessive

  4. Hysteresis in voltage-gated channels.

    PubMed

    Villalba-Galea, Carlos A

    2016-09-30

    Ion channels constitute a superfamily of membrane proteins found in all living creatures. Their activity allows fast translocation of ions across the plasma membrane down the ion's transmembrane electrochemical gradient, resulting in a difference in electrical potential across the plasma membrane, known as the membrane potential. A group within this superfamily, namely voltage-gated channels, displays activity that is sensitive to the membrane potential. The activity of voltage-gated channels is controlled by the membrane potential, while the membrane potential is changed by these channels' activity. This interplay produces variations in the membrane potential that have evolved into electrical signals in many organisms. These signals are essential for numerous biological processes, including neuronal activity, insulin release, muscle contraction, fertilization and many others. In recent years, the activity of the voltage-gated channels has been observed not to follow a simple relationship with the membrane potential. Instead, it has been shown that the activity of voltage-gated channel displays hysteresis. In fact, a growing number of evidence have demonstrated that the voltage dependence of channel activity is dynamically modulated by activity itself. In spite of the great impact that this property can have on electrical signaling, hysteresis in voltage-gated channels is often overlooked. Addressing this issue, this review provides examples of voltage-gated ion channels displaying hysteretic behavior. Further, this review will discuss how Dynamic Voltage Dependence in voltage-gated channels can have a physiological role in electrical signaling. Furthermore, this review will elaborate on the current thoughts on the mechanism underlying hysteresis in voltage-gated channels.

  5. Hyaluronan Does Not Affect Bupivacaine's Inhibitory Action on Voltage-Gated Potassium Channel Activities in Bovine Articular Chondrocytes

    PubMed Central

    Hester, William; Yang, Jinnan; Wang, Guo-Yong; Liu, Sen; O'Brien, Michael J.; Savoie, Felix H.; You, Zongbing

    2012-01-01

    Objectives. The objective of this paper is to determine if hyaluronan affects bupivacaine's anesthetic function. Methods. Whole cell patch clamp recordings were performed on bovine articular chondrocytes cultured in 60 mm dishes. The chondrocytes were treated with phosphate-buffered saline (control group), 7.5 mg/mL hyaluronan (Orthovisc), 0.25% bupivacaine, or a mixture of 7.5 mg/mL hyaluronan and 0.25% bupivacaine. Outward currents were elicited by step depolarization from −90 mV to 150 mV with 5 mV increments and holding for 200 ms. Results. The amplitude of outward currents elicited at 150 mV was 607.1 ± 135.4 pA (mean ± standard error) in the chondrocytes treated with phosphate buffered saline, 550.0 ± 194.9 pA in the chondrocytes treated with hyaluronan, 18.4 ± 8.3 pA in the chondrocytes treated with bupivacaine, and 12.8 ± 2.6 pA in the chondrocytes treated with a mixture of hyaluronan and bupivacaine. Conclusion. Hyaluronan does not affect bupivacaine's inhibitory action on the potassium channel activities in bovine articular chondrocytes. This finding suggests that intra-articular injection of a mixture of hyaluronan and bupivacaine may not affect the anesthetic effects of bupivacaine. PMID:22577566

  6. The gating of the CFTR channel.

    PubMed

    Moran, Oscar

    2017-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel expressed in the apical membrane of epithelia. Mutations in the CFTR gene are the cause of cystsic fibrosis. CFTR is the only ABC-protein that constitutes an ion channel pore forming subunit. CFTR gating is regulated in complex manner as phosphorylation is mandatory for channel activity and gating is directly regulated by binding of ATP to specific intracellular sites on the CFTR protein. This review covers our current understanding on the gating mechanism in CFTR and illustrates the relevance of alteration of these mechanisms in the onset of cystic fibrosis.

  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. Bubbles, Gating, and Anesthetics in Ion Channels

    PubMed Central

    Roth, Roland; Gillespie, Dirk; Nonner, Wolfgang; Eisenberg, Robert E.

    2008-01-01

    We suggest that bubbles are the bistable hydrophobic gates responsible for the on-off transitions of single channel currents. In this view, many types of channels gate by the same physical mechanism—dewetting by capillary evaporation—but different types of channels use different sensors to modulate hydrophobic properties of the channel wall and thereby trigger and control bubbles and gating. Spontaneous emptying of channels has been seen in many simulations. Because of the physics involved, such phase transitions are inherently sensitive, unstable threshold phenomena that are difficult to simulate reproducibly and thus convincingly. We present a thermodynamic analysis of a bubble gate using morphometric density functional theory of classical (not quantum) mechanics. Thermodynamic analysis of phase transitions is generally more reproducible and less sensitive to details than simulations. Anesthetic actions of inert gases—and their interactions with hydrostatic pressure (e.g., nitrogen narcosis)—can be easily understood by actions on bubbles. A general theory of gas anesthesia may involve bubbles in channels. Only experiments can show whether, or when, or which channels actually use bubbles as hydrophobic gates: direct observation of bubbles in channels is needed. Existing experiments show thin gas layers on hydrophobic surfaces in water and suggest that bubbles nearly exist in bulk water. PMID:18234836

  9. Lipid-dependent gating of a voltage-gated potassium channel

    PubMed Central

    Zheng, Hui; Liu, Weiran; Anderson, Lingyan Y.; Jiang, Qiu-Xing

    2011-01-01

    Recent studies hypothesized that phospholipids stabilize two voltage-sensing arginine residues of certain voltage-gated potassium channels in activated conformations. It remains unclear how lipids directly affect these channels. Here, by examining the conformations of the KvAP in different lipids, we showed that without voltage change, the voltage-sensor domains switched from the activated to the resting state when their surrounding lipids were changed from phospholipids to nonphospholipids. Such lipid-determined conformational change was coupled to the ion-conducting pore, suggesting that parallel to voltage gating, the channel is gated by its annular lipids. Our measurements recognized that the energetic cost of lipid-dependent gating approaches that of voltage gating, but kinetically it appears much slower. Our data support that a channel and its surrounding lipids together constitute a functional unit, and natural nonphospholipids such as cholesterol should exert strong effects on voltage-gated channels. Our first observation of lipid-dependent gating may have general implications to other membrane proteins. PMID:21427721

  10. Gating mechanisms of voltage-gated proton channels.

    PubMed

    Okamura, Yasushi; Fujiwara, Yuichiro; Sakata, Souhei

    2015-01-01

    Hv1 is a voltage-gated proton-selective channel that plays critical parts in host defense, sperm motility, and cancer progression. Hv1 contains a conserved voltage-sensor domain (VSD) that is shared by a large family of voltage-gated ion channels, but it lacks a pore domain. Voltage sensitivity and proton conductivity are conferred by a unitary VSD that consists of four transmembrane helices. The architecture of Hv1 differs from that of cation channels that form a pore in the center among multiple subunits (as in most cation channels) or homologous repeats (as in voltage-gated sodium and calcium channels). Hv1 forms a dimer in which a cytoplasmic coiled coil underpins the two protomers and forms a single, long helix that is contiguous with S4, the transmembrane voltage-sensing segment. The closed-state structure of Hv1 was recently solved using X-ray crystallography. In this article, we discuss the gating mechanism of Hv1 and focus on cooperativity within dimers and their sensitivity to metal ions.

  11. Deltamethrin affects the expression of voltage-gated calcium channel α1 subunits and the locomotion, egg-laying, foraging behavior of Caenorhabditis elegans.

    PubMed

    Zeng, Rune; Yu, Xing; Tan, Xing; Ye, Shan; Ding, Zhong

    2017-05-01

    Deltamethrin belongs to the class of synthetic pyrethroids, which are being widely used as insecticides in agricultural practices. Voltage-gated sodium channels (VGSCs) are the primary targets of these chemicals for toxicity to insects. Caenorhabditis elegans (C. elegans) does not have VGSCs but is susceptible to deltamethrin. Recent findings have suggested that pyrethroids can affect voltage-gated calcium channels (VGCCs). However, it remains elusive whether deltamethrin induces toxicity to C. elegans via modulating the activity of VGCCs. To identify the potential target of deltamethrin, we exposed C. elegans to different concentrations of deltamethrin and Ca(2+) channel blockers for different times, characterized the behavioral toxicity of deltamethrin on C. elegans, and determined the expression of egl-19, unc-2, and cca-1, which encode the α1-subunit of the L-, R/N/P/Q-, and T-type VGCC, respectively. We found that deltamethrin inhibited the locomotion, egg-laying and foraging ability of C. elegans in a concentration dependent manner. We also showed that body length of worms on agar plates containing 200mgL(-1) deltamethrin for 12h was not significantly different from controls, whereas the cholinesterase inhibitor carbofuran caused hypercontraction which is a characteristic of organophosphates and carbamates, suggesting that deltamethrin's mode of action is distinct from those nematicides. In addition, unc-2 was significantly up-regulated following 0.05mgL(-1) deltamethrin exposure for 24h; while egl-19 and cca-1 were significantly up-regulated following 5 and 50mgL(-1) deltamethrin exposure for 24h. Further tests of worms' sensitivity and expression of three α1-subunits of VGCC to Ca(2+) channel blockers indicate that deltamethrin may induce toxic behavior C. elegans via modulation of the expression of the α1-subunits of VGCC. This study provides insights into the linkage between deltamethrin-induced toxic behavior and the regulation of α1-subunits of VGCC

  12. Hair-bundle friction from transduction channels' gating forces

    NASA Astrophysics Data System (ADS)

    Bormuth, Volker; Barral, Jérémie; Joanny, Jean-François; Jülicher, Frank; Martin, Pascal

    2015-12-01

    Hearing starts when sound-evoked mechanical vibrations of the hair-cell bundle activate mechanosensitive ion channels, giving birth to an electrical signal. As for any mechanical system, friction impedes movements of the hair bundle and thus constrains the sensitivity and frequency selectivity of auditory transduction. We have shown recently that the opening and closing of the transduction channels produce internal frictional forces that can dominate viscous drag on the micrometer-sized hair bundle and thus provide a major source of damping [2]. We develop here a physical theory of passive hair-bundle mechanics that explains the origin of channel friction. We show that channel friction can be understood quantitatively by coupling the dynamics of the conformational change associated with channel gating to tip-link tension. As a result, varying channel properties affects friction, with faster channels producing smaller friction. The analysis emphasizes the dual role of transduction channels' gating forces, which affect both hair-bundle stiffness and drag. Friction originating from gating of ion channels is a general concept that is relevant to all mechanosensitive channels.

  13. 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.

  14. 1. UPPER SEGMENT OF SPILLWAY CHANNEL, DRUM GATES ALONG SIDE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. UPPER SEGMENT OF SPILLWAY CHANNEL, DRUM GATES ALONG SIDE OF CHANNEL, LOOKING SOUTH (up the channel) - Tieton Dam, Spillway & Drum Gates, South & East side of State Highway 12, Naches, Yakima County, WA

  15. Lack of conventional ATPase properties in CFTR chloride channel gating.

    PubMed

    Schultz, B D; Bridges, R J; Frizzell, R A

    1996-05-01

    CFTR shares structural homology with the ABC transporter superfamily of proteins which hydrolyze ATP to effect the transport of compounds across cell membranes. Some superfamily members are characterized as P-type ATPases because ATP-dependent transport is sensitive to the presence of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrolytic activity in channel gating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride channel activity under conditions known to inhibit the activity of ATPases; i.e., in the absence of divalent cations and in the presence of a variety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, reduced both the opening and closing rates of CFTR suggesting that Mg2+ plays a modulatory role in channel gating. However, channels continued to both open and close showing that Mg2+ is not an absolute requirement for channel activity. The nonselective P-type ATPase inhibitor, vanadate, did not alter the gating of CFTR when used at concentrations which completely inhibit the activity of other ABC transporters (1 mM). Higher concentrations of vanadate (10 mM) blocked the closing of CFTR, but did not affect the opening of the channel. As expected, more selective P-type (Sch28080, ouabain), V-type (bafilomycin A1, SCN-) and F-type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics. Vanadate inhibition of channel closure might suggest that a hydrolytic step is involved although the requirement for a high concentration raises the possibility of previously uncharacterized effects of this compound. Most conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for

  16. A comparison of 15 Hz sine on-line and off-line magnetic stimulation affecting the voltage-gated sodium channel currents of prefrontal cortex pyramidal neurons

    NASA Astrophysics Data System (ADS)

    Zheng, Yu; Dong, Lei; Gao, Yang; Dou, Jun-Rong; Li, Ze-yan

    2016-10-01

    Combined with the use of patch-clamp techniques, repetitive transcranial magnetic stimulation (rTMS) has proven to be a noninvasive neuromodulation tool that can inhibit or facilitate excitability of neurons after extensive research. The studies generally focused on the method: the neurons are first stimulated in an external standard magnetic exposure device, and then moved to the patch-clamp to record electrophysiological characteristics (off-line magnetic exposure). Despite its universality, real-time observation of the effects of magnetic stimulation on the neurons is more effective (on-line magnetic stimulation). In this study, we selected a standard exposure device for magnetic fields acting on mouse prefrontal cortex pyramidal neurons, and described a new method that a patch-clamp setup was modified to allow on-line magnetic stimulation. By comparing the off-line exposure and on-line stimulation of the same magnetic field intensity and frequency affecting the voltage-gated sodium channel currents, we succeeded in proving the feasibility of the new on-line stimulation device. We also demonstrated that the sodium channel currents of prefrontal cortex pyramidal neurons increased significantly under the 15 Hz sine 1 mT, and 2 mT off-line magnetic field exposure and under the 1 mT and 2 mT on-line magnetic stimulation, and the rate of acceleration was most significant on 2 mT on-line magnetic stimulation. This study described the development of a new on-line magnetic stimulator and successfully demonstrated its practicability for scientific stimulation of neurons.

  17. Voltage-gated proton channels: what' next?

    PubMed Central

    DeCoursey, Thomas E

    2008-01-01

    This review is an attempt to identify and place in context some of the many questions about voltage-gated proton channels that remain unsolved. As the gene was identified only 2 years ago, the situation is very different than in fields where the gene has been known for decades. For the proton channel, most of the obvious and less obvious structure–function questions are still wide open. Remarkably, the proton channel protein strongly resembles the voltage-sensing domain of many voltage-gated ion channels, and thus offers a novel approach to study gating mechanisms. Another surprise is that the proton channel appears to function as a dimer, with two separate conduction pathways. A number of significant biological questions remain in dispute, unanswered, or in some cases, not yet asked. This latter deficit is ascribable to the intrinsic difficulty in evaluating the importance of one component in a complex system, and in addition, to the lack, until recently, of a means of performing an unambiguous lesion experiment, that is, of selectively eliminating the molecule in question. We still lack a potent, selective pharmacological inhibitor, but the identification of the gene has allowed the development of powerful new tools including proton channel antibodies, siRNA and knockout mice. PMID:18801839

  18. Philosophy of voltage-gated proton channels

    PubMed Central

    DeCoursey, Thomas E.; Hosler, Jonathan

    2014-01-01

    In this review, voltage-gated proton channels are considered from a mainly teleological perspective. Why do proton channels exist? What good are they? Why did they go to such lengths to develop several unique hallmark properties such as extreme selectivity and ΔpH-dependent gating? Why is their current so minuscule? How do they manage to be so selective? What is the basis for our belief that they conduct H+ and not OH–? Why do they exist in many species as dimers when the monomeric form seems to work quite well? It is hoped that pondering these questions will provide an introduction to these channels and a way to logically organize their peculiar properties as well as to understand how they are able to carry out some of their better-established biological functions. PMID:24352668

  19. Gating the glutamate gate of CLC-2 chloride channel by pore occupancy.

    PubMed

    De Jesús-Pérez, José J; Castro-Chong, Alejandra; Shieh, Ru-Chi; Hernández-Carballo, Carmen Y; De Santiago-Castillo, José A; Arreola, Jorge

    2016-01-01

    CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage-sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H(+). Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl(-), Br(-), SCN(-), and I(-)) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pHe = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl(-)]i under unlikely protonation conditions (pHe = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H(+) plays a minor role in dislodging the glutamate gate.

  20. Gating the glutamate gate of CLC-2 chloride channel by pore occupancy

    PubMed Central

    De Jesús-Pérez, José J.; Castro-Chong, Alejandra; Shieh, Ru-Chi; Hernández-Carballo, Carmen Y.; De Santiago-Castillo, José A.

    2016-01-01

    CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage–sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H+. Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl−, Br−, SCN−, and I−) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pHe = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl−]i under unlikely protonation conditions (pHe = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H+ plays a minor role in dislodging the glutamate gate. PMID:26666914

  1. Optical control of an ion channel gate

    PubMed Central

    Lemoine, Damien; Habermacher, Chloé; Martz, Adeline; Méry, Pierre-François; Bouquier, Nathalie; Diverchy, Fanny; Taly, Antoine; Rassendren, François; Specht, Alexandre; Grutter, Thomas

    2013-01-01

    The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus. PMID:24297890

  2. Voltage-Gated Lipid Ion Channels

    PubMed Central

    Blicher, Andreas; Heimburg, Thomas

    2013-01-01

    Synthetic lipid membranes can display channel-like ion conduction events even in the absence of proteins. We show here that these events are voltage-gated with a quadratic voltage dependence as expected from electrostatic theory of capacitors. To this end, we recorded channel traces and current histograms in patch-experiments on lipid membranes. We derived a theoretical current-voltage relationship for pores in lipid membranes that describes the experimental data very well when assuming an asymmetric membrane. We determined the equilibrium constant between closed and open state and the open probability as a function of voltage. The voltage-dependence of the lipid pores is found comparable to that of protein channels. Lifetime distributions of open and closed events indicate that the channel open distribution does not follow exponential statistics but rather power law behavior for long open times. PMID:23823188

  3. siRNA screen for genes that affect Junín virus entry uncovers voltage-gated calcium channels as a therapeutic target

    PubMed Central

    Lavanya, Madakasira; Cuevas, Christian D.; Thomas, Monica; Cherry, Sara; Ross, Susan R.

    2014-01-01

    New world hemorrhagic fever arenaviruses infection of humans results in 15–30% mortality. We performed a high throughput siRNA screen with Junín virus glycoprotein-pseudotyped viruses to find potential host therapeutic targets. Voltage-gated calcium channels (VGCC) subunits, for which there are FDA-approved drugs, were identified in the screen. Knockdown of VGCC subunits or treatment with channel blockers diminished Junín virus-cell fusion and entry into cells and thereby decreased infection. Gabapentin, an FDA-approved drug used to treat neuropathic pain that targets the α2δ2 subunit, inhibited infection of mice by the Candid 1 vaccine strain of the virus. These findings demonstrate that VGCCs play a role in virus infection and have the potential to lead to therapeutic intervention of new world arenavirus infection. PMID:24068738

  4. Flufenamic acid decreases neuronal excitability through modulation of voltage-gated sodium channel gating.

    PubMed

    Yau, Hau-Jie; Baranauskas, Gytis; Martina, Marco

    2010-10-15

    The electrophysiological phenotype of individual neurons critically depends on the biophysical properties of the voltage-gated channels they express. Differences in sodium channel gating are instrumental in determining the different firing phenotypes of pyramidal cells and interneurons; moreover, sodium channel modulation represents an important mechanism of action for many widely used CNS drugs. Flufenamic acid (FFA) is a non-steroidal anti-inflammatory drug that has been long used as a blocker of calcium-dependent cationic conductances. Here we show that FFA inhibits voltage-gated sodium currents in hippocampal pyramidal neurons; this effect is dose-dependent with IC(50) = 189 μm. We used whole-cell and nucleated patch recordings to investigate the mechanisms of FFA modulation of TTX-sensitive voltage-gated sodium current. Our data show that flufenamic acid slows down the inactivation process of the sodium current, while shifting the inactivation curve ~10 mV toward more hyperpolarized potentials. The recovery from inactivation is also affected in a voltage-dependent way, resulting in slower recovery at hyperpolarized potentials. Recordings from acute slices demonstrate that FFA reduces repetitive- and abolishes burst-firing in CA1 pyramidal neurons. A computational model based on our data was employed to better understand the mechanisms of FFA action. Simulation data support the idea that FFA acts via a novel mechanism by reducing the voltage dependence of the sodium channel fast inactivation rates. These effects of FFA suggest that it may be an effective anti-epileptic drug.

  5. Voltage-Gated Calcium Channels in Nociception

    NASA Astrophysics Data System (ADS)

    Yasuda, Takahiro; Adams, David J.

    Voltage-gated calcium channels (VGCCs) are a large and functionally diverse group of membrane ion channels ubiquitously expressed throughout the central and peripheral nervous systems. VGCCs contribute to various physiological processes and transduce electrical activity into other cellular functions. This chapter provides an overview of biophysical properties of VGCCs, including regulation by auxiliary subunits, and their physiological role in neuronal functions. Subsequently, then we focus on N-type calcium (Cav2.2) channels, in particular their diversity and specific antagonists. We also discuss the role of N-type calcium channels in nociception and pain transmission through primary sensory dorsal root ganglion neurons (nociceptors). It has been shown that these channels are expressed predominantly in nerve terminals of the nociceptors and that they control neurotransmitter release. To date, important roles of N-type calcium channels in pain sensation have been elucidated genetically and pharmacologically, indicating that specific N-type calcium channel antagonists or modulators are particularly useful as therapeutic drugs targeting chronic and neuropathic pain.

  6. Normal mode gating motions of a ligand-gated ion channel persist in a fully hydrated lipid bilayer model.

    PubMed

    Bertaccini, Edward J; Trudell, James R; Lindahl, Erik

    2010-08-18

    We have previously used molecular modeling and normal-mode analyses combined with experimental data to visualize a plausible model of a transmembrane ligand-gated ion channel. We also postulated how the gating motion of the channel may be affected by the presence of various ligands, especially anesthetics. As is typical for normal-mode analyses, those studies were performed in vacuo to reduce the computational complexity of the problem. While such calculations constitute an efficient way to model the large scale structural flexibility of transmembrane proteins, they can be criticized for neglecting the effects of an explicit phospholipid bilayer or hydrated environment. Here, we show the successful calculation of normal-mode motions for our model of a glycine α-1 receptor, now suspended in a fully hydrated lipid bilayer. Despite the almost uniform atomic density, the introduction of water and lipid does not grossly distort the overall gating motion. Normal-mode analysis revealed that even a fully immersed glycine α-1 receptor continues to demonstrate an iris-like channel gating motion as a low-frequency, high-amplitude natural harmonic vibration consistent with channel gating. Furthermore, the introduction of periodic boundary conditions allows the examination of simultaneous harmonic vibrations of lipid in synchrony with the protein gating motions that are compatible with reasonable lipid bilayer perturbations. While these perturbations tend to influence the overall protein motion, this work provides continued support for the iris-like motion model that characterizes gating within the family of ligand-gated ion channels.

  7. 2. ALABAMA GATES LOOKING SOUTHEAST ALONG LINED CHANNEL, NOTE CHEMICAL ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. ALABAMA GATES LOOKING SOUTHEAST ALONG LINED CHANNEL, NOTE CHEMICAL PURIFICATION TANK IN DISTANCE FOR KEEPING DOWN GROWTH OF ALGAE - Los Angeles Aqueduct, Alabama Gates, Los Angeles, Los Angeles County, CA

  8. 4. SPILLWAY DRUM GATES AND CHANNEL, LOOKING NORTHEAST (upstream face ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. SPILLWAY DRUM GATES AND CHANNEL, LOOKING NORTHEAST (upstream face and Control House in background) - Tieton Dam, Spillway & Drum Gates, South & East side of State Highway 12, Naches, Yakima County, WA

  9. Identification and functional expression of a glutamate- and avermectin-gated chloride channel from Caligus rogercresseyi, a southern Hemisphere sea louse affecting farmed fish.

    PubMed

    Cornejo, Isabel; Andrini, Olga; Niemeyer, María Isabel; Marabolí, Vanessa; González-Nilo, F Danilo; Teulon, Jacques; Sepúlveda, Francisco V; Cid, L Pablo

    2014-09-01

    Parasitic sea lice represent a major sanitary threat to marine salmonid aquaculture, an industry accounting for 7% of world fish production. Caligus rogercresseyi is the principal sea louse species infesting farmed salmon and trout in the southern hemisphere. Most effective control of Caligus has been obtained with macrocyclic lactones (MLs) ivermectin and emamectin. These drugs target glutamate-gated chloride channels (GluCl) and act as irreversible non-competitive agonists causing neuronal inhibition, paralysis and death of the parasite. Here we report the cloning of a full-length CrGluClα receptor from Caligus rogercresseyi. Expression in Xenopus oocytes and electrophysiological assays show that CrGluClα is activated by glutamate and mediates chloride currents blocked by the ligand-gated anion channel inhibitor picrotoxin. Both ivermectin and emamectin activate CrGluClα in the absence of glutamate. The effects are irreversible and occur with an EC(50) value of around 200 nM, being cooperative (n(H) = 2) for ivermectin but not for emamectin. Using the three-dimensional structure of a GluClα from Caenorabditis elegans, the only available for any eukaryotic ligand-gated anion channel, we have constructed a homology model for CrGluClα. Docking and molecular dynamics calculations reveal the way in which ivermectin and emamectin interact with CrGluClα. Both drugs intercalate between transmembrane domains M1 and M3 of neighbouring subunits of a pentameric structure. The structure displays three H-bonds involved in this interaction, but despite similarity in structure only of two these are conserved from the C. elegans crystal binding site. Our data strongly suggest that CrGluClα is an important target for avermectins used in the treatment of sea louse infestation in farmed salmonids and open the way for ascertaining a possible mechanism of increasing resistance to MLs in aquaculture industry. Molecular modeling could help in the design of new, more efficient

  10. Identification and Functional Expression of a Glutamate- and Avermectin-Gated Chloride Channel from Caligus rogercresseyi, a Southern Hemisphere Sea Louse Affecting Farmed Fish

    PubMed Central

    Niemeyer, María Isabel; Marabolí, Vanessa; González-Nilo, F. Danilo; Teulon, Jacques; Sepúlveda, Francisco V.; Cid, L. Pablo

    2014-01-01

    Parasitic sea lice represent a major sanitary threat to marine salmonid aquaculture, an industry accounting for 7% of world fish production. Caligus rogercresseyi is the principal sea louse species infesting farmed salmon and trout in the southern hemisphere. Most effective control of Caligus has been obtained with macrocyclic lactones (MLs) ivermectin and emamectin. These drugs target glutamate-gated chloride channels (GluCl) and act as irreversible non-competitive agonists causing neuronal inhibition, paralysis and death of the parasite. Here we report the cloning of a full-length CrGluClα receptor from Caligus rogercresseyi. Expression in Xenopus oocytes and electrophysiological assays show that CrGluClα is activated by glutamate and mediates chloride currents blocked by the ligand-gated anion channel inhibitor picrotoxin. Both ivermectin and emamectin activate CrGluClα in the absence of glutamate. The effects are irreversible and occur with an EC50 value of around 200 nM, being cooperative (nH = 2) for ivermectin but not for emamectin. Using the three-dimensional structure of a GluClα from Caenorabditis elegans, the only available for any eukaryotic ligand-gated anion channel, we have constructed a homology model for CrGluClα. Docking and molecular dynamics calculations reveal the way in which ivermectin and emamectin interact with CrGluClα. Both drugs intercalate between transmembrane domains M1 and M3 of neighbouring subunits of a pentameric structure. The structure displays three H-bonds involved in this interaction, but despite similarity in structure only of two these are conserved from the C. elegans crystal binding site. Our data strongly suggest that CrGluClα is an important target for avermectins used in the treatment of sea louse infestation in farmed salmonids and open the way for ascertaining a possible mechanism of increasing resistance to MLs in aquaculture industry. Molecular modeling could help in the design of new, more efficient

  11. Molecular determinants of common gating of a ClC chloride channel.

    PubMed

    Bennetts, Brett; Parker, Michael W

    2013-01-01

    Uniquely, the ClC family harbours dissipative channels and anion/H(+) transporters that share unprecedented functional characteristics. ClC-1 channels are homodimers in which each monomer supports an identical pore carrying three anion-binding sites. Transient occupancy of the extracellular binding site by a conserved glutamate residue, E232, independently gates each pore. A common gate, the molecular basis of which is unknown, closes both pores simultaneously. Mutations affecting common gating underlie myotonia congenita in humans. Here we show that the common gate likely occludes the channel pore via interaction of E232 with a highly conserved tyrosine, Y578, at the central anion-binding site. We also identify structural linkages important for coordination of common gating between subunits and modulation by intracellular molecules. Our data reveal important molecular determinants of common gating of ClC channels and suggest that the molecular mechanism is an evolutionary vestige of coupled anion/H(+) transport.

  12. Allosteric Voltage Gating of Potassium Channels I

    PubMed Central

    Horrigan, Frank T.; Cui, Jianmin; Aldrich, Richard W.

    1999-01-01

    Activation of large conductance Ca2+-activated K+ channels is controlled by both cytoplasmic Ca2+ and membrane potential. To study the mechanism of voltage-dependent gating, we examined mSlo Ca2+-activated K+ currents in excised macropatches from Xenopus oocytes in the virtual absence of Ca2+ (<1 nM). In response to a voltage step, IK activates with an exponential time course, following a brief delay. The delay suggests that rapid transitions precede channel opening. The later exponential time course suggests that activation also involves a slower rate-limiting step. However, the time constant of IK relaxation [τ(IK)] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. τ(IK) increases weakly with voltage from −500 to −20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = −0.29 e). Similarly, the steady state GK–V relationship exhibits a maximum voltage dependence (z = 2 e) from 0 to +100 mV, and is weakly voltage dependent (z ≅ 0.4 e) at more negative voltages, where Po = 10−5–10−6. These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors. In the absence of Ca2+, this allosteric mechanism results in a gating scheme with five closed (C) and five open (O) states, where the majority of the channel's voltage dependence results from rapid C–C and O–O transitions, whereas the C–O transitions are rate limiting and weakly voltage dependent. These conclusions not only provide a framework for interpreting studies of large conductance Ca2+-activated K+ channel voltage gating, but also have important implications for understanding the mechanism of Ca2+ sensitivity. PMID:10436003

  13. Roscovitine, a cyclin-dependent kinase inhibitor, affects several gating mechanisms to inhibit cardiac L-type (Ca(V)1.2) calcium channels.

    PubMed

    Yarotskyy, V; Elmslie, K S

    2007-10-01

    L-type calcium channels (Ca((V))1.2) play an important role in cardiac contraction. Roscovitine, a cyclin-dependent kinase inhibitor and promising anticancer drug, has been shown to affect Ca((V))1.2 by inhibiting current amplitude and slowing activation. This research investigates the mechanism by which roscovitine inhibits Ca((V))1.2 channels. Ca((V))1.2 channels were transfected into HEK 293 cells, using the calcium phosphate precipitation method, and currents were measured using the whole-cell patch clamp technique. Roscovitine slows activation at all voltages, which precludes one previously proposed mechanism. In addition, roscovitine enhances voltage-dependent, but not calcium-dependent inactivation. This enhancement resulted from both an acceleration of inactivation and a slowing of the recovery from inactivation. Internally applied roscovitine failed to affect Ca((V))1.2 currents, which supports a kinase-independent mechanism and extracellular binding site. Unlike the dihydropyridines, closed state inactivation was not affected by roscovitine. Inactivation was enhanced in a dose-dependent manner with an IC(50)=29.5+/-12 microM, which is close to that for slow activation and inhibition. We conclude that roscovitine binds to an extracellular site on Ca((V))1.2 channels to inhibit current by both slowing activation and enhancing inactivation. Purine-based drugs could become a new option for treatment of diseases that benefit from L-channel inhibition such as cardiac arrhythmias and hypertension.

  14. Roscovitine, a cyclin-dependent kinase inhibitor, affects several gating mechanisms to inhibit cardiac L-type (Ca(V)1.2) calcium channels

    PubMed Central

    Yarotskyy, V; Elmslie, K S

    2007-01-01

    Background and purpose: L-type calcium channels (Ca (V)1.2) play an important role in cardiac contraction. Roscovitine, a cyclin-dependent kinase inhibitor and promising anticancer drug, has been shown to affect Ca (V)1.2 by inhibiting current amplitude and slowing activation. This research investigates the mechanism by which roscovitine inhibits Ca (V)1.2 channels. Experimental approach: Ca (V)1.2 channels were transfected into HEK 293 cells, using the calcium phosphate precipitation method, and currents were measured using the whole-cell patch clamp technique. Key results: Roscovitine slows activation at all voltages, which precludes one previously proposed mechanism. In addition, roscovitine enhances voltage-dependent, but not calcium-dependent inactivation. This enhancement resulted from both an acceleration of inactivation and a slowing of the recovery from inactivation. Internally applied roscovitine failed to affect Ca (V)1.2 currents, which supports a kinase-independent mechanism and extracellular binding site. Unlike the dihydropyridines, closed state inactivation was not affected by roscovitine. Inactivation was enhanced in a dose-dependent manner with an IC50=29.5±12 μM, which is close to that for slow activation and inhibition. Conclusions and implications: We conclude that roscovitine binds to an extracellular site on Ca (V)1.2 channels to inhibit current by both slowing activation and enhancing inactivation. Purine-based drugs could become a new option for treatment of diseases that benefit from L-channel inhibition such as cardiac arrhythmias and hypertension. PMID:17700718

  15. Deletion of cytosolic gating ring decreases gate and voltage sensor coupling in BK channels.

    PubMed

    Zhang, Guohui; Geng, Yanyan; Jin, Yakang; Shi, Jingyi; McFarland, Kelli; Magleby, Karl L; Salkoff, Lawrence; Cui, Jianmin

    2017-03-06

    Large conductance Ca(2+)-activated K(+) channels (BK channels) gate open in response to both membrane voltage and intracellular Ca(2+) The channel is formed by a central pore-gate domain (PGD), which spans the membrane, plus transmembrane voltage sensors and a cytoplasmic gating ring that acts as a Ca(2+) sensor. How these voltage and Ca(2+) sensors influence the common activation gate, and interact with each other, is unclear. A previous study showed that a BK channel core lacking the entire cytoplasmic gating ring (Core-MT) was devoid of Ca(2+) activation but retained voltage sensitivity (Budelli et al. 2013. Proc. Natl. Acad. Sci. USA http://dx.doi.org/10.1073/pnas.1313433110). In this study, we measure voltage sensor activation and pore opening in this Core-MT channel over a wide range of voltages. We record gating currents and find that voltage sensor activation in this truncated channel is similar to WT but that the coupling between voltage sensor activation and gating of the pore is reduced. These results suggest that the gating ring, in addition to being the Ca(2+) sensor, enhances the effective coupling between voltage sensors and the PGD. We also find that removal of the gating ring alters modulation of the channels by the BK channel's β1 and β2 subunits.

  16. Quasi-specific access of the potassium channel inactivation gate.

    PubMed

    Venkataraman, Gaurav; Srikumar, Deepa; Holmgren, Miguel

    2014-06-09

    Many voltage-gated potassium channels open in response to membrane depolarization and then inactivate within milliseconds. Neurons use these channels to tune their excitability. In Shaker K(+) channels, inactivation is caused by the cytoplasmic amino terminus, termed the inactivation gate. Despite having four such gates, inactivation is caused by the movement of a single gate into a position that occludes ion permeation. The pathway that this single inactivation gate takes into its inactivating position remains unknown. Here we show that a single gate threads through the intracellular entryway of its own subunit, but the tip of the gate has sufficient freedom to interact with all four subunits deep in the pore, and does so with equal probability. This pathway demonstrates that flexibility afforded by the inactivation peptide segment at the tip of the N-terminus is used to mediate function.

  17. Cnidarian Toxins Acting on Voltage-Gated Ion Channels

    PubMed Central

    Messerli, Shanta M.; Greenberg, Robert M.

    2006-01-01

    Voltage-gated ion channels generate electrical activity in excitable cells. As such, they are essential components of neuromuscular and neuronal systems, and are targeted by toxins from a wide variety of phyla, including the cnidarians. Here, we review cnidarian toxins known to target voltage-gated ion channels, the specific channel types targeted, and, where known, the sites of action of cnidarian toxins on different channels.

  18. Purifying selection and demographic expansion affect sequence diversity of the ligand-binding domain of a glutamate-gated chloride channel gene of Haemonchus placei.

    PubMed

    Mes, Ted H M

    2004-04-01

    Ninety-five genomic sequences of the ligand-binding domain of glutamate-gated chloride channel genes of three populations of the parasitic nematode H. placei were evaluated for patterns of diversity, demography, and selection. These genes code for subunits of ion channels, which are involved in the mode of action of the most commonly used antiparasitic drugs, the macrocyclic lactones. An extremely high frequency of unique segregating sites in exons and introns was observed, with significantly negative neutrality tests in each population for noncoding, synonymous, and nonsynonymous sites. Several tests indicated that support for balancing selection, positive selection, and hitchhiking was lacking. McDonald-Kreitman tests using H. contortus or C. elegans as an outgroup revealed an extreme excess of replacement polymorphism, consistent with weak purifying selection. Although these tests agree that negative selection may explain the excess of replacement changes, an alternative interpretation is required for the significantly negative Fu and Li's D statistics based on silent and noncoding sites. These include homogeneous forces such as background selection and demographic expansion. The lack of population subdivision and the negative values of Tajima's D for this outbreeding parasitic nematode render background selection less likely than demographic expansion. Comparison of D statistics based on different site types using neutral coalescent simulations supported this interpretation. Although this statistic was more negative for nonsynonymous sites than for synonymous sites, most comparisons of the D statistic were not significantly different between mutation classes. A few significant site comparisons were also consistent with demographic expansion, because the observed test statistic ( D(neutral) - D(selected)) were low relative to the neutral expectations. Finally, previous mitochondrial studies also identified a demographic expansion of this parasitic nematode

  19. Gating of the designed trimeric/tetrameric voltage-gated H+ channel

    PubMed Central

    Fujiwara, Yuichiro; Kurokawa, Tatsuki; Takeshita, Kohei; Nakagawa, Atsushi; Larsson, H Peter; Okamura, Yasushi

    2013-01-01

    The voltage-gated H+ channel functions as a dimer, a configuration that is different from standard tetrameric voltage-gated channels. Each channel protomer has its own permeation pathway. The C-terminal coiled-coil domain has been shown to be necessary for both dimerization and cooperative gating in the two channel protomers. Here we report the gating cooperativity in trimeric and tetrameric Hv channels engineered by altering the hydrophobic core sequence of the coiled-coil assembly domain. Trimeric and tetrameric channels exhibited more rapid and less sigmoidal kinetics of activation of H+ permeation than dimeric channels, suggesting that some channel protomers in trimers and tetramers failed to produce gating cooperativity observed in wild-type dimers. Multimerization of trimer and tetramer channels were confirmed by the biochemical analysis of proteins, including crystallography. These findings indicate that the voltage-gated H+ channel is optimally designed as a dimeric channel on a solid foundation of the sequence pattern of the coiled-coil core, with efficient cooperative gating that ensures sustained and steep voltage-dependent H+ conductance in blood cells. PMID:23165764

  20. Gated regulation of CRAC channel ion selectivity by STIM1

    PubMed Central

    McNally, Beth A.; Somasundaram, Agila; Yamashita, Megumi; Prakriya, Murali

    2011-01-01

    Two defining functional features of ion channels are ion selectivity and channel gating. Ion selectivity is generally considered an immutable property of the open channel structure, whereas gating involves transitions between open and closed channel states typically without changes in ion selectivity 1. In store-operated Ca2+ release-activated Ca2+ (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, STIM1 (stromal interaction molecule 1) remains unknown. CRAC channels are distinguished by an extraordinarily high Ca2+ selectivity and are instrumental in generating sustained [Ca2+]i elevations necessary for gene expression and effector function in many eukaryotic cells 2. Here, we probed the central features of the STIM1 gating mechanism in the CRAC channel protein, Orai1, and identified V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produced constitutively active CRAC channels that were open even in the absence of STIM1. Unexpectedly, although STIM1-free V102 mutant channels were not Ca2+-selective, their Ca2+ selectivity was dose-dependently boosted by interactions with STIM1. Similar enhancement of Ca2+ selectivity also occurred in wild-type (WT) Orai1 channels by increasing the number of STIM1 activation domains directly tethered to Orai1 channels. Thus, exquisite Ca2+ selectivity is not an intrinsic property of CRAC channels, but rather a tunable feature bestowed on otherwise non-selective Orai1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism wherein permeation and gating are closely coupled. PMID:22278058

  1. Evolutionarily conserved intracellular gate of voltage-dependent sodium channels

    NASA Astrophysics Data System (ADS)

    Oelstrom, Kevin; Goldschen-Ohm, Marcel P.; Holmgren, Miguel; Chanda, Baron

    2014-03-01

    Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

  2. Evolutionarily conserved intracellular gate of voltage-dependent sodium channels

    PubMed Central

    Oelstrom, Kevin; Goldschen-Ohm, Marcel P.; Holmgren, Miguel; Chanda, Baron

    2014-01-01

    Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily. PMID:24619022

  3. Emergence of ion channel modal gating from independent subunit kinetics.

    PubMed

    Bicknell, Brendan A; Goodhill, Geoffrey J

    2016-09-06

    Many ion channels exhibit a slow stochastic switching between distinct modes of gating activity. This feature of channel behavior has pronounced implications for the dynamics of ionic currents and the signaling pathways that they regulate. A canonical example is the inositol 1,4,5-trisphosphate receptor (IP3R) channel, whose regulation of intracellular Ca(2+) concentration is essential for numerous cellular processes. However, the underlying biophysical mechanisms that give rise to modal gating in this and most other channels remain unknown. Although ion channels are composed of protein subunits, previous mathematical models of modal gating are coarse grained at the level of whole-channel states, limiting further dialogue between theory and experiment. Here we propose an origin for modal gating, by modeling the kinetics of ligand binding and conformational change in the IP3R at the subunit level. We find good agreement with experimental data over a wide range of ligand concentrations, accounting for equilibrium channel properties, transient responses to changing ligand conditions, and modal gating statistics. We show how this can be understood within a simple analytical framework and confirm our results with stochastic simulations. The model assumes that channel subunits are independent, demonstrating that cooperative binding or concerted conformational changes are not required for modal gating. Moreover, the model embodies a generally applicable principle: If a timescale separation exists in the kinetics of individual subunits, then modal gating can arise as an emergent property of channel behavior.

  4. Emergence of ion channel modal gating from independent subunit kinetics

    PubMed Central

    Bicknell, Brendan A.

    2016-01-01

    Many ion channels exhibit a slow stochastic switching between distinct modes of gating activity. This feature of channel behavior has pronounced implications for the dynamics of ionic currents and the signaling pathways that they regulate. A canonical example is the inositol 1,4,5-trisphosphate receptor (IP3R) channel, whose regulation of intracellular Ca2+ concentration is essential for numerous cellular processes. However, the underlying biophysical mechanisms that give rise to modal gating in this and most other channels remain unknown. Although ion channels are composed of protein subunits, previous mathematical models of modal gating are coarse grained at the level of whole-channel states, limiting further dialogue between theory and experiment. Here we propose an origin for modal gating, by modeling the kinetics of ligand binding and conformational change in the IP3R at the subunit level. We find good agreement with experimental data over a wide range of ligand concentrations, accounting for equilibrium channel properties, transient responses to changing ligand conditions, and modal gating statistics. We show how this can be understood within a simple analytical framework and confirm our results with stochastic simulations. The model assumes that channel subunits are independent, demonstrating that cooperative binding or concerted conformational changes are not required for modal gating. Moreover, the model embodies a generally applicable principle: If a timescale separation exists in the kinetics of individual subunits, then modal gating can arise as an emergent property of channel behavior. PMID:27551100

  5. Ion channel gates: comparative analysis of energy barriers.

    PubMed

    Tai, Kaihsu; Haider, Shozeb; Grottesi, Alessandro; Sansom, Mark S P

    2009-04-01

    The energetic profile of an ion translated along the axis of an ion channel should reveal whether the structure corresponds to a functionally open or closed state of the channel. In this study, we explore the combined use of Poisson-Boltzmann electrostatic calculations and evaluation of van der Waals interactions between ion and pore to provide an initial appraisal of the gating state of a channel. This approach is exemplified by its application to the bacterial inward rectifier potassium channel KirBac3.1, where it reveals the closed gate to be formed by a ring of leucine (L124) side chains. We have extended this analysis to a comparative survey of gating profiles, including model hydrophobic nanopores, the nicotinic acetylcholine receptor, and a number of potassium channel structures and models. This enables us to identify three gating regimes, and to show the limitation of this computationally inexpensive method. For a (closed) gate radius of 0.4 nm < R < 0.8 nm, a hydrophobic gate may be present. For a gate radius of 0.2 nm < R < 0.4 nm, both electrostatic and van der Waals interactions will contribute to the barrier height. Below R = 0.2 nm, repulsive van der Waals interactions are likely to dominate, resulting in a sterically occluded gate. In general, the method is more useful when the channel is wider; for narrower channels, the flexibility of the protein may allow otherwise-unsurmountable energetic barriers to be overcome.

  6. Single-channel properties of ionic channels gated by cyclic nucleotides.

    PubMed Central

    Bucossi, G; Nizzari, M; Torre, V

    1997-01-01

    This paper presents an extensive analysis of single-channel properties of cyclic nucleotide gated (CNG) channels, obtained by injecting into Xenopus laevis oocytes the mRNA encoding for the alpha and beta subunits from bovine rods. When the alpha and beta subunits of the CNG channel are coexpressed, at least three types of channels with different properties are observed. One type of channel has well-resolved, multiple conductive levels at negative voltages, but not at positive voltages. The other two types of channel are characterized by flickering openings, but are distinguished because they have a low and a high conductance. The alpha subunit of CNG channels has a well-defined conductance of about 28 pS, but multiple conductive levels are observed in mutant channels E363D and T364M. The conductance of these open states is modulated by protons and the membrane voltage, and has an activation energy around 44 kJ/mol. The relative probability of occupying any of these open states is independent of the cGMP concentration, but depends on extracellular protons. The open probability in the presence of saturating cGMP was 0.78, 0.47, 0.5, and 0.007 in the w.t. and mutants E363D, T364M, and E363G, and its dependence on temperature indicates that the thermodynamics of the transition between the closed and open state is also affected by mutations in the pore region. These results suggest that CNG channels have different conductive levels, leading to the existence of multiple open states in homomeric channels and to the flickering behavior in heteromeric channels, and that the pore is an essential part of the gating of CNG channels. PMID:9138564

  7. Voltage-gated calcium channels of Paramecium cilia.

    PubMed

    Lodh, Sukanya; Yano, Junji; Valentine, Megan S; Van Houten, Judith L

    2016-10-01

    Paramecium cells swim by beating their cilia, and make turns by transiently reversing their power stroke. Reversal is caused by Ca(2+) entering the cilium through voltage-gated Ca(2+) (CaV) channels that are found exclusively in the cilia. As ciliary Ca(2+) levels return to normal, the cell pivots and swims forward in a new direction. Thus, the activation of the CaV channels causes cells to make a turn in their swimming paths. For 45 years, the physiological characteristics of the Paramecium ciliary CaV channels have been known, but the proteins were not identified until recently, when the P. tetraurelia ciliary membrane proteome was determined. Three CaVα1 subunits that were identified among the proteins were cloned and confirmed to be expressed in the cilia. We demonstrate using RNA interference that these channels function as the ciliary CaV channels that are responsible for the reversal of ciliary beating. Furthermore, we show that Pawn (pw) mutants of Paramecium that cannot swim backward for lack of CaV channel activity do not express any of the three CaV1 channels in their ciliary membrane, until they are rescued from the mutant phenotype by expression of the wild-type PW gene. These results reinforce the correlation of the three CaV channels with backward swimming through ciliary reversal. The PwB protein, found in endoplasmic reticulum fractions, co-immunoprecipitates with the CaV1c channel and perhaps functions in trafficking. The PwA protein does not appear to have an interaction with the channel proteins but affects their appearance in the cilia. © 2016. Published by The Company of Biologists Ltd.

  8. Voltage-gated calcium channels of Paramecium cilia

    PubMed Central

    Lodh, Sukanya; Valentine, Megan S.; Van Houten, Judith L.

    2016-01-01

    ABSTRACT Paramecium cells swim by beating their cilia, and make turns by transiently reversing their power stroke. Reversal is caused by Ca2+ entering the cilium through voltage-gated Ca2+ (CaV) channels that are found exclusively in the cilia. As ciliary Ca2+ levels return to normal, the cell pivots and swims forward in a new direction. Thus, the activation of the CaV channels causes cells to make a turn in their swimming paths. For 45 years, the physiological characteristics of the Paramecium ciliary CaV channels have been known, but the proteins were not identified until recently, when the P. tetraurelia ciliary membrane proteome was determined. Three CaVα1 subunits that were identified among the proteins were cloned and confirmed to be expressed in the cilia. We demonstrate using RNA interference that these channels function as the ciliary CaV channels that are responsible for the reversal of ciliary beating. Furthermore, we show that Pawn (pw) mutants of Paramecium that cannot swim backward for lack of CaV channel activity do not express any of the three CaV1 channels in their ciliary membrane, until they are rescued from the mutant phenotype by expression of the wild-type PW gene. These results reinforce the correlation of the three CaV channels with backward swimming through ciliary reversal. The PwB protein, found in endoplasmic reticulum fractions, co-immunoprecipitates with the CaV1c channel and perhaps functions in trafficking. The PwA protein does not appear to have an interaction with the channel proteins but affects their appearance in the cilia. PMID:27707864

  9. 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

  10. The cooperative voltage sensor motion that gates a potassium channel.

    PubMed

    Pathak, Medha; Kurtz, Lisa; Tombola, Francesco; Isacoff, Ehud

    2005-01-01

    The four arginine-rich S4 helices of a voltage-gated channel move outward through the membrane in response to depolarization, opening and closing gates to generate a transient ionic current. Coupling of voltage sensing to gating was originally thought to operate with the S4s moving independently from an inward/resting to an outward/activated conformation, so that when all four S4s are activated, the gates are driven to open or closed. However, S4 has also been found to influence the cooperative opening step (Smith-Maxwell et al., 1998a), suggesting a more complex mechanism of coupling. Using fluorescence to monitor structural rearrangements in a Shaker channel mutant, the ILT channel (Ledwell and Aldrich, 1999), that energetically isolates the steps of activation from the cooperative opening step, we find that opening is accompanied by a previously unknown and cooperative movement of S4. This gating motion of S4 appears to be coupled to the internal S6 gate and to two forms of slow inactivation. Our results suggest that S4 plays a direct role in gating. While large transmembrane rearrangements of S4 may be required to unlock the gating machinery, as proposed before, it appears to be the gating motion of S4 that drives the gates to open and close.

  11. Voltage-gated Calcium Channels and Autism Spectrum Disorders.

    PubMed

    Breitenkamp, Alexandra F; Matthes, Jan; Herzig, Stefan

    2015-01-01

    Autism spectrum disorder is a complex-genetic disease and its etiology is unknown for the majority of cases. So far, more than one hundred different susceptibility genes were detected. Voltage-gated calcium channels are among the candidates linked to autism spectrum disorder by results of genetic studies. Mutations of nearly all pore-forming and some auxiliary subunits of voltage gated calcium channels have been revealed from investigations of autism spectrum disorder patients and populations. Though there are only few electrophysiological characterizations of voltage-gated calcium channel mutations found in autistic patients these studies suggest their functional relevance. In summary, both genetic and functional data suggest a potential role of voltage-gated calcium channels in autism spectrum disorder. Future studies require refinement of the clinical and systems biological concepts of autism spectrum disorder and an appropriate holistic approach at the molecular level, e.g. regarding all facets of calcium channel functions.

  12. 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

  13. Cytoplasmic Domains and Voltage-Dependent Potassium Channel Gating

    PubMed Central

    Barros, Francisco; Domínguez, Pedro; de la Peña, Pilar

    2012-01-01

    The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered. PMID:22470342

  14. 3. INTAKE CHANNEL LOOKING WEST; DEBRIS FILTER SCREEN IN GATE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. INTAKE CHANNEL LOOKING WEST; DEBRIS FILTER SCREEN IN GATE 2. - Hondius Water Line, 1.6 miles Northwest of Park headquarters building & 1 mile Northwest of Beaver Meadows entrance station, Estes Park, Larimer County, CO

  15. 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

  16. Gating motions in voltage-gated potassium channels revealed by coarse-grained molecular dynamics simulations.

    PubMed

    Treptow, Werner; Marrink, Siewert-J; Tarek, Mounir

    2008-03-20

    Voltage-gated potassium (Kv) channels are ubiquitous transmembrane proteins involved in electric signaling of excitable tissues. A fundamental property of these channels is the ability to open or close in response to changes in the membrane potential. To date, their structure-based activation mechanism remains unclear, and there is a large controversy on how these gates function at the molecular level, in particular, how movements of the voltage sensor domain are coupled to channel gating. So far, all mechanisms proposed for this coupling are based on the crystal structure of the open voltage-gated Kv1.2 channel and structural models of the closed form based on electrophysiology experiments. Here, we use coarse-grain (CG) molecular dynamics simulations that allow conformational changes from the open to the closed form of the channel (embedded in its membrane environment) to be followed. Despite the low specificity of the CG force field, the obtained closed structure satisfies several experimental constraints. The overall results suggest a gating mechanism in which a lateral displacement the S4-S5 linker leads to a closing of the gate. Only a small up-down movement of the S4 helices is noticed. Additionally, the study suggests a peculiar upward motion of the intracellular tetramerization domain of the channel, hence providing a molecular view on how this domain may further regulate conduction in Kv channels.

  17. Biophysics, pathophysiology, and pharmacology of ion channel gating pores

    PubMed Central

    Moreau, Adrien; Gosselin-Badaroudine, Pascal; Chahine, Mohamed

    2014-01-01

    Voltage sensor domains (VSDs) are a feature of voltage gated ion channels (VGICs) and voltage sensitive proteins. They are composed of four transmembrane (TM) segments (S1–S4). Currents leaking through VSDs are called omega or gating pore currents. Gating pores are caused by mutations of the highly conserved positively charged amino acids in the S4 segment that disrupt interactions between the S4 segment and the gating charge transfer center (GCTC). The GCTC separates the intracellular and extracellular water crevices. The disruption of S4–GCTC interactions allows these crevices to communicate and create a fast activating and non-inactivating alternative cation-selective permeation pathway of low conductance, or a gating pore. Gating pore currents have recently been shown to cause periodic paralysis phenotypes. There is also increasing evidence that gating pores are linked to several other familial diseases. For example, gating pores in Nav1.5 and Kv7.2 channels may underlie mixed arrhythmias associated with dilated cardiomyopathy (DCM) phenotypes and peripheral nerve hyperexcitability (PNH), respectively. There is little evidence for the existence of gating pore blockers. Moreover, it is known that a number of toxins bind to the VSD of a specific domain of Na+ channels. These toxins may thus modulate gating pore currents. This focus on the VSD motif opens up a new area of research centered on developing molecules to treat a number of cell excitability disorders such as epilepsy, cardiac arrhythmias, and pain. The purpose of the present review is to summarize existing knowledge of the pathophysiology, biophysics, and pharmacology of gating pore currents and to serve as a guide for future studies aimed at improving our understanding of gating pores and their pathophysiological roles. PMID:24772081

  18. Biophysical properties of the voltage gated proton channel HV1

    PubMed Central

    Musset, Boris; DeCoursey, Thomas

    2012-01-01

    The biophysical properties of the voltage gated proton channel (HV1) are the key elements of its physiological function. The voltage gated proton channel is a unique molecule that in contrast to all other ion channels is exclusively selective for protons. Alone among proton channels, it has voltage and time dependent gating like other “classical” ion channels. HV1 is furthermore a sensor for the pH in the cell and the surrounding media. Its voltage dependence is strictly coupled to the pH gradient across the membrane. This regulation restricts opening of the channel to specific voltages at any given pH gradient, therefore allowing HV1 to perform its physiological task in the tissue it is expressed in. For HV1 there is no known blocker. The most potent channel inhibitor is zinc (Zn2+) which prevents channel opening. An additional characteristic of HV1 is its strong temperature dependence of both gating and conductance. In contrast to single-file water filled pores like the gramicidin channel, HV1 exhibits pronounced deuterium effects and temperature effects on conduction, consistent with a different conduction mechanism than other ion channels. These properties may be explained by the recent identification of an aspartate in the pore of HV1 that is essential to its proton selectivity. PMID:23050239

  19. Animal Toxins Influence Voltage-Gated Sodium Channel Function

    PubMed Central

    Gilchrist, John

    2017-01-01

    Voltage-gated sodium (Nav) channels are essential contributors to neuronal excitability, making them the most commonly targeted ion channel family by toxins found in animal venoms. These molecules can be used to probe the functional aspects of Nav channels on a molecular level and to explore their physiological role in normal and diseased tissues. This chapter summarizes our existing knowledge of the mechanisms by which animal toxins influence Nav channels as well as their potential application in designing therapeutic drugs. PMID:24737238

  20. Monte Carlo study of gating and selection in potassium channels

    NASA Astrophysics Data System (ADS)

    Andreucci, Daniele; Bellaveglia, Dario; Cirillo, Emilio N. M.; Marconi, Silvia

    2011-08-01

    The study of selection and gating in potassium channels is a very important issue in modern biology. Indeed, such structures are known in essentially all types of cells in all organisms where they play many important functional roles. The mechanism of gating and selection of ionic species is not clearly understood. In this paper we study a model in which gating is obtained via an affinity-switching selectivity filter. We discuss the dependence of selectivity and efficiency on the cytosolic ionic concentration and on the typical pore open state duration. We demonstrate that a simple modification in the way in which the selectivity filter is modeled yields larger channel efficiency.

  1. Decavanadate modulates gating of TRPM4 cation channels.

    PubMed

    Nilius, Bernd; Prenen, Jean; Janssens, Annelies; Voets, Thomas; Droogmans, Guy

    2004-11-01

    We have tested the effects of decavanadate (DV), a compound known to interfere with ATP binding in ATP-dependent transport proteins, on TRPM4, a Ca(2+)-activated, voltage-dependent monovalent cation channel, whose activity is potently blocked by intracellular ATP(4-). Application of micromolar Ca(2+) concentrations to the cytoplasmic side of inside-out patches led to immediate current activation followed by rapid current decay, which can be explained by an at least 30-fold decreased apparent affinity for Ca(2+). Subsequent application of DV (10 microm) strongly affected the voltage-dependent gating of the channel, resulting in large sustained currents over the voltage range between -180 and +140 mV. The effect of DV was half-maximal at a concentration of 1.9 microm. The Ca(2+)- and voltage-dependent gating of the channel was well described by a sequential kinetic scheme in which Ca(2+) binding precedes voltage-dependent gating. The effects of DV could be explained by an action on the voltage-dependent closing step. Surprisingly, DV did not antagonize the effect of ATP(4-) on TRPM4, but caused a nearly 10-fold increase in the sensitivity of the ATP(4-) block. TRPM5, which is the most homologous channel to TRPM4, was not modulated by DV. The effect of DV was lost in a TRPM4 chimera in which the C-terminus was substituted with that of TRPM5. Deletion of a cluster in the C-terminus of TRPM4 containing positively charged amino acid residues with a high homology to part of the decavanadate binding site in SERCA pumps, completely abolished the DV effect but also accelerated desensitization. Deletion of a similar site in the N-terminus had no effects on DV responses. These results indicate that the C-terminus of TRPM4 is critically involved in mediating the DV effects. In conclusion, decavanadate modulates TRPM4, but not TRPM5, by inhibiting voltage-dependent closure of the channel.

  2. 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.

  3. 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.

  4. Voltage Gated Ion Channel Function: Gating, Conduction, and the Role of Water and Protons

    PubMed Central

    Kariev, Alisher M.; Green, Michael E.

    2012-01-01

    Ion channels, which are found in every biological cell, regulate the concentration of electrolytes, and are responsible for multiple biological functions, including in particular the propagation of nerve impulses. The channels with the latter function are gated (opened) by a voltage signal, which allows Na+ into the cell and K+ out. These channels have several positively charged amino acids on a transmembrane domain of their voltage sensor, and it is generally considered, based primarily on two lines of experimental evidence, that these charges move with respect to the membrane to open the channel. At least three forms of motion, with greatly differing extents and mechanisms of motion, have been proposed. There is a “gating current”, a capacitative current preceding the channel opening, that corresponds to several charges (for one class of channel typically 12–13) crossing the membrane field, which may not require protein physically crossing a large fraction of the membrane. The coupling to the opening of the channel would in these models depend on the motion. The conduction itself is usually assumed to require the “gate” of the channel to be pulled apart to allow ions to enter as a section of the protein partially crosses the membrane, and a selectivity filter at the opposite end of the channel determines the ion which is allowed to pass through. We will here primarily consider K+ channels, although Na+ channels are similar. We propose that the mechanism of gating differs from that which is generally accepted, in that the positively charged residues need not move (there may be some motion, but not as gating current). Instead, protons may constitute the gating current, causing the gate to open; opening consists of only increasing the diameter at the gate from approximately 6 Å to approximately 12 Å. We propose in addition that the gate oscillates rather than simply opens, and the ion experiences a barrier to its motion across the channel that is tuned

  5. Allosteric gating mechanism underlies the flexible gating of KCNQ1 potassium channels.

    PubMed

    Osteen, Jeremiah D; Barro-Soria, Rene; Robey, Seth; Sampson, Kevin J; Kass, Robert S; Larsson, H Peter

    2012-05-01

    KCNQ1 (Kv7.1) is a unique member of the superfamily of voltage-gated K(+) channels in that it displays a remarkable range of gating behaviors tuned by coassembly with different β subunits of the KCNE family of proteins. To better understand the basis for the biophysical diversity of KCNQ1 channels, we here investigate the basis of KCNQ1 gating in the absence of β subunits using voltage-clamp fluorometry (VCF). In our previous study, we found the kinetics and voltage dependence of voltage-sensor movements are very similar to those of the channel gate, as if multiple voltage-sensor movements are not required to precede gate opening. Here, we have tested two different hypotheses to explain KCNQ1 gating: (i) KCNQ1 voltage sensors undergo a single concerted movement that leads to channel opening, or (ii) individual voltage-sensor movements lead to channel opening before all voltage sensors have moved. Here, we find that KCNQ1 voltage sensors move relatively independently, but that the channel can conduct before all voltage sensors have activated. We explore a KCNQ1 point mutation that causes some channels to transition to the open state even in the absence of voltage-sensor movement. To interpret these results, we adopt an allosteric gating scheme wherein KCNQ1 is able to transition to the open state after zero to four voltage-sensor movements. This model allows for widely varying gating behavior, depending on the relative strength of the opening transition, and suggests how KCNQ1 could be controlled by coassembly with different KCNE family members.

  6. Charge movement in gating-locked HCN channels reveals weak coupling of voltage sensors and gate.

    PubMed

    Ryu, Sujung; Yellen, Gary

    2012-11-01

    HCN (hyperpolarization-activated cyclic nucleotide gated) pacemaker channels have an architecture similar to that of voltage-gated K(+) channels, but they open with the opposite voltage dependence. HCN channels use essentially the same positively charged voltage sensors and intracellular activation gates as K(+) channels, but apparently these two components are coupled differently. In this study, we examine the energetics of coupling between the voltage sensor and the pore by using cysteine mutant channels for which low concentrations of Cd(2+) ions freeze the open-closed gating machinery but still allow the sensors to move. We were able to lock mutant channels either into open or into closed states by the application of Cd(2+) and measure the effect on voltage sensor movement. Cd(2+) did not immobilize the gating charge, as expected for strict coupling, but rather it produced shifts in the voltage dependence of voltage sensor charge movement, consistent with its effect of confining transitions to either closed or open states. From the magnitude of the Cd(2+)-induced shifts, we estimate that each voltage sensor produces a roughly three- to sevenfold effect on the open-closed equilibrium, corresponding to a coupling energy of ∼1.3-2 kT per sensor. Such coupling is not only opposite in sign to the coupling in K(+) channels, but also much weaker.

  7. Interaction of the BKCa channel gating ring with dendrotoxins

    PubMed Central

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

    2014-01-01

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

  8. Surfaces and boundaries in the mechanosensitive channel gating

    NASA Astrophysics Data System (ADS)

    Sukharev, Sergei

    2009-03-01

    Mechanosensitive (MS) channels are gated by tension transmitted through the surrounding lipid bilayer. Inorganic ions or amphipathic modifiers that interact with the bilayer surface alter the packing of lipids and perturb the lateral pressure. We describe the effects of lanthanide ions, fluorinated alcohols and esters of parabenzoic acid as potent modifiers of MS channel gating. The other boundary that plays a critical role in channel gating is the water-vapor interface resulting from capillary dewetting of the hydrophobic gate. Molecular simulations predict two alternate positions for this boundary in the pore of the mechanosensitive channel MscS. We approached this problem experimentally by hydrophilizing the outer segment of the pore to resolve if it is `dry' in the closed state. We observed a reduction in activating tension, substantial changes in MscS kinetics and complete removal of gating hysteresis. The kinetic treatment of channel traces recorded in response to steps of tension suggested the sequence of events that leads to the channel opening implying that pore hydration and dewetting are the rate-limiting steps in MscS transitions.

  9. HMJ-53A accelerates slow inactivation gating of voltage-gated K+ channels in mouse neuroblastoma N2A cells.

    PubMed

    Chao, Chia-Chia; Shieh, Jeffrey; Kuo, Sheng-Chu; Wu, Bor-Tsang; Hour, Mann-Jen; Leung, Yuk-Man

    2008-06-01

    Voltage-gated K(+) (Kv) channels are important in repolarization of excitable cells such as neurons and endocrine cells. Kv channel gating exhibits slow inactivation (slow current decay) during continuous depolarization. The molecular mechanism involved in such slow inactivation is not completely understood, but evidence has suggested that it involves a restriction of the outer channel pore surrounding the selectivity filter. Pharmacological tools probing this slow inactivation process are scarce. In this work we reported that bath application of HMJ-53A (30 microM), a novel compound, could drastically speed up the slow decay (decay tau=1677+/-120 ms and 85.6+/-7.7 ms, respectively, in the absence and presence of HMJ-53A) of Kv currents in neuroblastoma N2A cells. HMJ-53A also significantly left-shifted the steady-state inactivation curve by 12 mV. HMJ-53A, however, did not affect voltage-dependence of activation and the kinetics of channel activation. Intracellular application of this drug through patch pipette dialysis was ineffective at all in accelerating the slow current decay, suggesting that HMJ-53A acted extracellularly. Blockade of currents by HMJ-53A did not require an open state of channels. In addition, the inactivation time constants and percentage block of Kv currents in the presence of HMJ-53A were independent of the (i) degree of depolarization and (ii) intracellular K(+) concentration. Therefore, this drug did not appear to directly occlude the outer channel pore during stimulation (depolarization). Taken together, our results suggest that HMJ-53A selectively affected (accelerated) the slow inactivation gating process of Kv channels, and could thus be a selective and novel probe for the inactivation gate.

  10. Gating the Selectivity Filter in ClC Chloride Channels

    NASA Astrophysics Data System (ADS)

    Dutzler, Raimund; Campbell, Ernest B.; MacKinnon, Roderick

    2003-04-01

    ClC channels conduct chloride (Cl-) ions across cell membranes and thereby govern the electrical activity of muscle cells and certain neurons, the transport of fluid and electrolytes across epithelia, and the acidification of intracellular vesicles. The structural basis of ClC channel gating was studied. Crystal structures of wild-type and mutant Escherichia coli ClC channels bound to a monoclonal Fab fragment reveal three Cl- binding sites within the 15-angstrom neck of an hourglass-shaped pore. The Cl- binding site nearest the extracellular solution can be occupied either by a Cl- ion or by a glutamate carboxyl group. Mutations of this glutamate residue in Torpedo ray ClC channels alter gating in electrophysiological assays. These findings reveal a form of gating in which the glutamate carboxyl group closes the pore by mimicking a Cl- ion.

  11. Comparative study of the gating motif and C-type inactivation in prokaryotic voltage-gated sodium channels.

    PubMed

    Irie, Katsumasa; Kitagawa, Kazuya; Nagura, Hitoshi; Imai, Tomoya; Shimomura, Takushi; Fujiyoshi, Yoshinori

    2010-02-05

    Prokaryotic voltage-gated sodium channels (Na(V)s) are homotetramers and are thought to inactivate through a single mechanism, named C-type inactivation. Here we report the voltage dependence and inactivation rate of the NaChBac channel from Bacillus halodurans, the first identified prokaryotic Na(V), as well as of three new homologues cloned from Bacillus licheniformis (Na(V)BacL), Shewanella putrefaciens (Na(V)SheP), and Roseobacter denitrificans (Na(V)RosD). We found that, although activated by a lower membrane potential, Na(V)BacL inactivates as slowly as NaChBac. Na(V)SheP and Na(V)RosD inactivate faster than NaChBac. Mutational analysis of helix S6 showed that residues corresponding to the "glycine hinge" and "PXP motif" in voltage-gated potassium channels are not obligatory for channel gating in these prokaryotic Na(V)s, but mutations in the regions changed the inactivation rates. Mutation of the region corresponding to the glycine hinge in Na(V)BacL (A214G), Na(V)SheP (A216G), and NaChBac (G219A) accelerated inactivation in these channels, whereas mutation of glycine to alanine in the lower part of helix S6 in NaChBac (G229A), Na(V)BacL (G224A), and Na(V)RosD (G217A) reduced the inactivation rate. These results imply that activation gating in prokaryotic Na(V)s does not require gating motifs and that the residues of helix S6 affect C-type inactivation rates in these channels.

  12. Redox Regulation of Neuronal Voltage-Gated Calcium Channels

    PubMed Central

    Jevtovic-Todorovic, Vesna

    2014-01-01

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

  13. Sea anemone venom as a source of insecticidal peptides acting on voltage-gated Na+ channels

    PubMed Central

    Bosmans, Frank; Tytgat, Jan

    2007-01-01

    Sea anemones produce a myriad of toxic peptides and proteins of which a large group acts on voltage-gated Na+ channels. However, in comparison to other organisms, their venoms and toxins are poorly studied. Most of the known voltage-gated Na+ channel toxins isolated from sea anemone venoms act on neurotoxin receptor site 3 and inhibit the inactivation of these channels. Furthermore, it seems that most of these toxins have a distinct preference for crustaceans. Given the close evolutionary relationship between crustaceans and insects, it is not surprising that sea anemone toxins also profoundly affect insect voltage-gated Na+ channels, which constitutes the scope of this review. For this reason, these peptides can be considered as insecticidal lead compounds in the development of insecticides. PMID:17224168

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

    PubMed Central

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

    2013-01-01

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

  15. On the Evolution of Voltage Gated Ion Channels

    NASA Astrophysics Data System (ADS)

    Brenner, Michael

    2006-03-01

    This talk summarizes some ideas, calculations and data analysis/collection surrounding the structure and evolution of ion channels, in particular voltage gated sodium channels. The great advantage of ion channels is that they are individual proteins whose function has long been known and is readily inferred through voltage measurements. Their evolution can be tracked through the growing data base of sequences. Kinetic data is readily available, showing important differences between nearly identical channels. I will discuss our efforts to collate available functional data on voltage gated sodium channels into an 'ion channel property space' . We then use this dataset to infer underlying kinetic models, and to create evolutionary trees based on the function of the channels. Finally, I will discuss our endeavors to how ion channels evolved to be the way they are: Examples of questions we would like to answer include: to what extent do design principles dictate the details of the kinetic schemes of ion channels, such as (a) the symmetry of the sodium and potassium channels (or lack thereof), as reflected in their kinetic schemes ; (b) the coupling of sodium channel kinetics to potassium channel kinetics; or (c) activation/inactivation of the channels themselves.

  16. Desensitization mechanism in prokaryotic ligand-gated ion channel.

    PubMed

    Velisetty, Phanindra; Chakrapani, Sudha

    2012-05-25

    Crystal structures of Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated prokaryotic homologue of pentameric ligand-gated ion channel (LGIC) from G. violaceus, have provided high-resolution models of the channel architecture and its role in selective ion conduction and drug binding. However, it is still unclear which functional states of the LGIC gating scheme these crystal structures represent. Much of this uncertainty arises from a lack of thorough understanding of the functional properties of these prokaryotic channels. To elucidate the molecular events that constitute gating, we have carried out an extensive characterization of GLIC function and dynamics in reconstituted proteoliposomes by patch clamp measurements and EPR spectroscopy. We find that GLIC channels show rapid activation upon jumps to acidic pH followed by a time-dependent loss of conductance because of desensitization. GLIC desensitization is strongly coupled to activation and is modulated by voltage, permeant ions, pore-blocking drugs, and membrane cholesterol. Many of these properties are parallel to functions observed in members of eukaryotic LGIC. Conformational changes in loop C, measured by site-directed spin labeling and EPR spectroscopy, reveal immobilization during desensitization analogous to changes in LGIC and acetylcholine binding protein. Together, our studies suggest conservation of mechanistic aspects of desensitization among LGICs of prokaryotic and eukaryotic origin.

  17. Voltage-gated proton channel is expressed on phagosomes

    SciTech Connect

    Okochi, Yoshifumi; Sasaki, Mari; Iwasaki, Hirohide; Okamura, Yasushi

    2009-05-01

    Voltage-gated proton channel has been suggested to help NADPH oxidase activity during respiratory burst of phagocytes through its activities of compensating charge imbalance and regulation of pH. In phagocytes, robust production of reactive oxygen species occurs in closed membrane compartments, which are called phagosomes. However, direct evidence for the presence of voltage-gated proton channels in phagosome has been lacking. In this study, the expression of voltage-gated proton channels was studied by Western blot with the antibody specific to the voltage-sensor domain protein, VSOP/Hv1, that has recently been identified as the molecular correlate for the voltage-gated proton channel. Phagosomal membranes of neutrophils contain VSOP/Hv1 in accordance with subunits of NADPH oxidases, gp91, p22, p47 and p67. Superoxide anion production upon PMA activation was significantly reduced in neutrophils from VSOP/Hv1 knockout mice. These are consistent with the idea that voltage-gated proton channels help NADPH oxidase in phagocytes to produce reactive oxygen species.

  18. Voltage-gated proton channel is expressed on phagosomes.

    PubMed

    Okochi, Yoshifumi; Sasaki, Mari; Iwasaki, Hirohide; Okamura, Yasushi

    2009-05-01

    Voltage-gated proton channel has been suggested to help NADPH oxidase activity during respiratory burst of phagocytes through its activities of compensating charge imbalance and regulation of pH. In phagocytes, robust production of reactive oxygen species occurs in closed membrane compartments, which are called phagosomes. However, direct evidence for the presence of voltage-gated proton channels in phagosome has been lacking. In this study, the expression of voltage-gated proton channels was studied by Western blot with the antibody specific to the voltage-sensor domain protein, VSOP/Hv1, that has recently been identified as the molecular correlate for the voltage-gated proton channel. Phagosomal membranes of neutrophils contain VSOP/Hv1 in accordance with subunits of NADPH oxidases, gp91, p22, p47 and p67. Superoxide anion production upon PMA activation was significantly reduced in neutrophils from VSOP/Hv1 knockout mice. These are consistent with the idea that voltage-gated proton channels help NADPH oxidase in phagocytes to produce reactive oxygen species.

  19. Voltage-gated sodium channels: biophysics, pharmacology, and related channelopathies.

    PubMed

    Savio-Galimberti, Eleonora; Gollob, Michael H; Darbar, Dawood

    2012-01-01

    Voltage-gated sodium channels (VGSC) are multi-molecular protein complexes expressed in both excitable and non-excitable cells. They are primarily formed by a pore-forming multi-spanning integral membrane glycoprotein (α-subunit) that can be associated with one or more regulatory β-subunits. The latter are single-span integral membrane proteins that modulate the sodium current (I(Na)) and can also function as cell adhesion molecules. In vitro some of the cell-adhesive functions of the β-subunits may play important physiological roles independently of the α-subunits. Other endogenous regulatory proteins named "channel partners" or "channel interacting proteins" (ChiPs) like caveolin-3 and calmodulin/calmodulin kinase II (CaMKII) can also interact and modulate the expression and/or function of VGSC. In addition to their physiological roles in cell excitability and cell adhesion, VGSC are the site of action of toxins (like tetrodotoxin and saxitoxin), and pharmacologic agents (like antiarrhythmic drugs, local anesthetics, antiepileptic drugs, and newly developed analgesics). Mutations in genes that encode α- and/or β-subunits as well as the ChiPs can affect the structure and biophysical properties of VGSC, leading to the development of diseases termed sodium "channelopathies".  This review will outline the structure, function, and biophysical properties of VGSC as well as their pharmacology and associated channelopathies and highlight some of the recent advances in this field.

  20. Potassium channels and their evolving gates.

    PubMed

    Jan, L Y; Jan, Y N

    1994-09-08

    Potassium channels allow potassium ions to flow across the membrane and play a key role in maintaining membrane potential. Recent research has begun to reveal how these channels transport potassium in preference to other ions, how their activity is controlled, and how they are related to other channels.

  1. The gating mechanism of the large mechanosensitive channel MscL

    NASA Technical Reports Server (NTRS)

    Sukharev, S.; Betanzos, M.; Chiang, C. S.; Guy, H. R.

    2001-01-01

    The mechanosensitive channel of large conductance, MscL, is a ubiquitous membrane-embedded valve involved in turgor regulation in bacteria. The crystal structure of MscL from Mycobacterium tuberculosis provides a starting point for analysing molecular mechanisms of tension-dependent channel gating. Here we develop structural models in which a cytoplasmic gate is formed by a bundle of five amino-terminal helices (S1), previously unresolved in the crystal structure. When membrane tension is applied, the transmembrane barrel expands and pulls the gate apart through the S1-M1 linker. We tested these models by substituting cysteines for residues predicted to be near each other only in either the closed or open conformation. Our results demonstrate that S1 segments form the bundle when the channel is closed, and crosslinking between S1 segments prevents opening. S1 segments interact with M2 when the channel is open, and crosslinking of S1 to M2 impedes channel closing. Gating is affected by the length of the S1-M1 linker in a manner consistent with the model, revealing critical spatial relationships between the domains that transmit force from the lipid bilayer to the channel gate.

  2. Multimeric nature of voltage-gated proton channels.

    PubMed

    Koch, Hans P; Kurokawa, Tatsuki; Okochi, Yoshifumi; Sasaki, Mari; Okamura, Yasushi; Larsson, H Peter

    2008-07-01

    Voltage-gated potassium channels are comprised of four subunits, and each subunit has a pore domain and a voltage-sensing domain (VSD). The four pore domains assemble to form one single central pore, and the four individual VSDs control the gate of the pore. Recently, a family of voltage-gated proton channels, such as H(V) or voltage sensor only protein (VSOP), was discovered that contain a single VSD but no pore domain. It has been assumed that VSOP channels are monomeric and contain a single VSD that functions as both the VSD and the pore domain. It remains unclear, however, how a protein that contains only a VSD and no pore domain can conduct ions. Using fluorescence measurements and immunoprecipitation techniques, we show here that VSOP channels are expressed as multimeric channels. Further, FRET experiments on constructs with covalently linked subunits show that VSOP channels are dimers. Truncation of the cytoplasmic regions of VSOP reduced the dimerization, suggesting that the dimerization is caused mainly by cytoplasmic protein-protein interactions. However, these N terminus- and C terminus-deleted channels displayed large proton currents. Therefore, we conclude that even though VSOP channels are expressed mainly as dimers in the cell membrane, single VSOP subunits could function independently as proton channels.

  3. Multimeric nature of voltage-gated proton channels

    PubMed Central

    Koch, Hans P.; Kurokawa, Tatsuki; Okochi, Yoshifumi; Sasaki, Mari; Okamura, Yasushi; Larsson, H. Peter

    2008-01-01

    Voltage-gated potassium channels are comprised of four subunits, and each subunit has a pore domain and a voltage-sensing domain (VSD). The four pore domains assemble to form one single central pore, and the four individual VSDs control the gate of the pore. Recently, a family of voltage-gated proton channels, such as HV or voltage sensor only protein (VSOP), was discovered that contain a single VSD but no pore domain. It has been assumed that VSOP channels are monomeric and contain a single VSD that functions as both the VSD and the pore domain. It remains unclear, however, how a protein that contains only a VSD and no pore domain can conduct ions. Using fluorescence measurements and immunoprecipitation techniques, we show here that VSOP channels are expressed as multimeric channels. Further, FRET experiments on constructs with covalently linked subunits show that VSOP channels are dimers. Truncation of the cytoplasmic regions of VSOP reduced the dimerization, suggesting that the dimerization is caused mainly by cytoplasmic protein–protein interactions. However, these N terminus- and C terminus-deleted channels displayed large proton currents. Therefore, we conclude that even though VSOP channels are expressed mainly as dimers in the cell membrane, single VSOP subunits could function independently as proton channels. PMID:18583477

  4. Hydrophobic plug functions as a gate in voltage-gated proton channels.

    PubMed

    Chamberlin, Adam; Qiu, Feng; Rebolledo, Santiago; Wang, Yibo; Noskov, Sergei Y; Larsson, H Peter

    2014-01-14

    Voltage-gated proton (Hv1) channels play important roles in the respiratory burst, in pH regulation, in spermatozoa, in apoptosis, and in cancer metastasis. Unlike other voltage-gated cation channels, the Hv1 channel lacks a centrally located pore formed by the assembly of subunits. Instead, the proton permeation pathway in the Hv1 channel is within the voltage-sensing domain of each subunit. The gating mechanism of this pathway is still unclear. Mutagenic and fluorescence studies suggest that the fourth transmembrane (TM) segment (S4) functions as a voltage sensor and that there is an outward movement of S4 during channel activation. Using thermodynamic mutant cycle analysis, we find that the conserved positively charged residues in S4 are stabilized by countercharges in the other TM segments both in the closed and open states. We constructed models of both the closed and open states of Hv1 channels that are consistent with the mutant cycle analysis. These structural models suggest that electrostatic interactions between TM segments in the closed state pull hydrophobic residues together to form a hydrophobic plug in the center of the voltage-sensing domain. Outward S4 movement during channel activation induces conformational changes that remove this hydrophobic plug and instead insert protonatable residues in the center of the channel that, together with water molecules, can form a hydrogen bond chain across the channel for proton permeation. This suggests that salt bridge networks and the hydrophobic plug function as the gate in Hv1 channels and that outward movement of S4 leads to the opening of this gate.

  5. Afterbay, looking west at the discharge channels and hydraulic gate ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Afterbay, looking west at the discharge channels and hydraulic gate check cylinders. The outlet at left without a hydraulic cylinder is the outlet for the ca. 1974-1975 outdoor regulatory pumps. The gate box for the spillback is visible at the far left on the west side of the canal - Wellton-Mohawk Irrigation System, Pumping Plant No. 1, Bounded by Gila River & Union Pacific Railroad, Wellton, Yuma County, AZ

  6. The voltage-gated potassium channels and their relatives.

    PubMed

    Yellen, Gary

    2002-09-05

    The voltage-gated potassium channels are the prototypical members of a family of membrane signalling proteins. These protein-based machines have pores that pass millions of ions per second across the membrane with astonishing selectivity, and their gates snap open and shut in milliseconds as they sense changes in voltage or ligand concentration. The architectural modules and functional components of these sophisticated signalling molecules are becoming clear, but some important links remain to be elucidated.

  7. Excitability constraints on voltage-gated sodium channels.

    PubMed

    Angelino, Elaine; Brenner, Michael P

    2007-09-01

    We study how functional constraints bound and shape evolution through an analysis of mammalian voltage-gated sodium channels. The primary function of sodium channels is to allow the propagation of action potentials. Since Hodgkin and Huxley, mathematical models have suggested that sodium channel properties need to be tightly constrained for an action potential to propagate. There are nine mammalian genes encoding voltage-gated sodium channels, many of which are more than approximately 90% identical by sequence. This sequence similarity presumably corresponds to similarity of function, consistent with the idea that these properties must be tightly constrained. However, the multiplicity of genes encoding sodium channels raises the question: why are there so many? We demonstrate that the simplest theoretical constraints bounding sodium channel diversity--the requirements of membrane excitability and the uniqueness of the resting potential--act directly on constraining sodium channel properties. We compare the predicted constraints with functional data on mammalian sodium channel properties collected from the literature, including 172 different sets of measurements from 40 publications, wild-type and mutant, under a variety of conditions. The data from all channel types, including mutants, obeys the excitability constraint; on the other hand, channels expressed in muscle tend to obey the constraint of a unique resting potential, while channels expressed in neuronal tissue do not. The excitability properties alone distinguish the nine sodium channels into four different groups that are consistent with phylogenetic analysis. Our calculations suggest interpretations for the functional differences between these groups.

  8. Multiple modalities converge on a common gate to control K2P channel function.

    PubMed

    Bagriantsev, Sviatoslav N; Peyronnet, Rémi; Clark, Kimberly A; Honoré, Eric; Minor, Daniel L

    2011-07-15

    Members of the K(2P) potassium channel family regulate neuronal excitability and are implicated in pain, anaesthetic responses, thermosensation, neuroprotection, and mood. Unlike other potassium channels, K(2P)s are gated by remarkably diverse stimuli that include chemical, thermal, and mechanical modalities. It has remained unclear whether the various gating inputs act through separate or common channel elements. Here, we show that protons, heat, and pressure affect activity of the prototypical, polymodal K(2P), K(2P)2.1 (KCNK2/TREK-1), at a common molecular gate that comprises elements of the pore-forming segments and the N-terminal end of the M4 transmembrane segment. We further demonstrate that the M4 gating element is conserved among K(2P)s and is employed regardless of whether the gating stimuli are inhibitory or activating. Our results define a unique gating mechanism shared by K(2P) family members and suggest that their diverse sensory properties are achieved by coupling different molecular sensors to a conserved core gating apparatus.

  9. Multiple modalities converge on a common gate to control K2P channel function

    PubMed Central

    Bagriantsev, Sviatoslav N; Peyronnet, Rémi; Clark, Kimberly A; Honoré, Eric; Minor, Daniel L

    2011-01-01

    Members of the K2P potassium channel family regulate neuronal excitability and are implicated in pain, anaesthetic responses, thermosensation, neuroprotection, and mood. Unlike other potassium channels, K2Ps are gated by remarkably diverse stimuli that include chemical, thermal, and mechanical modalities. It has remained unclear whether the various gating inputs act through separate or common channel elements. Here, we show that protons, heat, and pressure affect activity of the prototypical, polymodal K2P, K2P2.1 (KCNK2/TREK-1), at a common molecular gate that comprises elements of the pore-forming segments and the N-terminal end of the M4 transmembrane segment. We further demonstrate that the M4 gating element is conserved among K2Ps and is employed regardless of whether the gating stimuli are inhibitory or activating. Our results define a unique gating mechanism shared by K2P family members and suggest that their diverse sensory properties are achieved by coupling different molecular sensors to a conserved core gating apparatus. PMID:21765396

  10. Sensing bilayer tension: bacterial mechanosensitive channels and their gating mechanisms.

    PubMed

    Booth, Ian R; Rasmussen, Tim; Edwards, Michelle D; Black, Susan; Rasmussen, Akiko; Bartlett, Wendy; Miller, Samantha

    2011-06-01

    Mechanosensitive channels sense and respond to changes in bilayer tension. In many respects, this is a unique property: the changes in membrane tension gate the channel, leading to the transient formation of open non-selective pores. Pore diameter is also high for the bacterial channels studied, MscS and MscL. Consequently, in cells, gating has severe consequences for energetics and homoeostasis, since membrane depolarization and modification of cytoplasmic ionic composition is an immediate consequence. Protection against disruption of cellular integrity, which is the function of the major channels, provides a strong evolutionary rationale for possession of such disruptive channels. The elegant crystal structures for these channels has opened the way to detailed investigations that combine molecular genetics with electrophysiology and studies of cellular behaviour. In the present article, the focus is primarily on the structure of MscS, the small mechanosensitive channel. The description of the structure is accompanied by discussion of the major sites of channel-lipid interaction and reasoned, but limited, speculation on the potential mechanisms of tension sensing leading to gating.

  11. Gating of a mechanosensitive channel due to cellular flows

    PubMed Central

    Pak, On Shun; Young, Y.-N.; Marple, Gary R.; Veerapaneni, Shravan; Stone, Howard A.

    2015-01-01

    A multiscale continuum model is constructed for a mechanosensitive (MS) channel gated by tension in a lipid bilayer membrane under stresses due to fluid flows. We illustrate that for typical physiological conditions vesicle hydrodynamics driven by a fluid flow may render the membrane tension sufficiently large to gate a MS channel open. In particular, we focus on the dynamic opening/closing of a MS channel in a vesicle membrane under a planar shear flow and a pressure-driven flow across a constriction channel. Our modeling and numerical simulation results quantify the critical flow strength or flow channel geometry for intracellular transport through a MS channel. In particular, we determine the percentage of MS channels that are open or closed as a function of the relevant measure of flow strength. The modeling and simulation results imply that for fluid flows that are physiologically relevant and realizable in microfluidic configurations stress-induced intracellular transport across the lipid membrane can be achieved by the gating of reconstituted MS channels, which can be useful for designing drug delivery in medical therapy and understanding complicated mechanotransduction. PMID:26216988

  12. Gating of a mechanosensitive channel due to cellular flows.

    PubMed

    Pak, On Shun; Young, Y-N; Marple, Gary R; Veerapaneni, Shravan; Stone, Howard A

    2015-08-11

    A multiscale continuum model is constructed for a mechanosensitive (MS) channel gated by tension in a lipid bilayer membrane under stresses due to fluid flows. We illustrate that for typical physiological conditions vesicle hydrodynamics driven by a fluid flow may render the membrane tension sufficiently large to gate a MS channel open. In particular, we focus on the dynamic opening/closing of a MS channel in a vesicle membrane under a planar shear flow and a pressure-driven flow across a constriction channel. Our modeling and numerical simulation results quantify the critical flow strength or flow channel geometry for intracellular transport through a MS channel. In particular, we determine the percentage of MS channels that are open or closed as a function of the relevant measure of flow strength. The modeling and simulation results imply that for fluid flows that are physiologically relevant and realizable in microfluidic configurations stress-induced intracellular transport across the lipid membrane can be achieved by the gating of reconstituted MS channels, which can be useful for designing drug delivery in medical therapy and understanding complicated mechanotransduction.

  13. Distributed structures underlie gating differences between the kin channel KAT1 and the Kout channel SKOR.

    PubMed

    Riedelsberger, Janin; Sharma, Tripti; Gonzalez, Wendy; Gajdanowicz, Pawel; Morales-Navarro, Samuel Elías; Garcia-Mata, Carlos; Mueller-Roeber, Bernd; González-Nilo, Fernando Danilo; Blatt, Michael R; Dreyer, Ingo

    2010-01-01

    The family of voltage-gated (Shaker-like) potassium channels in plants includes both inward-rectifying (K(in)) channels that allow plant cells to accumulate K(+) and outward-rectifying (K(out)) channels that mediate K(+) efflux. Despite their close structural similarities, K(in) and K(out) channels differ in their gating sensitivity towards voltage and the extracellular K(+) concentration. We have carried out a systematic program of domain swapping between the K(out) channel SKOR and the K(in) channel KAT1 to examine the impacts on gating of the pore regions, the S4, S5, and the S6 helices. We found that, in particular, the N-terminal part of the S5 played a critical role in KAT1 and SKOR gating. Our findings were supported by molecular dynamics of KAT1 and SKOR homology models. In silico analysis revealed that during channel opening and closing, displacement of certain residues, especially in the S5 and S6 segments, is more pronounced in KAT1 than in SKOR. From our analysis of the S4-S6 region, we conclude that gating (and K(+)-sensing in SKOR) depend on a number of structural elements that are dispersed over this approximately 145-residue sequence and that these place additional constraints on configurational rearrangement of the channels during gating.

  14. Mechanism of electromechanical coupling in voltage-gated potassium channels.

    PubMed

    Blunck, Rikard; Batulan, Zarah

    2012-01-01

    Voltage-gated ion channels play a central role in the generation of action potentials in the nervous system. They are selective for one type of ion - sodium, calcium, or potassium. Voltage-gated ion channels are composed of a central pore that allows ions to pass through the membrane and four peripheral voltage sensing domains that respond to changes in the membrane potential. Upon depolarization, voltage sensors in voltage-gated potassium channels (Kv) undergo conformational changes driven by positive charges in the S4 segment and aided by pairwise electrostatic interactions with the surrounding voltage sensor. Structure-function relations of Kv channels have been investigated in detail, and the resulting models on the movement of the voltage sensors now converge to a consensus; the S4 segment undergoes a combined movement of rotation, tilt, and vertical displacement in order to bring 3-4e(+) each through the electric field focused in this region. Nevertheless, the mechanism by which the voltage sensor movement leads to pore opening, the electromechanical coupling, is still not fully understood. Thus, recently, electromechanical coupling in different Kv channels has been investigated with a multitude of techniques including electrophysiology, 3D crystal structures, fluorescence spectroscopy, and molecular dynamics simulations. Evidently, the S4-S5 linker, the covalent link between the voltage sensor and pore, plays a crucial role. The linker transfers the energy from the voltage sensor movement to the pore domain via an interaction with the S6 C-termini, which are pulled open during gating. In addition, other contact regions have been proposed. This review aims to provide (i) an in-depth comparison of the molecular mechanisms of electromechanical coupling in different Kv channels; (ii) insight as to how the voltage sensor and pore domain influence one another; and (iii) theoretical predictions on the movement of the cytosolic face of the Kv channels during gating.

  15. Mechanism of Electromechanical Coupling in Voltage-Gated Potassium Channels

    PubMed Central

    Blunck, Rikard; Batulan, Zarah

    2012-01-01

    Voltage-gated ion channels play a central role in the generation of action potentials in the nervous system. They are selective for one type of ion – sodium, calcium, or potassium. Voltage-gated ion channels are composed of a central pore that allows ions to pass through the membrane and four peripheral voltage sensing domains that respond to changes in the membrane potential. Upon depolarization, voltage sensors in voltage-gated potassium channels (Kv) undergo conformational changes driven by positive charges in the S4 segment and aided by pairwise electrostatic interactions with the surrounding voltage sensor. Structure-function relations of Kv channels have been investigated in detail, and the resulting models on the movement of the voltage sensors now converge to a consensus; the S4 segment undergoes a combined movement of rotation, tilt, and vertical displacement in order to bring 3–4e+ each through the electric field focused in this region. Nevertheless, the mechanism by which the voltage sensor movement leads to pore opening, the electromechanical coupling, is still not fully understood. Thus, recently, electromechanical coupling in different Kv channels has been investigated with a multitude of techniques including electrophysiology, 3D crystal structures, fluorescence spectroscopy, and molecular dynamics simulations. Evidently, the S4–S5 linker, the covalent link between the voltage sensor and pore, plays a crucial role. The linker transfers the energy from the voltage sensor movement to the pore domain via an interaction with the S6 C-termini, which are pulled open during gating. In addition, other contact regions have been proposed. This review aims to provide (i) an in-depth comparison of the molecular mechanisms of electromechanical coupling in different Kv channels; (ii) insight as to how the voltage sensor and pore domain influence one another; and (iii) theoretical predictions on the movement of the cytosolic face of the Kv channels during

  16. A Gate Hinge Controls the Epithelial Calcium Channel TRPV5

    PubMed Central

    van der Wijst, Jenny; Leunissen, Elizabeth H.; Blanchard, Maxime G.; Venselaar, Hanka; Verkaart, Sjoerd; Paulsen, Candice E.; Bindels, René J.; Hoenderop, Joost G.

    2017-01-01

    TRPV5 is unique within the large TRP channel family for displaying a high Ca2+ selectivity together with Ca2+-dependent inactivation. Our study aims to uncover novel insights into channel gating through in-depth structure-function analysis. We identify an exceptional tryptophan (W583) at the terminus of the intracellular pore that is unique for TRPV5 (and TRPV6). A combination of site-directed mutagenesis, biochemical and electrophysiological analysis, together with homology modeling, demonstrates that W583 is part of the gate for Ca2+ permeation. The W583 mutants show increased cell death due to profoundly enhanced Ca2+ influx, resulting from altered channel function. A glycine residue above W583 might act as flexible linker to rearrange the tryptophan gate. Furthermore, we hypothesize functional crosstalk between the pore region and carboxy terminus, involved in Ca2+-calmodulin-mediated inactivation. This study proposes a unique channel gating mechanism and delivers detailed molecular insight into the Ca2+ permeation pathway that can be extrapolated to other Ca2+-selective channels. PMID:28374795

  17. From toxins targeting ligand gated ion channels to therapeutic molecules.

    PubMed

    Nasiripourdori, Adak; Taly, Valérie; Grutter, Thomas; Taly, Antoine

    2011-03-01

    Ligand-gated ion channels (LGIC) play a central role in inter-cellular communication. This key function has two consequences: (i) these receptor channels are major targets for drug discovery because of their potential involvement in numerous human brain diseases; (ii) they are often found to be the target of plant and animal toxins. Together this makes toxin/receptor interactions important to drug discovery projects. Therefore, toxins acting on LGIC are presented and their current/potential therapeutic uses highlighted.

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

    PubMed Central

    Elinder, Fredrik; Liin, Sara I.

    2017-01-01

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

  19. Cyclic nucleotide-gated channels in non-sensory organs.

    PubMed

    Kraus-Friedmann, N

    2000-03-01

    Cyclic nucleotide-gated channels represent a class of ion channels activated directly by the binding of either cyclic-GMP or cyclic-AMP. They carry both mono and divalent cations, but select calcium over sodium. In the majority of the cases studied, binding of cyclic nucleotides to the channel results in the opening of the channel and the influx of calcium. As a consequence, cytosolic free calcium levels increase leading to the modifications of calcium-dependent processes. This represents and important link in the chain of events leading to the physiological response. Cyclic nucleotide-gated channels were discovered in sensory cell types, in the retina, and in olfactory cells, and were extensively studied in those cells. However, it is becoming increasingly evident that such channels are present not only in sensory systems, but in most, if not all, cell types where cyclic nucleotides play a role in signal transduction. A hypothesis is presented here which attributes physiological importance to these channels in non-sensory organs. Four examples of such channels in non-sensory cells are discussed in detail: those in the liver, in the heart, in the brain, and in the testis with the emphasis on the possible physiological roles that these channels might have in these organs.

  20. Inactivation gating determines nicotine blockade of human HERG channels.

    PubMed

    Wang, H Z; Shi, H; Liao, S J; Wang, Z

    1999-09-01

    We have previously found that nicotine blocked multiple K+ currents, including the rapid component of delayed rectifier K+ currents (IKr), by interacting directly with the channels. To shed some light on the mechanisms of interaction between nicotine and channels, we performed detailed analysis on the human ether-à-go-go-related gene (HERG) channels, which are believed to be equivalent to the native I(Kr) when expressed in Xenopus oocytes. Nicotine suppressed the HERG channels in a concentration-dependent manner with greater potency with voltage protocols, which favor channel inactivation. Nicotine caused dramatic shifts of the voltage-dependent inactivation curve to more negative potentials and accelerated the inactivation process. Conversely, maneuvers that weakened the channel inactivation gating considerably relieved the blockade. Elevating the extracellular K+ concentration from 5 to 20 mM increased the nicotine concentration (by approximately 100-fold) needed to achieve the same degree of inhibition. Moreover, nicotine lost its ability to block the HERG channels when a single mutation was introduced to a residue located after transmembrane domain 6 (S631A) to remove the rapid channel inactivation. Our data suggest that the inactivation gating determines nicotine blockade of the HERG channels.

  1. Seizure suppression through manipulating splicing of a voltage-gated sodium channel.

    PubMed

    Lin, Wei-Hsiang; He, Miaomiao; Baines, Richard A

    2015-04-01

    Seizure can result from increased voltage-gated persistent sodium current expression. Although many clinically-approved antiepileptic drugs target voltage-gated persistent sodium current, none exclusively repress this current without also adversely affecting the transient voltage-gated sodium current. Achieving a more selective block has significant potential for the treatment of epilepsy. Recent studies show that voltage-gated persistent sodium current amplitude is regulated by alternative splicing offering the possibility of a novel route for seizure control. In this study we identify 291 splicing regulators that, on knockdown, alter splicing of the Drosophila voltage-gated sodium channel to favour inclusion of exon K, rather than the mutually exclusive exon L. This change is associated with both a significant reduction in voltage-gated persistent sodium current, without change to transient voltage-gated sodium current, and to rescue of seizure in this model insect. RNA interference mediated knock-down, in two different seizure mutants, shows that 95 of these regulators are sufficient to significantly reduce seizure duration. Moreover, most suppress seizure activity in both mutants, indicative that they are part of well conserved pathways and likely, therefore, to be optimal candidates to take forward to mammalian studies. We provide proof-of-principle for such studies by showing that inhibition of a selection of regulators, using small molecule inhibitors, is similarly effective to reduce seizure. Splicing of the Drosophila sodium channel shows many similarities to its mammalian counterparts, including altering the amplitude of voltage-gated persistent sodium current. Our study provides the impetus to investigate whether manipulation of splicing of mammalian voltage-gated sodium channels may be exploitable to provide effective seizure control. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain.

  2. Seizure suppression through manipulating splicing of a voltage-gated sodium channel

    PubMed Central

    Lin, Wei-Hsiang; He, Miaomiao

    2015-01-01

    Seizure can result from increased voltage-gated persistent sodium current expression. Although many clinically-approved antiepileptic drugs target voltage-gated persistent sodium current, none exclusively repress this current without also adversely affecting the transient voltage-gated sodium current. Achieving a more selective block has significant potential for the treatment of epilepsy. Recent studies show that voltage-gated persistent sodium current amplitude is regulated by alternative splicing offering the possibility of a novel route for seizure control. In this study we identify 291 splicing regulators that, on knockdown, alter splicing of the Drosophila voltage-gated sodium channel to favour inclusion of exon K, rather than the mutually exclusive exon L. This change is associated with both a significant reduction in voltage-gated persistent sodium current, without change to transient voltage-gated sodium current, and to rescue of seizure in this model insect. RNA interference mediated knock-down, in two different seizure mutants, shows that 95 of these regulators are sufficient to significantly reduce seizure duration. Moreover, most suppress seizure activity in both mutants, indicative that they are part of well conserved pathways and likely, therefore, to be optimal candidates to take forward to mammalian studies. We provide proof-of-principle for such studies by showing that inhibition of a selection of regulators, using small molecule inhibitors, is similarly effective to reduce seizure. Splicing of the Drosophila sodium channel shows many similarities to its mammalian counterparts, including altering the amplitude of voltage-gated persistent sodium current. Our study provides the impetus to investigate whether manipulation of splicing of mammalian voltage-gated sodium channels may be exploitable to provide effective seizure control. PMID:25681415

  3. Suppression of voltage-gated Na(+) channels and neuronal excitability by imperatorin.

    PubMed

    Wu, King-Chuen; Chen, Yi-Hung; Cheng, Ka-Shun; Kuo, Yueh-Hsiung; Yang, Chin-Tsang; Wong, Kar-Lok; Tu, Yuan-Kun; Chan, Paul; Leung, Yuk-Man

    2013-12-05

    Imperatorin is a naturally occurring furocoumarin compound isolated from plants such as Angelica archangelica and Cnidium monnieri. It has multiple pharmacological effects including anticonvulsant effects. Here we determined the effects of imperatorin on voltage-gated Na(+) channels (VGSC) using whole-cell patch clamp techniques in differentiated neuronal NG108-15 cells. We showed that imperatorin inhibited VGSC; such inhibition did not show state-dependence. Imperatorin caused a left shift in the steady-state inactivation curve without affecting activation gating. The inhibition of VGSC by imperatorin displayed a mild frequency-dependence. Imperatorin was also shown to inhibit VGSC and action potential amplitude without affecting voltage-gated K(+) channels in rat hippocampal CA1 neurons. In conclusion, our results suggest that imperatorin dampens neuronal excitability by inhibiting VGSC.

  4. Claudin-2-dependent paracellular channels are dynamically gated

    PubMed Central

    Weber, Christopher R; Liang, Guo Hua; Wang, Yitang; Das, Sudipto; Shen, Le; Yu, Alan S L; Nelson, Deborah J; Turner, Jerrold R

    2015-01-01

    Intercellular tight junctions form selectively permeable barriers that seal the paracellular space. Trans-tight junction flux has been measured across large epithelial surfaces, but conductance across individual channels has never been measured. We report a novel trans-tight junction patch clamp technique that detects flux across individual claudin-2 channels within the tight junction of cultured canine renal tubule or human intestinal epithelial monolayers. In both cells, claudin-2 channels display conductances of ~90 pS. The channels are gated, strictly dependent on claudin-2 expression, and display size- and charge-selectivity typical of claudin-2. Kinetic analyses indicate one open and two distinct closed states. Conductance is symmetrical and reversible, characteristic of a passive, paracellular process, and blocked by reduced temperature or site-directed mutagenesis and chemical derivatization of the claudin-2 pore. We conclude that claudin-2 forms gated paracellular channels and speculate that modulation of tight junction channel gating kinetics may be an unappreciated mechanism of barrier regulation. DOI: http://dx.doi.org/10.7554/eLife.09906.001 PMID:26568313

  5. Resurgent current of voltage-gated Na+ channels

    PubMed Central

    Lewis, Amanda H; Raman, Indira M

    2014-01-01

    Resurgent Na+ current results from a distinctive form of Na+ channel gating, originally identified in cerebellar Purkinje neurons. In these neurons, the tetrodotoxin-sensitive voltage-gated Na+ channels responsible for action potential firing have specialized mechanisms that reduce the likelihood that they accumulate in fast inactivated states, thereby shortening refractory periods and permitting rapid, repetitive, and/or burst firing. Under voltage clamp, step depolarizations evoke transient Na+ currents that rapidly activate and quickly decay, and step repolarizations elicit slower channel reopening, or a ‘resurgent’ current. The generation of resurgent current depends on a factor in the Na+ channel complex, probably a subunit such as NaVβ4 (Scn4b), which blocks open Na+ channels at positive voltages, competing with the fast inactivation gate, and unblocks at negative voltages, permitting recovery from an open channel block along with a flow of current. Following its initial discovery, resurgent Na+ current has been found in nearly 20 types of neurons. Emerging research suggests that resurgent current is preferentially increased in a variety of clinical conditions associated with altered cellular excitability. Here we review the biophysical, molecular and structural mechanisms of resurgent current and their relation to the normal functions of excitable cells as well as pathophysiology. PMID:25172941

  6. Modulation of the slow/common gating of CLC channels by intracellular cadmium

    PubMed Central

    Yu, Yawei; Tsai, Ming-Feng; Yu, Wei-Ping

    2015-01-01

    Members of the CLC family of Cl− channels and transporters are homodimeric integral membrane proteins. Two gating mechanisms control the opening and closing of Cl− channels in this family: fast gating, which regulates opening and closing of the individual pores in each subunit, and slow (or common) gating, which simultaneously controls gating of both subunits. Here, we found that intracellularly applied Cd2+ reduces the current of CLC-0 because of its inhibition on the slow gating. We identified CLC-0 residues C229 and H231, located at the intracellular end of the transmembrane domain near the dimer interface, as the Cd2+-coordinating residues. The inhibition of the current of CLC-0 by Cd2+ was greatly enhanced by mutation of I225W and V490W at the dimer interface. Biochemical experiments revealed that formation of a disulfide bond within this Cd2+-binding site is also affected by mutation of I225W and V490W, indicating that these two mutations alter the structure of the Cd2+-binding site. Kinetic studies showed that Cd2+ inhibition appears to be state dependent, suggesting that structural rearrangements may occur in the CLC dimer interface during Cd2+ modulation. Mutations of I290 and I556 of CLC-1, which correspond to I225 and V490 of CLC-0, respectively, have been shown previously to cause malfunction of CLC-1 Cl− channel by altering the common gating. Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0. The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd2+ likely depends on their alteration of subunit interactions. PMID:26621774

  7. Modulation of the slow/common gating of CLC channels by intracellular cadmium.

    PubMed

    Yu, Yawei; Tsai, Ming-Feng; Yu, Wei-Ping; Chen, Tsung-Yu

    2015-12-01

    Members of the CLC family of Cl(-) channels and transporters are homodimeric integral membrane proteins. Two gating mechanisms control the opening and closing of Cl(-) channels in this family: fast gating, which regulates opening and closing of the individual pores in each subunit, and slow (or common) gating, which simultaneously controls gating of both subunits. Here, we found that intracellularly applied Cd(2+) reduces the current of CLC-0 because of its inhibition on the slow gating. We identified CLC-0 residues C229 and H231, located at the intracellular end of the transmembrane domain near the dimer interface, as the Cd(2+)-coordinating residues. The inhibition of the current of CLC-0 by Cd(2+) was greatly enhanced by mutation of I225W and V490W at the dimer interface. Biochemical experiments revealed that formation of a disulfide bond within this Cd(2+)-binding site is also affected by mutation of I225W and V490W, indicating that these two mutations alter the structure of the Cd(2+)-binding site. Kinetic studies showed that Cd(2+) inhibition appears to be state dependent, suggesting that structural rearrangements may occur in the CLC dimer interface during Cd(2+) modulation. Mutations of I290 and I556 of CLC-1, which correspond to I225 and V490 of CLC-0, respectively, have been shown previously to cause malfunction of CLC-1 Cl(-) channel by altering the common gating. Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0. The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd(2+) likely depends on their alteration of subunit interactions.

  8. Molecular biology and biophysical properties of ion channel gating pores.

    PubMed

    Moreau, Adrien; Gosselin-Badaroudine, Pascal; Chahine, Mohamed

    2014-11-01

    The voltage sensitive domain (VSD) is a pivotal structure of voltage-gated ion channels (VGICs) and plays an essential role in the generation of electrochemical signals by neurons, striated muscle cells, and endocrine cells. The VSD is not unique to VGICs. Recent studies have shown that a VSD regulates a phosphatase. Similarly, Hv1, a voltage-sensitive protein that lacks an apparent pore domain, is a self-contained voltage sensor that operates as an H⁺ channel. VSDs are formed by four transmembrane helices (S1-S4). The S4 helix is positively charged due to the presence of arginine and lysine residues. It is surrounded by two water crevices that extend into the membrane from both the extracellular and intracellular milieus. A hydrophobic septum disrupts communication between these water crevices thus preventing the permeation of ions. The septum is maintained by interactions between the charged residues of the S4 segment and the gating charge transfer center. Mutating the charged residue of the S4 segment allows the water crevices to communicate and generate gating pore or omega pore. Gating pore currents have been reported to underlie several neuronal and striated muscle channelopathies. Depending on which charged residue on the S4 segment is mutated, gating pores are permeant either at depolarized or hyperpolarized voltages. Gating pores are cation selective and seem to converge toward Eisenmann's first or second selectivity sequences. Most gating pores are blocked by guanidine derivatives as well as trivalent and quadrivalent cations. Gating pores can be used to study the movement of the voltage sensor and could serve as targets for novel small therapeutic molecules.

  9. Afterbay, showing four discharge channels and four hydraulic gate check ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Afterbay, showing four discharge channels and four hydraulic gate check cylinders, one for each discharge pipe opening. The fifth bay at the left without a hydraulic cylinder is the outlet for the regulatory pumps added in 1972. The still well is visible at right - Wellton-Mohawk Irrigation System, Pumping Plant No. 3, South of Interstate 8, Wellton, Yuma County, AZ

  10. Afterbay, showing the six discharge channels and six hydraulic gate ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Afterbay, showing the six discharge channels and six hydraulic gate check cylinders, one for each of the discharge pipes. A stilling well is in the right foreground, and the Pumping Plant is visible in the background. View to the north - Wellton-Mohawk Irrigation System, Pumping Plant No. 1, Bounded by Gila River & Union Pacific Railroad, Wellton, Yuma County, AZ

  11. Afterbay, showing five discharge channels and five hydraulic gate check ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Afterbay, showing five discharge channels and five hydraulic gate check cylinders, one for each discharge pipe opening. The spillback inlet is visible in the left foreground. View to the west - Wellton-Mohawk Irrigation System, Pumping Plant No. 2, Bounded by Interstate 8 to south, Wellton, Yuma County, AZ

  12. 6. GATES 3, 4, AND 5, INTAKE CHANNEL LOOKING EAST; ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. GATES 3, 4, AND 5, INTAKE CHANNEL LOOKING EAST; WATER THAT PASSED INTO PIPES ENTERED SETTLING VAULT. - Hondius Water Line, 1.6 miles Northwest of Park headquarters building & 1 mile Northwest of Beaver Meadows entrance station, Estes Park, Larimer County, CO

  13. Phospholemman Modulates the Gating of Cardiac L-Type Calcium Channels

    PubMed Central

    Wang, Xianming; Gao, Guofeng; Guo, Kai; Yarotskyy, Viktor; Huang, Congxin; Elmslie, Keith S.; Peterson, Blaise Z.

    2010-01-01

    Ca2+ entry through L-type calcium channels (CaV1.2) is critical in shaping the cardiac action potential and initiating cardiac contraction. Modulation of CaV1.2 channel gating directly affects myocyte excitability and cardiac function. We have found that phospholemman (PLM), a member of the FXYD family and regulator of cardiac ion transport, coimmunoprecipitates with CaV1.2 channels from guinea pig myocytes, which suggests PLM is an endogenous modulator. Cotransfection of PLM in HEK293 cells slowed CaV1.2 current activation at voltages near the threshold for activation, slowed deactivation after long and strong depolarizing steps, enhanced the rate and magnitude of voltage-dependent inactivation (VDI), and slowed recovery from inactivation. However, Ca2+-dependent inactivation was not affected. Consistent with slower channel closing, PLM significantly increased Ca2+ influx via CaV1.2 channels during the repolarization phase of a human cardiac action potential waveform. Our results support PLM as an endogenous regulator of CaV1.2 channel gating. The enhanced VDI induced by PLM may help protect the heart under conditions such as ischemia or tachycardia where the channels are depolarized for prolonged periods of time and could induce Ca2+ overload. The time and voltage-dependent slowed deactivation could represent a gating shift that helps maintain Ca2+ influx during the cardiac action potential waveform plateau phase. PMID:20371314

  14. Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels.

    PubMed

    Tian, Lijun; Jeffries, Owen; McClafferty, Heather; Molyvdas, Adam; Rowe, Iain C M; Saleem, Fozia; Chen, Lie; Greaves, Jennifer; Chamberlain, Luke H; Knaus, Hans-Guenther; Ruth, Peter; Shipston, Michael J

    2008-12-30

    Large conductance calcium- and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the pore-forming alpha-subunits. However, the molecular mechanism(s) by which phosphorylation of the alpha-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single alpha-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.

  15. Drastic reduction of the slow gate of human muscle chloride channel (ClC-1) by mutation C277S.

    PubMed

    Accardi, A; Ferrera, L; Pusch, M

    2001-08-01

    1. Single channel measurements suggest that the human muscle chloride channel ClC-1 presumably has a double barrelled structure, with a fast single protopore gate and a slow common pore gate similar to that of ClC-0, the chloride channel from Torpedo. The single point mutation C212S has been shown to abolish the slow gating of ClC-0 locking the slow gate in the open state. In order to test the hypothesis that the slow gating process found in ClC-1 corresponds to the well characterised slow gate found in ClC-0 we investigated the gating effects in ClC-1 of the homologous mutation corresponding to C212S, C277S. 2. We found that the mutation C277S strongly reduced the slow component of macroscopic gating relaxations at negative and at positive voltages. 3. Time constants of the fast gating relaxations were not affected by the mutation but the minimal open probability of the fast gate at negative voltages was slightly reduced to 0.08 compared with the WT value of 0.22. 4. Additionally, we characterised the block of WT ClC-1 and mutant C277S by the S(-) enantiomer of CPB (2-(p-chlorophenoxy) butyric acid), and found that the block is practically unaffected by the mutation suggesting that CPB does not interact with the slow gate of ClC-1. 5. We conclude that the slow and fast gating processes of ClC-1, respectively, reflect the slow common pore gate and the single protopore gate of the double-barrelled ClC-1 channel.

  16. Drastic reduction of the slow gate of human muscle chloride channel (ClC-1) by mutation C277S

    PubMed Central

    Accardi, Alessio; Ferrera, Loretta; Pusch, Michael

    2001-01-01

    Single channel measurements suggest that the human muscle chloride channel ClC-1 presumably has a double barrelled structure, with a fast single protopore gate and a slow common pore gate similar to that of ClC-0, the chloride channel from Torpedo. The single point mutation C212S has been shown to abolish the slow gating of ClC-0 locking the slow gate in the open state. In order to test the hypothesis that the slow gating process found in ClC-1 corresponds to the well characterised slow gate found in ClC-0 we investigated the gating effects in ClC-1 of the homologous mutation corresponding to C212S, C277S. We found that the mutation C277S strongly reduced the slow component of macroscopic gating relaxations at negative and at positive voltages. Time constants of the fast gating relaxations were not affected by the mutation but the minimal open probability of the fast gate at negative voltages was slightly reduced to 0.08 compared with the WT value of 0.22. Additionally, we characterised the block of WT ClC-1 and mutant C277S by the S(—) enantiomer of CPB (2-(p-chlorophenoxy) butyric acid), and found that the block is practically unaffected by the mutation suggesting that CPB does not interact with the slow gate of ClC-1. We conclude that the slow and fast gating processes of ClC-1, respectively, reflect the slow common pore gate and the single protopore gate of the double-barrelled ClC-1 channel. PMID:11483705

  17. Analysis of gate underlap channel double gate MOS transistor for electrical detection of bio-molecules

    NASA Astrophysics Data System (ADS)

    Ajay; Narang, Rakhi; Saxena, Manoj; Gupta, Mridula

    2015-12-01

    In this paper, an analytical model for gate drain underlap channel Double-Gate Metal-Oxide-Semiconductor Field-Effect Transistor (DG-MOSFET) for label free electrical detection of biomolecules has been proposed. The conformal mapping technique has been used to derive the expressions for surface potential, lateral electric field, energy bands (i.e. conduction and valence band) and threshold voltage (Vth). Subsequently a full drain current model to analyze the sensitivity of the biosensor has been developed. The shift in the threshold voltage and drain current (after the biomolecules interaction with the gate underlap channel region of the MOS transistor) has been used as a sensing metric. All the characteristic trends have been verified through ATLAS (SILVACO) device simulation results.

  18. Analytical modeling of the direct tunneling current through high-k gate stacks for long-channel cylindrical surrounding-gate MOSFETs

    NASA Astrophysics Data System (ADS)

    Lina, Shi; Yiqi, Zhuang; Cong, Li; Dechang, Li

    2014-03-01

    An analytical direct tunneling gate current model for cylindrical surrounding gate (CSG) MOSFETs with high-k gate stacks is developed. It is found that the direct tunneling gate current is a strong function of the gate's oxide thickness, but that it is less affected by the change in channel radius. It is also revealed that when the thickness of the equivalent oxide is constant, the thinner the first layer, the smaller the direct tunneling gate current. Moreover, it can be seen that the dielectric with a higher dielectric constant shows a lower tunneling current than expected. The accuracy of the analytical model is verified by the good agreement of its results with those obtained by the three-dimensional numerical device simulator ISE.

  19. 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

  20. Neurological perspectives on voltage-gated sodium channels

    PubMed Central

    Linley, John E.; Baker, Mark D.; Minett, Michael S.; Cregg, Roman; Werdehausen, Robert; Rugiero, François

    2012-01-01

    The activity of voltage-gated sodium channels has long been linked to disorders of neuronal excitability such as epilepsy and chronic pain. Recent genetic studies have now expanded the role of sodium channels in health and disease, to include autism, migraine, multiple sclerosis, cancer as well as muscle and immune system disorders. Transgenic mouse models have proved useful in understanding the physiological role of individual sodium channels, and there has been significant progress in the development of subtype selective inhibitors of sodium channels. This review will outline the functions and roles of specific sodium channels in electrical signalling and disease, focusing on neurological aspects. We also discuss recent advances in the development of selective sodium channel inhibitors. PMID:22961543

  1. Gated Ion Channel-Based Biosensor Device

    NASA Astrophysics Data System (ADS)

    Separovic, Frances; Cornell, Bruce A.

    A biosensor device based on the ion channel gramicidin A (gA) incorporated into a bilayer membrane is described. This generic immunosensing device utilizes gA coupled to an antibody and assembled in a lipid membrane. The membrane is chemically tethered to a gold electrode, which reports on changes in the ionic conduction of the lipid bilayer. Binding of a target molecule in the bathing solution to the antibody causes the gramicidin channels to switch from predominantly conducting dimers to predominantly nonconducting monomers. Conventional a.c. impedance spectroscopy between the gold and a counter electrode in the bathing solution is used to measure changes in the ionic conductivity of the membrane. This approach permits the quantitative detection of a range of target species, including bacteria, proteins, toxins, DNA sequences, and drug molecules.

  2. Split gate SOI trench LDMOS with low-resistance channel

    NASA Astrophysics Data System (ADS)

    Ying-Wang; Wang, Yi-fan; Liu, Yan-juan; Yang-Wang

    2017-02-01

    A split gate SOI trench LDMOSFET (SGT-LDMOS) structure is proposed and the low-resistance channel is introduced to further reduces the specific on-state resistance (Ron,sp). The split gate SOI trench LDMOS with low on-resistance channel (SGTL-LDMOS) structure shows a reduction in specific on-state resistance (Ron,sp) compared to that of a conventional SOI trench LDMOS (CT-LDMOS) and SGT-LDMOS structures. This is due to the increased N-type concentration in the drift region and the lower channel resistance. In addition, the split-gate floating structure in the SGTL-LDMOS also reduces the specific gate-charge (Qg,sp) and increases the breakdown voltage as compared to the CT-LDMOS. As a result, the breakdown voltage (BV) of the SGTL-LDMOS increases from 183 V of the CT-LDMOS to 227 V, the Ron,sp decreases from 43.4 mΩ cm2 to 9.3 mΩ cm2, and the Qg,sp decreases from 78.4 nC/cm2 to 50.0 nC/cm2.

  3. Intramembrane congestion effects on lysenin channel voltage-induced gating.

    PubMed

    Krueger, Eric; Bryant, Sheenah; Shrestha, Nisha; Clark, Tyler; Hanna, Charles; Pink, David; Fologea, Daniel

    2016-03-01

    All cell membranes are packed with proteins. The ability to investigate the regulatory mechanisms of protein channels in experimental conditions mimicking their congested native environment is crucial for understanding the environmental physicochemical cues that may fundamentally contribute to their functionality in natural membranes. Here we report on investigations of the voltage-induced gating of lysenin channels in congested conditions experimentally achieved by increasing the number of channels inserted into planar lipid membranes. Typical electrophysiology measurements reveal congestion-induced changes to the voltage-induced gating, manifested as a significant reduction of the response to external voltage stimuli. Furthermore, we demonstrate a similar diminished voltage sensitivity for smaller populations of channels by reducing the amount of sphingomyelin in the membrane. Given lysenin's preference for targeting lipid rafts, this result indicates the potential role of the heterogeneous organization of the membrane in modulating channel functionality. Our work indicates that local congestion within membranes may alter the energy landscape and the kinetics of conformational changes of lysenin channels in response to voltage stimuli. This level of understanding may be extended to better characterize the role of the specific membrane environment in modulating the biological functionality of protein channels in health and disease.

  4. Intramembrane congestion effects on lysenin channel voltage-induced gating

    PubMed Central

    Krueger, Eric; Bryant, Sheenah; Shrestha, Nisha; Clark, Tyler; Hanna, Charles; Pink, David; Fologea, Daniel

    2016-01-01

    All cell membranes are packed with proteins. The ability to investigate the regulatory mechanisms of protein channels in experimental conditions mimicking their congested native environment is crucial for understanding the environmental physicochemical cues that may fundamentally contribute to their functionality in natural membranes. Here we report on investigations of the voltage-induced gating of lysenin channels in congested conditions experimentally achieved by increasing the number of channels inserted into planar lipid membranes. Typical electrophysiology measurements reveal congestion-induced changes to the voltage-induced gating, manifested as a significant reduction of the response to external voltage stimuli. Furthermore, we demonstrate a similar diminished voltage sensitivity for smaller populations of channels by reducing the amount of sphingomyelin in the membrane. Given lysenin’s preference for targeting lipid rafts, this result indicates the potential role of the heterogeneous organization of the membrane in modulating channel functionality. Our work indicates that local congestion within membranes may alter the energy landscape and the kinetics of conformational changes of lysenin channels in response to voltage stimuli. This level of understanding may be extended to better characterize the role of the specific membrane environment in modulating the biological functionality of protein channels in health and disease. PMID:26695013

  5. Physiological roles of voltage-gated proton channels in leukocytes

    PubMed Central

    Demaurex, Nicolas; El Chemaly, Antoun

    2010-01-01

    Voltage-gated proton channels are designed to extrude large quantities of cytosolic acid in response to depolarising voltages. The discovery of the Hvcn1 gene and the generation of mice lacking the channel molecule have confirmed several postulated functions of proton channels in leukocytes. In neutrophils and macrophages, proton channels are required for high-level production of superoxide anions by the phagocytic NADPH oxidase, a bactericidal enzyme essential for host defence against infections. In B lymphocytes, proton channels are required for low-level production of superoxide that boosts the production of antibodies. Proton channels sustain the activity of immune cells in several ways. By extruding excess cytosolic acid, proton channels prevent deleterious acidification of the cytosol and at the same time deliver protons required for chemical conversion of the superoxide secreted by membrane oxidases. By moving positive charges across membranes, proton channels limit the depolarisation of the plasma membrane, promoting the electrogenic activity of NADPH oxidases and the entry of calcium ions into cells. Acid extrusion by proton channels is not restricted to leukocytes but also mediates the intracellular alkalinisation required for the activation of spermatozoids. Proton channels are therefore multitalented channels that control male fertility as well as our innate and adaptive immunity. PMID:20693294

  6. A stochastic four-state model of contingent gating of gap junction channels containing two "fast" gates sensitive to transjunctional voltage.

    PubMed

    Paulauskas, Nerijus; Pranevicius, Mindaugas; Pranevicius, Henrikas; Bukauskas, Feliksas F

    2009-05-20

    Connexins, a family of membrane proteins, form gap junction (GJ) channels that provide a direct pathway for electrical and metabolic signaling between cells. We developed a stochastic four-state model describing gating properties of homotypic and heterotypic GJ channels each composed of two hemichannels (connexons). GJ channel contain two "fast" gates (one per hemichannel) oriented opposite in respect to applied transjunctional voltage (V(j)). The model uses a formal scheme of peace-linear aggregate and accounts for voltage distribution inside the pore of the channel depending on the state, unitary conductances and gating properties of each hemichannel. We assume that each hemichannel can be in the open state with conductance gamma(h,o) and in the residual state with conductance gamma(h,res), and that both gamma(h,o) and gamma(h,res) rectifies. Gates can exhibit the same or different gating polarities. Gating of each hemichannel is determined by the fraction of V(j) that falls across the hemichannel, and takes into account contingent gating when gating of one hemichannel depends on the state of apposed hemichannel. At the single-channel level, the model revealed the relationship between unitary conductances of hemichannels and GJ channels and how this relationship is affected by gamma(h,o) and gamma(h,res) rectification. Simulation of junctions containing up to several thousands of homotypic or heterotypic GJs has been used to reproduce experimentally measured macroscopic junctional current and V(j)-dependent gating of GJs formed from different connexin isoforms. V(j)-gating was simulated by imitating several frequently used experimental protocols: 1), consecutive V(j) steps rising in amplitude, 2), slowly rising V(j) ramps, and 3), series of V(j) steps of high frequency. The model was used to predict V(j)-gating of heterotypic GJs from characteristics of corresponding homotypic channels. The model allowed us to identify the parameters of V(j)-gates under which

  7. 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

  8. Positive allosteric modulators of α7 nicotinic acetylcholine receptors affect neither the function of other ligand- and voltage-gated ion channels and acetylcholinesterase, nor β-amyloid content.

    PubMed

    Arias, Hugo R; Ravazzini, Federica; Targowska-Duda, Katarzyna M; Kaczor, Agnieszka A; Feuerbach, Dominik; Boffi, Juan C; Draczkowski, Piotr; Montag, Dirk; Brown, Brandon M; Elgoyhen, Ana Belén; Jozwiak, Krzysztof; Puia, Giulia

    2016-07-01

    The activity of positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (AChRs), including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and 3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) α7, rat (r) α9α10, hα3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] and voltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and β-amyloid (Aβ) content. The functional results indicate that PAM-2 inhibits hα3-containing AChRs (IC50=26±6μM) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitive GluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE, and PAM-4 does not affect agonist-activated rα9α10 AChRs. Relevant clinical concentrations of PAM-2-4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 and GluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of Aβ42 in an Alzheimer's disease mouse model (i.e., 5XFAD). The molecular docking and dynamics results using the hα7 model suggest that the active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. These results support our previous study showing that these PAMs are selective for the α7 AChR, and clarify that the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets.

  9. On the gating of mechanosensitive channels by fluid shear stress

    NASA Astrophysics Data System (ADS)

    Peng, Zhangli; Pak, On Shun; Feng, Zhe; Liu, Allen P.; Young, Yuan-Nan

    2016-12-01

    Mechanosensation is an important process in biological fluid-structure interaction. To understand the biophysics underlying mechanosensation, it is essential to quantify the correlation between membrane deformation, membrane tension, external fluid shear stress, and conformation of mechanosensitive (MS) channels. Smoothed dissipative particle dynamics (SDPD) simulations of vesicle/cell in three types of flow configurations are conducted to calculate the tension in lipid membrane due to fluid shear stress from the surrounding viscous flow. In combination with a simple continuum model for an MS channel, SDPD simulation results suggest that shearing adhered vesicles/cells is more effective to induce membrane tension sufficient to stretch MS channels open than a free shear flow or a constrictive channel flow. In addition, we incorporate the bilayer-cytoskeletal interaction in a two-component model to probe the effects of a cytoskeletal network on the gating of MS channels.

  10. 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

  11. The Extracellular Linker of Muscle Acetylcholine Receptor Channels Is a Gating Control Element

    PubMed Central

    Grosman, Claudio; Salamone, Frank N.; Sine, Steven M.; Auerbach, Anthony

    2000-01-01

    We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2–M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the α subunit (αS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the αS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an ∼92-fold increased gating equilibrium constant, which is consistent with an ∼10-fold decreased EC50 in the presence of ACh. With choline, this mutation accelerates channel opening ∼28-fold, slows channel closing ∼3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, αS269I acetylcholine receptors open at a rate of ∼1.4 × 106 s−1 and close at a rate of ∼760 s−1. These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of ∼140 s−1. Ile mutations at positions flanking αS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the α subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the αS269I mutation, Ile mutations at equivalent positions of the β, ε, and δ subunits do not affect apparent open-channel lifetimes. However, in β and ε, shifting the mutation one residue to the NH2-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction

  12. Mechanosensitive behavior of bacterial cyclic nucleotide gated (bCNG) ion channels: Insights into the mechanism of channel gating in the mechanosensitive channel of small conductance superfamily.

    PubMed

    Malcolm, Hannah R; Elmore, Donald E; Maurer, Joshua A

    2012-01-20

    We have recently identified and characterized the bacterial cyclic nucleotide gated (bCNG) subfamily of the larger mechanosensitive channel of small conductance (MscS) superfamily of ion channels. The channel domain of bCNG channels exhibits significant sequence homology to the mechanosensitive subfamily of MscS in the regions that have previously been used as a hallmark for channels that gate in response to mechanical stress. However, we have previously demonstrated that three of these channels are unable to rescue Escherichiacoli from osmotic downshock. Here, we examine an additional nine bCNG homologues and further demonstrate that the full-length bCNG channels are unable to rescue E. coli from hypoosmotic stress. However, limited mechanosensation is restored upon removal of the cyclic nucleotide binding domain. This indicates that the C-terminal domain of the MscS superfamily can drive channel gating and further highlight the ability of a superfamily of ion channels to be gated by multiple stimuli.

  13. Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating.

    PubMed

    Wang, Wuyang; Linsdell, Paul

    2012-03-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are controlled by ATP binding and hydrolysis by its nucleotide binding domains (NBDs). This is presumed to control opening of a single "gate" within the permeation pathway, however, the location of such a gate has not been described. We used patch clamp recording to monitor access of cytosolic cysteine reactive reagents to cysteines introduced into different transmembrane (TM) regions in a cysteine-less form of CFTR. The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)(2)(-) ions was reduced when ATP concentration was reduced from 1mM to 10μM, and modification by MTSES was accelerated when 2mM pyrophosphate was applied to prevent channel closure. Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). The rate of modification of Q98C and I344C by both MTSES and Au(CN)(2)(-) was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. These results suggest that access from the cytoplasm to K95 and V345 is similar in open and closed channels. In contrast, modifying ATP-dependent channel gating alters access to Q98 and I344, located further into the pore. We propose that ATP-dependent gating of CFTR is associated with the opening and closing of a gate within the permeation pathway at the level of these pore-lining amino acids.

  14. ATP-gated channels in vascular smooth muscle cells.

    PubMed

    Benham, C D

    1990-01-01

    ATP acting through P2x-purinoceptors activates cation channels with some similarities to the activation of channels gated by acetylcholine and glutamate (channels that can also act as fast excitatory transmitters). These experiments clearly demonstrate an ATP-mediated Ca2+ influx through agonist-gated channels and a consequent elevation of [Ca2+]i in these single vascular smooth muscle cells. The combination of the ability to hold these cells under voltage-clamp and to measure [Ca2+]i simultaneously has allowed us to exclude other possible explanations for the rise in [Ca2+]i under these conditions. Thus, although the major cation entering through the channels is Na+, ATP receptor activation will also generate subtle, localized increases in [Ca2+]. These increases might directly activate contractile proteins or, if insufficient to do this, might upregulate other Ca2(+)-dependent enzymes modulating the contractile process and provide an enhanced source of Ca2+ for uptake into internal Ca2+ stores. Further understanding of the physiological role of this conductance pathway may require the development of specific receptor antagonists or channel blockers.

  15. Design of a Gated Molecular Proton Channel

    SciTech Connect

    Gu, Wei; Zhou, Bo; Geyer, Tihamer; Hutter, Michael C.; Fang, Haiping; Helms, Volkhard H.

    2011-01-17

    The generation of an electrochemical pH gradient across biological membranes using energy from photosynthesis and respiration provides the universal driving force in cells for the production of adenosine triphosphate (ATP), the energy unit of life. Creating such an electrochemical potential requires the transportation of protons against a thermodynamic gradient. In biological proton pumps, chemical energy is used to induce protein conformational changes during each catalytic cycle where one or a few protons are pumped against a proton concentration gradient across the membrane. On the other hand, membrane channels also exist that mediate continuous particle exchange and may be switched between open and closed states. Being able to design nanochannels with similar functions would be of great importance for creating novel molecular devices with a wide range of applications such as molecular motors, fuel cells, rechargeable nanobatteries that provide energy to other nanomachines, and the generation of locally and temporally controlled pH jumps on microfluidic chips.

  16. 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

  17. Subtype-selective targeting of voltage-gated sodium channels

    PubMed Central

    England, Steve; de Groot, Marcel J

    2009-01-01

    Voltage-gated sodium channels are key to the initiation and propagation of action potentials in electrically excitable cells. Molecular characterization has shown there to be nine functional members of the family, with a high degree of sequence homology between the channels. This homology translates into similar biophysical and pharmacological properties. Confidence in some of the channels as drug targets has been boosted by the discovery of human mutations in the genes encoding a number of them, which give rise to clinical conditions commensurate with the changes predicted from the altered channel biophysics. As a result, they have received much attention for their therapeutic potential. Sodium channels represent well-precedented drug targets as antidysrhythmics, anticonvulsants and local anaesthetics provide good clinical efficacy, driven through pharmacology at these channels. However, electrophysiological characterization of clinically useful compounds in recombinant expression systems shows them to be weak, with poor selectivity between channel types. This has led to the search for subtype-selective modulators, which offer the promise of treatments with improved clinical efficacy and better toleration. Despite developments in high-throughput electrophysiology platforms, this has proven very challenging. Structural biology is beginning to offer us a greater understanding of the three-dimensional structure of voltage-gated ion channels, bringing with it the opportunity to do real structure-based drug design in the future. This discipline is still in its infancy, but developments with the expression and purification of prokaryotic sodium channels offer the promise of structure-based drug design in the not too distant future. PMID:19845672

  18. Monocyte Chemotactic Protein-1 Regulates Voltage-Gated K+ Channels and Macrophage Transmigration

    PubMed Central

    Gendelman, Howard E.; Ding, Shengyuan; Gong, Nan; Liu, Jianuo; Ramirez, Servio H.; Persidsky, Yuri; Mosley, R. Lee; Wang, Tong; Volsky, David J.; Xiong, Huangui

    2009-01-01

    Progressive human immunodeficiency virus (HIV)-1 infection and virus-induced neuroinflammatory responses effectuates monocyte-macrophage transmigration across the blood-brain barrier (BBB). A key factor in mediating these events is monocyte chemotactic protein-1 (MCP-1). Upregulated glial-derived MCP-1 in HIV-1 infected brain tissues generates a gradient for monocyte recruitment into the nervous system. We posit that the inter-relationships between MCP-1, voltage gated ion channels, cell shape and volume, and cell mobility underlie monocyte transmigration across the BBB. In this regard, MCP-1 serves both as a chemoattractant and an inducer of monocyte-macrophage ion flux affecting cell shape and mobility. To address this hypothesis, MCP-1 treated bone marrow derived macrophages (BMM) were analyzed for gene and protein expression, electrophysiology, and capacity to migrate across a laboratory constructed BBB. MCP-1 enhanced K+ channel gene (KCNA3) and channel protein expression. Electrophysiological studies revealed that MCP-1 increased outward K+ currents in a dose dependent manner. In vitro studies demonstrated that MCP-1 increased BMM migration across an artificial BBB and the MCP-1-induced BMM migration was blocked by tetraethylammonium, a voltage-gated K+ channel blocker. Together these data demonstrated that MCP-1 affects macrophage migratory movement through regulation of voltage-gated K+ channels and as such, provides a novel therapeutic strategy for neuroAIDS PMID:19034671

  19. 21. OUTLET PIPE AND CONCRETE CHANNEL FOR THE HEAD GATE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    21. OUTLET PIPE AND CONCRETE CHANNEL FOR THE HEAD GATE PICTURED IN CO-43-A-20. - Highline Canal, Sand Creek Lateral, Beginning at intersection of Peoria Street & Highline Canal in Arapahoe County (City of Aurora), Sand Creek lateral Extends 15 miles Northerly through Araphoe County, City & County of Denver, & Adams County to its end point, approximately 1/4 mile Southest of intersectioin of D Street & Ninth Avenue in Adams County (Rocky Mountain Arsenal, Commerce City Vicinity), Commerce City, Adams County, CO

  20. From Toxins Targeting Ligand Gated Ion Channels to Therapeutic Molecules

    PubMed Central

    Nasiripourdori, Adak; Taly, Valérie; Grutter, Thomas; Taly, Antoine

    2011-01-01

    Ligand-gated ion channels (LGIC) play a central role in inter-cellular communication. This key function has two consequences: (i) these receptor channels are major targets for drug discovery because of their potential involvement in numerous human brain diseases; (ii) they are often found to be the target of plant and animal toxins. Together this makes toxin/receptor interactions important to drug discovery projects. Therefore, toxins acting on LGIC are presented and their current/potential therapeutic uses highlighted. PMID:22069709

  1. Analysis and functional implications of phosphorylation of neuronal voltage-gated potassium channels

    PubMed Central

    Cerda, Oscar; Trimmer, James S.

    2012-01-01

    Phosphorylation is the most common and abundant posttranslational modification to eukaryotic proteins, regulating a plethora of dynamic cellular processes. Here, we review and discuss recent advances in our knowledge of the breadth and importance of reversible phosphorylation in regulating the expression, localization and function of mammalian neuronal voltage-gated potassium (Kv) channels, key regulators of neuronal function. We highlight the role of modern mass spectrometric techniques and phosphospecific antibodies that reveal the extent and nature of phosphorylation at specific sites in Kv channels. We also emphasize the role of reversible phosphorylation in dynamically regulating diverse aspects of Kv channel biology. Finally, we discuss as important future directions the determination of the mechanistic basis for how altering phosphorylation state affects Kv channel expression, localization and function, the nature of macromolecular signaling complexes containing Kv channels and enzymes regulating their phosphorylation state, and the specific role of Kv channel phosphorylation in regulating neuronal function during physiological and pathophysiological events. PMID:20600597

  2. Modelling modal gating of ion channels with hierarchical Markov models

    PubMed Central

    Fackrell, Mark; Crampin, Edmund J.; Taylor, Peter

    2016-01-01

    Many ion channels spontaneously switch between different levels of activity. Although this behaviour known as modal gating has been observed for a long time it is currently not well understood. Despite the fact that appropriately representing activity changes is essential for accurately capturing time course data from ion channels, systematic approaches for modelling modal gating are currently not available. In this paper, we develop a modular approach for building such a model in an iterative process. First, stochastic switching between modes and stochastic opening and closing within modes are represented in separate aggregated Markov models. Second, the continuous-time hierarchical Markov model, a new modelling framework proposed here, then enables us to combine these components so that in the integrated model both mode switching as well as the kinetics within modes are appropriately represented. A mathematical analysis reveals that the behaviour of the hierarchical Markov model naturally depends on the properties of its components. We also demonstrate how a hierarchical Markov model can be parametrized using experimental data and show that it provides a better representation than a previous model of the same dataset. Because evidence is increasing that modal gating reflects underlying molecular properties of the channel protein, it is likely that biophysical processes are better captured by our new approach than in earlier models. PMID:27616917

  3. THE CRYSTAL STRUCTURE OF A VOLTAGE-GATED SODIUM CHANNEL

    PubMed Central

    Payandeh, Jian; Scheuer, Todd; Zheng, Ning; Catterall, William A.

    2011-01-01

    Voltage-gated sodium channels initiate electrical signaling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity, and drug block is unknown. Here, we report the crystal structure of a voltage-gated Na+-channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage-sensors at 2.7 Å resolution. The arginine gating charges make multiple hydrophilic interactions within the voltage-sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures suggest that the voltage-sensor domains and the S4-S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ~6.5 Å wide, and water-filled, with four acidic side-chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high field-strength anionic coordination site, which confers Na+-selectivity through partial dehydration via direct interaction with glutamate side-chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs. PMID:21743477

  4. Marine Toxins That Target Voltage-gated Sodium Channels

    PubMed Central

    Al-Sabi, Ahmed; McArthur, Jeff; Ostroumov, Vitaly; French, Robert J.

    2006-01-01

    Eukaryotic, voltage-gated sodium (NaV) channels are large membrane proteins which underlie generation and propagation of rapid electrical signals in nerve, muscle and heart. Nine different NaV receptor sites, for natural ligands and/or drugs, have been identified, based on functional analyses and site-directed mutagenesis. In the marine ecosystem, numerous toxins have evolved to disrupt NaV channel function, either by inhibition of current flow through the channels, or by modifying the activation and inactivation gating processes by which the channels open and close. These toxins function in their native environment as offensive or defensive weapons in prey capture or deterrence of predators. In composition, they range from organic molecules of varying size and complexity to peptides consisting of ~10–70 amino acids. We review the variety of known NaV-targeted marine toxins, outlining, where known, their sites of interaction with the channel protein and their functional effects. In a number of cases, these natural ligands have the potential applications as drugs in clinical settings, or as models for drug development.

  5. A single amino acid gates the KcsA channel

    SciTech Connect

    Hirano, Minako; Okuno, Daichi; Onishi, Yukiko; Ide, Toru

    2014-08-08

    Highlights: • pH-dependent gating of the KcsA channel is regulated by the CPD. • E146 is the most essential amino acid for pH sensing by the KcsA. • The protonated-mimicking mutant, E146Q, is constitutively open independent of pH. • Minimal rearrangement of the CPD is sufficient for opening of the KcsA. - Abstract: The KcsA channel is a proton-activated potassium channel. We have previously shown that the cytoplasmic domain (CPD) acts as a pH-sensor, and the charged states of certain negatively charged amino acids in the CPD play an important role in regulating the pH-dependent gating. Here, we demonstrate the KcsA channel is constitutively open independent of pH upon mutating E146 to a neutrally charged amino acid. In addition, we found that rearrangement of the CPD following this mutation was not large. Our results indicate that minimal rearrangement of the CPD, particularly around E146, is sufficient for opening of the KcsA channel.

  6. Hysteresis of gating underlines sensitization of TRPV3 channels.

    PubMed

    Liu, Beiying; Yao, Jing; Zhu, Michael X; Qin, Feng

    2011-11-01

    Vanilloid receptors of the transient receptor potential family have functions in thermal sensation and nociception. Among them, transient receptor potential vanilloid (TRPV)3 displays a unique property by which the repeated stimulation causes successive increases in its activity. The property has been known as sensitization and is observed in both native cells and cells heterologously expressing TRPV3. Transient increases in intracellular calcium levels have been implicated to play a key role in this process by mediating interaction of calmodulin with the channel. In support of the mechanism, BAPTA, a fast calcium chelator, accelerates the sensitization, whereas the slow chelator EGTA is ineffectual. Here, we show that the sensitization of TRPV3 also occurred independently of Ca(2+). It was observed in both inside-out and outside-out membrane patches. BAPTA, but not EGTA, has a direct potentiation effect on channel activation. Analogues of BAPTA lacking Ca(2+)-buffering capability were similarly effective. The stimulation-induced sensitization and the potentiation by BAPTA are distinguishable in reversibility. We conclude that the sensitization of TRPV3 is intrinsic to the channel itself and occurs as a result of hysteresis of channel gating. BAPTA accelerates the sensitization process by potentiating the gating of the channel.

  7. Gating mechanosensitive channels in bacteria with an atomic force microscope

    NASA Astrophysics Data System (ADS)

    Garces, Renata; Miller, Samantha; Schmidt, Christoph F.; Third Institute of Physics Team; School of Medical Sciences Collaboration

    The regulation of growth and integrity of bacteria is critically linked to mechanical stress. Bacteria typically maintain a high difference of osmotic pressure (turgor pressure) with respect to the environment. This pressure difference (on the order of 1 atm) is supported by the cell envelope, a composite of lipid membranes and a rigid cell wall. Turgor pressure is controlled by the ratio of osmolytes inside and outside bacteria and thus, can abruptly increase upon osmotic downshock. For structural integrity bacteria rely on the mechanical stability of the cell wall and on the action of mechanosensitive (MS) channels: membrane proteins that release solutes in response to stress in the cell envelope. We here present experimental data on MS channels gating. We activate channels by indenting living bacteria with the cantilever of an atomic force microscope (AFM). We compare responses of wild-type and mutant bacteria in which some or all MS channels have been eliminated.

  8. Voltage-gated Potassium Channels as Therapeutic Drug Targets

    PubMed Central

    Wulff, Heike; Castle, Neil A.; Pardo, Luis A.

    2009-01-01

    The human genome contains 40 voltage-gated potassium channels (KV) which are involved in diverse physiological processes ranging from repolarization of neuronal or cardiac action potentials, over regulating calcium signaling and cell volume, to driving cellular proliferation and migration. KV channels offer tremendous opportunities for the development of new drugs for cancer, autoimmune diseases and metabolic, neurological and cardiovascular disorders. This review first discusses pharmacological strategies for targeting KV channels with venom peptides, antibodies and small molecules and then highlights recent progress in the preclinical and clinical development of drugs targeting KV1.x, KV7.x (KCNQ), KV10.1 (EAG1) and KV11.1 (hERG) channels. PMID:19949402

  9. A single amino acid gates the KcsA channel.

    PubMed

    Hirano, Minako; Okuno, Daichi; Onishi, Yukiko; Ide, Toru

    2014-08-08

    The KcsA channel is a proton-activated potassium channel. We have previously shown that the cytoplasmic domain (CPD) acts as a pH-sensor, and the charged states of certain negatively charged amino acids in the CPD play an important role in regulating the pH-dependent gating. Here, we demonstrate the KcsA channel is constitutively open independent of pH upon mutating E146 to a neutrally charged amino acid. In addition, we found that rearrangement of the CPD following this mutation was not large. Our results indicate that minimal rearrangement of the CPD, particularly around E146, is sufficient for opening of the KcsA channel.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. Cardiac Mechano-Gated Ion Channels and Arrhythmias

    PubMed Central

    Peyronnet, Remi; Nerbonne, Jeanne M.; Kohl, Peter

    2015-01-01

    Mechanical forces will have been omnipresent since the origin of life, and living organisms have evolved mechanisms to sense, interpret and respond to mechanical stimuli. The cardiovascular system in general, and the heart in particular, are exposed to constantly changing mechanical signals, including stretch, compression, bending, and shear. The heart adjusts its performance to the mechanical environment, modifying electrical, mechanical, metabolic, and structural properties over a range of time scales. Many of the underlying regulatory processes are encoded intra-cardially, and are thus maintained even in heart transplant recipients. Although mechano-sensitivity of heart rhythm has been described in the medical literature for over a century, its molecular mechanisms are incompletely understood. Thanks to modern biophysical and molecular technologies, the roles of mechanical forces in cardiac biology are being explored in more detail, and detailed mechanisms of mechano-transduction have started to emerge. Mechano-gated ion channels are cardiac mechano-receptors. They give rise to mechano-electric feedback, thought to contribute to normal function, disease development, and, potentially, therapeutic interventions. In this review, we focus on acute mechanical effects on cardiac electrophysiology, explore molecular candidates underlying observed responses, and discuss their pharmaceutical regulation. From this, we identify open research questions and highlight emerging technologies that may help in addressing them. Cardiac electrophysiology is acutely affected by the heart’s mechanical environment. Mechano-electric feedback affects excitability, conduction, and electrical load, and remains an underestimated player in arrhythmogenesis. The utility of therapeutic interventions targeting acute mechano-electrical transduction is an open field worthy of further study. PMID:26838316

  15. Animal toxins acting on voltage-gated potassium channels.

    PubMed

    Mouhat, Stéphanie; Andreotti, Nicolas; Jouirou, Besma; Sabatier, Jean-Marc

    2008-01-01

    Animal venoms are rich natural sources of bioactive compounds, including peptide toxins acting on the various types of ion channels, i.e. K(+), Na(+), Cl(-) and Ca(2+). Among K+ channel-acting toxins, those selective for voltage-gated K(+) (Kv) channels are widely represented and have been isolated from the venoms of numerous animal species, such as scorpions, sea anemones, snakes, marine cone snails and spiders. The toxins characterized hitherto contain between 22 and 60 amino acid residues, and are cross-linked by two to four disulfide bridges. Depending on their types of fold, toxins can be classified in eight structural categories, which showed a combination of beta-strands, helices, or a mixture of both. The main architectural motifs thereof are referred to as alpha/beta scaffold and inhibitor cystine knot (ICK). A detailed analysis of toxin structures and pharmacological selectivities indicates that toxins exhibiting a similar type of fold can exert their action on several subtypes of Kv channels, whereas a particular Kv channel can be targeted by toxins that possess unrelated folds. Therefore, it appears that the ability of structurally divergent toxins to interact with a particular Kv channel relies onto a similar spatial distribution of amino acid residues that are key to the toxin-channel interaction (rather than the type of toxin fold). The diversity of Kv channel blockers and their therapeutic value in the potential treatment of a number of specific human diseases, especially autoimmune disorders, inflammatory neuropathies and cancer, are reviewed.

  16. Catalyst-like modulation of transition states for CFTR channel opening and closing: new stimulation strategy exploits nonequilibrium gating.

    PubMed

    Csanády, László; Töröcsik, Beáta

    2014-02-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is the chloride ion channel mutated in cystic fibrosis (CF) patients. It is an ATP-binding cassette protein, and its resulting cyclic nonequilibrium gating mechanism sets it apart from most other ion channels. The most common CF mutation (ΔF508) impairs folding of CFTR but also channel gating, reducing open probability (Po). This gating defect must be addressed to effectively treat CF. Combining single-channel and macroscopic current measurements in inside-out patches, we show here that the two effects of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) on CFTR, pore block and gating stimulation, are independent, suggesting action at distinct sites. Furthermore, detailed kinetic analysis revealed that NPPB potently increases Po, also of ΔF508 CFTR, by affecting the stability of gating transition states. This finding is unexpected, because for most ion channels, which gate at equilibrium, altering transition-state stabilities has no effect on Po; rather, agonists usually stimulate by stabilizing open states. Our results highlight how for CFTR, because of its unique cyclic mechanism, gating transition states determine Po and offer strategic targets for potentiator compounds to achieve maximal efficacy.

  17. Gating the bacterial mechanosensitive channel MscL in vivo

    PubMed Central

    Batiza, Ann Finney; Kuo, Mario Meng-Chiang; Yoshimura, Kenjiro; Kung, Ching

    2002-01-01

    YggB and MscL are the major mechanosensitive channels in Escherichia coli, and each can rescue the double knockout mutant from osmotic downshock. However, the role of MscL in wild-type bacteria is in question, not only because cells without MscL survive severe osmotic downshocks, but because 1.8 times more suction is required to gate MscL than YggB under patch clamp. Here, we extend previous evidence [Ajouz, B., Berrier, C., Garrigues, A., Besnard, M. & Ghazi, A. (1998) J. Biol. Chem. 273, 26670–26674] to show that downshock gates MscL in vivo even in the presence of YggB. We have made this determination by engineering a channel we can structurally modify in vivo (Leu-19→Cys MscL). MscLs with charges in their constrictions are known to open easily and transiently to substates and stop cell growth. In this study, we use downshock to stretch this region open to allow attachment of a charged thiosulfonate reagent MTSET+, thereby creating a toxic channel. Therefore, channel opening can be monitored by loss of colony forming units. By this measure, we find that an ≈800 mmol/kg downshock from 1,200 mmol/kg medium opens Leu-19→Cys MscL in the presence of YggB, but a downshock of only ≈400 mmol/kg is required in the absence of YggB. In parallel, Leu-19→Cys MscL, stretched open by large sustained suction in the presence of MTSET+ in voltage-clamped patches, subsequently flickers open with little suction. These observations show that MscL opening is triggered by a specific downshock, even in the presence of YggB, that YggB buffers MscL gating in vivo, and that residue 19 becomes exposed upon channel opening. PMID:11960017

  18. Transduction channels' gating can control friction on vibrating hair-cell bundles in the ear.

    PubMed

    Bormuth, Volker; Barral, Jérémie; Joanny, Jean-François; Jülicher, Frank; Martin, Pascal

    2014-05-20

    Hearing starts when sound-evoked mechanical vibrations of the hair-cell bundle activate mechanosensitive ion channels, giving birth to an electrical signal. As for any mechanical system, friction impedes movements of the hair bundle and thus constrains the sensitivity and frequency selectivity of auditory transduction. Friction is generally thought to result mainly from viscous drag by the surrounding fluid. We demonstrate here that the opening and closing of the transduction channels produce internal frictional forces that can dominate viscous drag on the micrometer-sized hair bundle. We characterized friction by analyzing hysteresis in the force-displacement relation of single hair-cell bundles in response to periodic triangular stimuli. For bundle velocities high enough to outrun adaptation, we found that frictional forces were maximal within the narrow region of deflections that elicited significant channel gating, plummeted upon application of a channel blocker, and displayed a sublinear growth for increasing bundle velocity. At low velocity, the slope of the relation between the frictional force and velocity was nearly fivefold larger than the hydrodynamic friction coefficient that was measured when the transduction machinery was decoupled from bundle motion by severing tip links. A theoretical analysis reveals that channel friction arises from coupling the dynamics of the conformational change associated with channel gating to tip-link tension. Varying channel properties affects friction, with faster channels producing smaller friction. We propose that this intrinsic source of friction may contribute to the process that sets the hair cell's characteristic frequency of responsiveness.

  19. Coherent beam combining in atmospheric channels using gated backscatter.

    PubMed

    Naeh, Itay; Katzir, Abraham

    2016-02-01

    This paper introduces the concept of atmospheric channels and describes a possible approach for the coherent beam combining of lasers of an optical phased array (OPA) in a turbulent atmosphere. By using the recently introduced sparse spectrum harmonic augmentation method, a comprehensive simulative investigation was performed and the exceptional properties of the atmospheric channels were numerically demonstrated. Among the interesting properties are the ability to guide light in a confined manner in a refractive channel, the ability to gather different sources to the same channel, and the ability to maintain a constant relative phase within the channel between several sources. The newly introduced guiding properties combined with a suggested method for channel probing and phase measurement by aerosol backscattered radiation allows coherence improvement of the phased array's elements and energy refocusing at the location of the channel in order to increase power in the bucket without feedback from the target. The method relies on the electronic focusing, electronic scanning, and time gating of the OPA, combined with elements of the relative phase measurements.

  20. Calmodulin regulation (calmodulation) of voltage-gated calcium channels

    PubMed Central

    Ben-Johny, Manu

    2014-01-01

    Calmodulin regulation (calmodulation) of the family of voltage-gated CaV1-2 channels comprises a prominent prototype for ion channel regulation, remarkable for its powerful Ca2+ sensing capabilities, deep in elegant mechanistic lessons, and rich in biological and therapeutic implications. This field thereby resides squarely at the epicenter of Ca2+ signaling biology, ion channel biophysics, and therapeutic advance. This review summarizes the historical development of ideas in this field, the scope and richly patterned organization of Ca2+ feedback behaviors encompassed by this system, and the long-standing challenges and recent developments in discerning a molecular basis for calmodulation. We conclude by highlighting the considerable synergy between mechanism, biological insight, and promising therapeutics. PMID:24863929

  1. Ion selectivity and gating mechanisms of FNT channels

    PubMed Central

    Waight, Andrew B.; Czyzewski, Bryan K.; Wang, Da-Neng

    2013-01-01

    The phospholipid bilayer has evolved to be a protective and selective barrier by which the cell maintains high concentrations of life sustaining organic and inorganic material. As gatekeepers responsible for an immense amount of bidirectional chemical traffic between the cytoplasm and extracellular milieu, ion channels have been studied in detail since their postulated existence nearly three-quarters of a century ago. Over the past fifteen years, we have begun to understand how selective permeability can be achieved for both cationic and anionic ions. Our mechanistic knowledge has expanded recently with studies of a large family of anion channels, the Formate Nitrite Transport (FNT) family. This family has proven amenable to structural studies at a resolution high enough to reveal intimate details of ion selectivity and gating. With five representative members having yielded a total of 15 crystal structures, this family represents one of the richest sources of structural information for anion channels. PMID:23773802

  2. TAURINE REGULATION OF VOLTAGE-GATED CHANNELS IN RETINAL NEURONS

    PubMed Central

    Rowan, Matthew JM; Bulley, Simon; Purpura, Lauren; Ripps, Harris; Shen, Wen

    2017-01-01

    Taurine activates not only Cl−-permeable ionotropic receptors, but also receptors that mediate metabotropic responses. The metabotropic property of taurine was revealed in electrophysiological recordings obtained after fully blocking Cl−-permeable receptors with an inhibitory “cocktail” consisting of picrotoxin, SR95531, and strychnine. We found that taurine’s metabotropic effects regulate voltage-gated channels in retinal neurons. After applying the inhibitory cocktail, taurine enhanced delayed outward rectifier K+ channels preferentially in Off-bipolar cells, and the effect was completely blocked by the specific PKC inhibitor, GF109203X. Additionally, taurine also acted through a metabotropic pathway to suppress both L- and N-type Ca2+ channels in retinal neurons, which were insensitive to the potent GABAB receptor inhibitor, CGP55845. This study reinforces our previous finding that taurine in physiological concentrations produces a multiplicity of metabotropic effects that precisely govern the integration of signals being transmitted from the retina to the brain. PMID:23392926

  3. Side-gated ultrathin-channel nanopore FET sensors

    NASA Astrophysics Data System (ADS)

    Yanagi, Itaru; Oura, Takeshi; Haga, Takanobu; Ando, Masahiko; Yamamoto, Jiro; Mine, Toshiyuki; Ishida, Takeshi; Hatano, Toshiyuki; Akahori, Rena; Yokoi, Takahide; Anazawa, Takashi

    2016-03-01

    A side-gated, ultrathin-channel nanopore FET (SGNAFET) is proposed for fast and label-free DNA sequencing. The concept of the SGNAFET comprises the detection of changes in the channel current during DNA translocation through a nanopore and identifying the four types of nucleotides as a result of these changes. To achieve this goal, both p- and n-type SGNAFETs with a channel thicknesses of 2 or 4 nm were fabricated, and the stable transistor operation of both SGNAFETs in air, water, and a KCl buffer solution were confirmed. In addition, synchronized current changes were observed between the ionic current through the nanopore and the SGNAFET’s drain current during DNA translocation through the nanopore.

  4. Voltage-gated sodium channels and pain-related disorders.

    PubMed

    Kanellopoulos, Alexandros H; Matsuyama, Ayako

    2016-12-01

    Voltage-gated sodium channels (VGSCs) are heteromeric transmembrane protein complexes. Nine homologous members, SCN1A-11A, make up the VGSC gene family. Sodium channel isoforms display a wide range of kinetic properties endowing different neuronal types with distinctly varied firing properties. Among the VGSCs isoforms, Nav1.7, Nav1.8 and Nav1.9 are preferentially expressed in the peripheral nervous system. These isoforms are known to be crucial in the conduction of nociceptive stimuli with mutations in these channels thought to be the underlying cause of a variety of heritable pain disorders. This review provides an overview of the current literature concerning the role of VGSCs in the generation of pain and heritable pain disorders. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  5. Taurine regulation of voltage-gated channels in retinal neurons.

    PubMed

    Rowan, Matthew J M; Bulley, Simon; Purpura, Lauren A; Ripps, Harris; Shen, Wen

    2013-01-01

    Taurine activates not only Cl(-)-permeable ionotropic receptors but also receptors that mediate metabotropic responses. The metabotropic property of taurine was revealed in electrophysiological recordings obtained after fully blocking Cl(-)-permeable receptors with an inhibitory "cocktail" consisting of picrotoxin, SR95531, and strychnine. We found that taurine's metabotropic effects regulate voltage-gated channels in retinal neurons. After applying the inhibitory cocktail, taurine enhanced delayed outward rectifier K(+) channels preferentially in Off-bipolar cells, and the effect was completely blocked by the specific PKC inhibitor, GF109203X. Additionally, taurine also acted through a metabotropic pathway to suppress both L- and N-type Ca(2+) channels in retinal neurons, which were insensitive to the potent GABA(B) receptor inhibitor, CGP55845. This study reinforces our previous finding that taurine in physiological concentrations produces a multiplicity of metabotropic effects that precisely govern the integration of signals being transmitted from the retina to the brain.

  6. Mechanisms of modulation by internal protons of cyclic nucleotide-gated channels cloned from sensory receptor cells.

    PubMed Central

    Gavazzo, P; Picco, C; Menini, A

    1997-01-01

    We have examined the modulation by internal protons of cyclic nucleotide-gated (CNG) channels cloned from bovine olfactory receptor cells and retinal rods. CNG channels were studied in excised inside-out membrane patches from Xenopus laevis oocytes previously injected with the mRNA encoding for the subunit 1 of olfactory or rod channels. Channels were activated by cGMP or cAMP, and currents as a function of cyclic nucleotide concentrations were measured as pHi varied between 7.6 and 5.0. Increasing internal proton concentrations caused a partial blockage of the single-channel current, consistent with protonation of a single acidic site with a pK1 of 4.5-4.7, both in rod and in olfactory CNG channels. Channel gating properties were also affected by internal protons. The open probability at low cyclic nucleotide concentrations was greatly increased by lowering pHi, and the increase was larger when channels were activated by cAMP than by cGMP. Therefore, internal protons affected both channel permeation and gating properties, causing a reduction in single-channel current and an increase in open probability. These effects are likely to be caused by different titratable groups on the channel. PMID:9308192

  7. The β Subunit of Voltage-Gated Ca2+ Channels

    PubMed Central

    Buraei, Zafir; Yang, Jian

    2015-01-01

    Calcium regulates a wide spectrum of physiological processes such as heartbeat, muscle contraction, neuronal communication, hormone release, cell division, and gene transcription. Major entry-ways for Ca2+ in excitable cells are high-voltage activated (HVA) Ca2+channels. These are plasma membrane proteins composed of several subunits, including α1, α2δ, β, and γ. Although the principal α1 subunit (Cavα1) contains the channel pore, gating machinery and most drug binding sites, the cytosolic auxiliary β subunit (Cavβ) plays an essential role in regulating the surface expression and gating properties of HVA Ca2+ channels. Cavβ is also crucial for the modulation of HVA Ca2+ channels by G proteins, kinases, and the Ras-related RGK GTPases. New proteins have emerged in recent years that modulate HVA Ca2+ channels by binding to Cavβ. There are also indications that Cavβ may carry out Ca2+ channel-independent functions, including directly regulating gene transcription. All four subtypes of Cavβ, encoded by different genes, have a modular organization, consisting of three variable regions, a conserved guanylate kinase (GK) domain, and a conserved Src-homology 3 (SH3) domain, placing them into the membrane-associated guanylate kinase (MAGUK) protein family. Crystal structures of Cavβs reveal how they interact with Cavα1, open new research avenues, and prompt new inquiries. In this article, we review the structure and various biological functions of Cavβ, with both a historical perspective as well as an emphasis on recent advances. PMID:20959621

  8. Cyclic Nucleotide-Gated Channel Block by Hydrolysis-Resistant Tetracaine Derivatives

    PubMed Central

    Andrade, Adriana L.; Melich, Kenneth; Whatley, G. Gregory; Kirk, Sarah R.; Karpen, Jeffrey W.

    2011-01-01

    To meet a pressing need for better cyclic nucleotide-gated (CNG) channel antagonists, we have increased the biological stability of tetracaine-based blockers by synthesizing amide and thioamide linkage substitutions of tetracaine (1) and a higher affinity octyl tail derivative (5). We report the apparent KD values, the mechanism of block, and the in vitro hydrolysis rates for these compounds. The ester linkage substitutions did not adversely affect CNG channel block; unexpectedly, thioamide substitution in 1 (compound 8) improved block significantly. Furthermore, the ester linkage substitutions did not appear to affect the mechanism of block in terms of the strong state preference for closed channels. All ester substituted compounds, especially the thioamide substitutions, were more resistant to hydrolysis by serum cholinesterase than their ester counterparts. These findings have implications for dissecting the physiological roles of CNG channels, treating certain forms of retinal degeneration, and possibly the current clinical uses of compound 1. PMID:21634421

  9. Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels

    NASA Astrophysics Data System (ADS)

    Lau, Carus H. Y.; King, Glenn F.; Mobli, Mehdi

    2016-09-01

    Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most spider-venom peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the spider-venom peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating, and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels.

  10. Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels

    PubMed Central

    Lau, Carus H. Y.; King, Glenn F.; Mobli, Mehdi

    2016-01-01

    Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most spider-venom peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the spider-venom peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating, and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels. PMID:27677715

  11. Mechanism of gating of T-type calcium channels

    PubMed Central

    1990-01-01

    We have analyzed the gating kinetics of T-type Ca channels in 3T3 fibroblasts. Our results show that channel closing, inactivation, and recovery from inactivation each include a voltage-independent step which becomes rate limiting at extreme potentials. The data require a cyclic model with a minimum of two closed, one open, and two inactivated states. Such a model can produce good fits to our data even if the transitions between closed states are the only voltage-dependent steps in the activating pathway leading from closed to inactivated states. Our analysis suggests that the channel inactivation step, as well as the direct opening and closing transitions, are not intrinsically voltage sensitive. Single-channel recordings are consistent with this scheme. As expected, each channel produces a single burst per opening and then inactivates. Comparison of the kinetics of T-type Ca current in fibroblasts and neuronal cells reveals significant differences which suggest that different subtypes of T-type Ca channels are expressed differentially in a tissue specific manner. PMID:2172443

  12. Mechanosensitive channels: multiplicity of families and gating paradigms.

    PubMed

    Sukharev, Sergei; Corey, David P

    2004-02-03

    Mechanosensitive ion channels are the primary transducers that convert mechanical force into an electrical or chemical signal in hearing, touch, and other mechanical senses. Unlike vision, olfaction, and some types of taste, which all use similar kinds of primary heterotrimeric GTP-binding protein-coupled receptors, mechanosensation relies on diverse types of transducer molecules. Unrelated types of channels can be used for the perception of various mechanical stimuli, not only in distant groups of organisms, but also in separate locations of the same organism. The extreme sensitivity of the transduction mechanism in the auditory system, which relies on an elaborate structure of rigid cilia, filamentous links, and molecular motors to focus force on transduction channels, contrasts with that of the bacterial channel MscL, which is opened by high lateral tension in the membrane and fulfills a safety-valve rather than a sensory function. The spatial scales of conformational movement and force in these two systems are described, and are shown to be consistent with a general physical description of mechanical channel gating. We outline the characteristics of several types of mechanosensitive channels and the functional contexts in which they participate in signaling and cellular regulation in sensory and nonsensory cells.

  13. Conserved Gating Elements in TRPC4 and TRPC5 Channels*

    PubMed Central

    Beck, Andreas; Speicher, Tilman; Stoerger, Christof; Sell, Thomas; Dettmer, Viviane; Jusoh, Siti A.; Abdulmughni, Ammar; Cavalié, Adolfo; Philipp, Stephan E.; Zhu, Michael X.; Helms, Volkhard; Wissenbach, Ulrich; Flockerzi, Veit

    2013-01-01

    TRPC4 and TRPC5 proteins share 65% amino acid sequence identity and form Ca2+-permeable nonselective cation channels. They are activated by stimulation of receptors coupled to the phosphoinositide signaling cascade. Replacing a conserved glycine residue within the cytosolic S4–S5 linker of both proteins by a serine residue forces the channels into an open conformation. Expression of the TRPC4G503S and TRPC5G504S mutants causes cell death, which could be prevented by buffering the Ca2+ of the culture medium. Current-voltage relationships of the TRPC4G503S and TRPC5G504S mutant ion channels resemble that of fully activated TRPC4 and TRPC5 wild-type channels, respectively. Modeling the structure of the transmembrane domains and the pore region (S4-S6) of TRPC4 predicts a conserved serine residue within the C-terminal sequence of the predicted S6 helix as a potential interaction site. Introduction of a second mutation (S623A) into TRPC4G503S suppressed the constitutive activation and partially rescued its function. These results indicate that the S4–S5 linker is a critical constituent of TRPC4/C5 channel gating and that disturbance of its sequence allows channel opening independent of any sensor domain. PMID:23677990

  14. 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.

  15. Cytoskeletal Actin Gates a Cl− Channel in Neocortical Astrocytes

    PubMed Central

    Lascola, Christopher D.; Nelson, Deborah J.; Kraig, Richard P.

    2009-01-01

    Increases in astroglial Cl− conductance accompany changes in cell morphology and disassembly of cytoskeletal actin, but Cl− channels underlying these conductance increases have not been described. We characterize an outwardly rectifying Cl− channel in rodent neocortical cultured astrocytes and describe how cell shape and cytoskeletal actin modulate channel gating. In inside-out patch-clamp recordings from cultured astrocytes, outwardly rectifying Cl− channels either were spontaneously active or inducible in quiescent patches by depolarizing voltage steps. Average single-channel conductance was 36 pS between −60 and −80 mV and was 75 pS between 60 and 80 mV in symmetrical (150 mm NaCl) solutions. The permeability ratio (PNa/PCl) was 0.14 at lower ionic strength but increased at higher salt concentrations. Both ATP and 4,4-diisothiocyanostilbene-2,2′-disulfonic acid produced a flicker block, whereas Zn2+ produced complete inhibition of channel activity. The frequency of observing both spontaneous and inducible Cl− channel activity was markedly higher in stellate than in flat, polygonally shaped astrocytes. In addition, cytoskeletal actin modulated channel open-state probability (PO) and conductance at negative membrane potentials, controlling the degree of outward rectification. Direct application of phalloidin, which stabilizes actin, preserved low PO and promoted lower conductance levels at negative potentials. Lower PO also was induced by direct application of polymerized actin. The actions of phalloidin and actin were reversed by coapplication of gelsolin and cytochalasin D, respectively. These results provide the first report of an outwardly rectifying Cl− channel in neocortical astrocytes and demonstrate how changes in cell shape and cytoskeletal actin may control Cl− conductance in these cells. PMID:9464993

  16. Cytoskeletal actin gates a Cl- channel in neocortical astrocytes.

    PubMed

    Lascola, C D; Nelson, D J; Kraig, R P

    1998-03-01

    Increases in astroglial Cl- conductance accompany changes in cell morphology and disassembly of cytoskeletal actin, but Cl- channels underlying these conductance increases have not been described. We characterize an outwardly rectifying Cl- channel in rodent neocortical cultured astrocytes and describe how cell shape and cytoskeletal actin modulate channel gating. In inside-out patch-clamp recordings from cultured astrocytes, outwardly rectifying Cl- channels either were spontaneously active or inducible in quiescent patches by depolarizing voltage steps. Average single-channel conductance was 36 pS between -60 and -80 mV and was 75 pS between 60 and 80 mV in symmetrical (150 mM NaCl) solutions. The permeability ratio (PNa/PCl) was 0.14 at lower ionic strength but increased at higher salt concentrations. Both ATP and 4, 4-diisothiocyanostilbene-2,2'-disulfonic acid produced a flicker block, whereas Zn2+ produced complete inhibition of channel activity. The frequency of observing both spontaneous and inducible Cl- channel activity was markedly higher in stellate than in flat, polygonally shaped astrocytes. In addition, cytoskeletal actin modulated channel open-state probability (PO) and conductance at negative membrane potentials, controlling the degree of outward rectification. Direct application of phalloidin, which stabilizes actin, preserved low PO and promoted lower conductance levels at negative potentials. Lower PO also was induced by direct application of polymerized actin. The actions of phalloidin and actin were reversed by coapplication of gelsolin and cytochalasin D, respectively. These results provide the first report of an outwardly rectifying Cl- channel in neocortical astrocytes and demonstrate how changes in cell shape and cytoskeletal actin may control Cl- conductance in these cells.

  17. Voltage-Gated Na+ Channels: Not Just for Conduction.

    PubMed

    Kruger, Larisa C; Isom, Lori L

    2016-06-01

    Voltage-gated sodium channels (VGSCs), composed of a pore-forming α subunit and up to two associated β subunits, are critical for the initiation of the action potential (AP) in excitable tissues. Building on the monumental discovery and description of sodium current in 1952, intrepid researchers described the voltage-dependent gating mechanism, selectivity of the channel, and general structure of the VGSC channel. Recently, crystal structures of bacterial VGSC α subunits have confirmed many of these studies and provided new insights into VGSC function. VGSC β subunits, first cloned in 1992, modulate sodium current but also have nonconducting roles as cell-adhesion molecules and function in neurite outgrowth and neuronal pathfinding. Mutations in VGSC α and β genes are associated with diseases caused by dysfunction of excitable tissues such as epilepsy. Because of the multigenic and drug-resistant nature of some of these diseases, induced pluripotent stem cells and other novel approaches are being used to screen for new drugs and further understand how mutations in VGSC genes contribute to pathophysiology.

  18. Structure and gating of CLC channels and exchangers

    PubMed Central

    Accardi, Alessio

    2015-01-01

    Abstract Since their serendipitous discovery the CLC family of Cl− transporting proteins has been a never ending source of surprises. From their double-barrelled architecture to their complex structure and divergence as channels and transporters, the CLCs never cease to amaze biophysicists, biochemists and physiologists alike. These unusual functional properties allow the CLCs to fill diverse physiological niches, regulating processes that range from muscle contraction to acidification of intracellular organelles, nutrient accumulation and survival of bacteria to environmental stresses. Over the last 15 years, the availability of atomic-level information on the structure of the CLCs, coupled to the discovery that the family is divided into passive channels and secondary active transporters, has revolutionized our understanding of their function. These breakthroughs led to the identification of the key structural elements regulating gating, transport, selectivity and regulation by ligands. Unexpectedly, many lines of evidence indicate that the CLC exchangers function according to a non-conventional transport mechanism that defies the fundamental tenets of the alternating-access paradigm for exchange transport, paving the way for future unexpected insights into the principles underlying active transport and channel gating. PMID:26148215

  19. Cholesterol Influences Voltage-Gated Calcium Channels and BK-Type Potassium Channels in Auditory Hair Cells

    PubMed Central

    Purcell, Erin K.; Liu, Liqian; Thomas, Paul V.; Duncan, R. Keith

    2011-01-01

    The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitabilityare unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassiumcurrent by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (∼30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology. PMID:22046269

  20. Conformational dynamics of the inner pore helix of voltage-gated potassium channels

    NASA Astrophysics Data System (ADS)

    Choe, Seungho; Grabe, Michael

    2009-06-01

    Voltage-gated potassium (Kv) channels control the electrical excitability of neurons and muscles. Despite this key role, how these channels open and close or gate is not fully understood. Gating is usually attributed to the bending and straightening of pore-lining helices at glycine and proline residues. In this work we focused on the role of proline in the Pro-Val-Pro (PVP) motif of the inner S6 helix in the Kv1.2 channel. We started by developing a simple hinged-rod model to fully explore the configurational space of bent helices and we related these configurations to the degree of pore opening. We then carried out fully atomistic simulations of the S6 helices and compared these simulations to the hinged-rod model. Both methods suggest that Kv1 channels are not tightly closed when the inner helices are straight, unlike what is seen in the non-PVP containing channels KcsA and KirBac. These results invite the possibility that the S6 helices may be kinked when Kv1 channels are closed. Our simulations indicate that the wild-type helix adopts multiple spatially distinct configurations, which is consistent with its role in adopting a closed state and an open state. The two most dominant configurational basins correspond to a 6 Å movement of the helix tail accompanied by the PVP region undergoing a local α-helix to 310-helix transition. We explored how single point mutations affect the propensity of the S6 helix to adopt particular configurations. Interestingly, mutating the first proline, P405 (P473 in Shaker), to alanine completely removed the bistable nature of the S6 helix possibly explaining why this mutation compromises the channel. Next, we considered four other mutations in the area known to affect channel gating and we saw similarly dramatic changes to the helix's dynamics and range of motion. Our results suggest a possible mechanism of helix pore closure and they suggest differences in the closed state of glycine-only channels, like KcsA, and PVP containing

  1. Voltage Gated Proton Channels Find Their Dream Job Managing the Respiratory Burst in Phagocytes

    PubMed Central

    DeCoursey, Thomas E.

    2011-01-01

    The voltage gated proton channel bears surprising resemblance to the voltage-sensing domain (S1–S4) of other voltage gated ion channels, but is a dimer with two conduction pathways. The proton channel seems designed for efficient proton extrusion from cells. In phagocytes, it facilitates the production of reactive oxygen species by NADPH oxidase. PMID:20134026

  2. 2,4-Toluene diisocyanate suppressed the calcium signaling of ligand gated ion channel receptors.

    PubMed

    Liu, Pei-Shan; Chiung, Yin-Mei; Kao, Yi-Yun; Chen, Han-Ting

    2006-02-15

    Toluene diisocyanate (TDI) is widely used as a chemical intermediate in the production of polyurethane. TDI-induced asthma is related to its disturbance of acetylcholine activity in most affected workers, but the relevant mechanisms are unclear. Toluene diamine (TDA) is the main metabolite of TDI. TDI and TDA have in common the basic toluene structure. Toluene is an abused solvent affecting neuronal signal transduction by influencing the function of ligand gated ion channel receptors, including nicotinic acetylcholine receptors (nAChR), P2X purinoceptors, [gamma]-aminobutyric acid type A (GABAA) receptors, etc. To understand the actions of TDI and TDA on ligand gated ion channels, we investigated their effects on the changes of cytosolic calcium concentration ([Ca2+]c) while stimulating nAChR in human neuroblastoma SH-SY5Y cells, P2 purinoceptors in PC12 cells, and GABAA receptors in bovine adrenal chromaffin cells. Our results showed that both TDI and TDA suppressed the [Ca2+]c rise induced by the potent nicotinic ligand, epibatidine, in human SH-SY5Y cells. Similar but stronger suppression of ATP-induced [Ca2+]c rise occurred in PC12 cells. TDI and TDA also partially suppressed the [Ca2+] c rise induced by GABA in bovine adrenal chromaffin cells. We conclude that TDI and TDA can act on ligand gated ion channel receptors. Our findings suggest that TDI and TDA might have some neurotoxicity that will need to be investigated.

  3. Transcriptional regulation of voltage-gated Ca(2+) channels.

    PubMed

    González-Ramírez, Ricardo; Felix, Ricardo

    2017-03-31

    The transcriptional regulation of voltage-gated Ca(2+) (CaV ) channels is an emerging research area that promises to improve our understanding of how many relevant physiological events are shaped in the central nervous system, the skeletal muscle, and other tissues. Interestingly, a picture of how transcription of CaV channel subunit genes is controlled is evolving with the identification of the promoter regions required for tissue-specific expression, and the identification of transcription factors that control their expression. These promoters share several characteristics that include multiple transcriptional start sites, lack of a TATA box, and the presence of elements conferring tissue-selective expression. Likewise, changes in CaV channel expression occur throughout development, following ischemia, seizures, or chronic drug administration. This review focuses on insights achieved regarding the control of CaV channel gene expression. To further understand the complexities of expression and to increase the possibilities of detecting CaV channel alterations causing human disease, a deeper knowledge on the structure of the 5' upstream regions of the genes encoding these remarkable proteins will be necessary. This article is protected by copyright. All rights reserved.

  4. Gating, Regulation, and Structure in K2P K+ Channels: In Varietate Concordia?

    PubMed

    Niemeyer, María Isabel; Cid, L Pablo; González, Wendy; Sepúlveda, Francisco V

    2016-09-01

    K2P K(+) channels with two pore domains in tandem associate as dimers to produce so-called background conductances that are regulated by a variety of stimuli. Whereas gating in K2P channels has been poorly understood, recent developments have provided important clues regarding the gating mechanism for this family of proteins. Two modes of gating present in other K(+) channels have been considered. The first is the so-called activation gating that occurs by bundle crossing and the splaying apart of pore-lining helices commanding ion passage. The second mode involves a change in conformation at the selectivity filter (SF), which impedes ion flow at this narrow portion of the conduction pathway and accounts for extracellular pH modulation of several K2P channels. Although some evidence supports the existence of an activation gate in K2P channels, recent results suggest that perhaps all stimuli, even those sensed at a distant location in the protein, are also mediated by SF gating. Recently resolved crystal structures of K2P channels in conductive and nonconductive conformations revealed that the nonconductive state is reached by blockade by a lipid acyl chain that gains access to the channel cavity through intramembrane fenestrations. Here we discuss whether this novel type of gating, proposed so far only for membrane tension gating, might mediate gating in response to other stimuli or whether SF gating is the only type of opening/closing mechanism present in K2P channels.

  5. Effect of lanthanum on voltage-dependent gating of a cloned mammalian neuronal potassium channel.

    PubMed

    Tytgat, J; Daenens, P

    1997-02-28

    The effect of the trivalent cation lanthanum (La3+) on voltage-dependent gating of a cloned mammalian neuronal Kv1.1 potassium channel was studied under whole-cell voltage-clamp conditions in oocytes of Xenopus laevis. La3+ (100 microM) was found to decrease the potassium currents at all test potentials and to shift the midpoint of the fraction open channels/membrane voltage curve by approximately +20 mV. The opening and closing time constants of Kv1.1 channels were empirically fitted with a 4th power Hodgkin-Huxley formalism, or with mono- and multi-exponentials. It was found that La3+ slowed down the kinetics of activation, speeded up those of deactivation, and shifted the opening kinetics by approximately + 60 mV. Interestingly, all these parameters of channel gating were not affected equally by La3+. Furthermore, amplitudes of the inward tail currents evoked at potentials more negative than the potassium equilibrium potential (E(K+)) were more strongly inhibited by La3+ than those of the outward tail currents evoked at potentials more positive than E(K+). This suggests voltage-dependent block and binding of La3+ to the Kv1.1 channel protein. We conclude that these actions cannot be explained in terms of surface charge considerations alone. Our results provide evidence for a direct interaction with the potassium channel protein, shedding new light on the mechanism of action of this lanthanide.

  6. Age-related homeostatic mid-channel proteolysis of neuronal L-type voltage-gated Ca2+ channels

    PubMed Central

    Michailidis, Ioannis E.; Abele-Henckels, Kathryn; Zhang, Wei K.; Lin, Bochao; Yu, Yong; Geyman, Larry; Ehlers, Michael D.; Pnevmatikakis, Eftychios A.; Yang, Jian

    2014-01-01

    SUMMARY Neural circuitry and brain activity depend critically on proper function of voltage-gated calcium channels (VGCCs), whose activity must be tightly controlled. We show that the main body of the pore-forming α1 subunit of neuronal L-type VGCCs, Cav1.2, is proteolytically cleaved, resulting in Cav1.2 fragment-channels that separate but remain on the plasma membrane. This “gmid-channel” proteolysis is regulated by channel activity, involves the Ca2+-dependent protease calpain and the ubiquitin-proteasome system, and causes attenuation and biophysical alterations of VGCC currents. Recombinant Cav1.2 fragment-channels mimicking the products of mid-channel proteolysis do not form active channels on their own, but when properly paired, produce currents with distinct biophysical properties. Mid-channel proteolysis increases dramatically with age and can be attenuated with an L-type VGCC blocker in vivo. Mid-channel proteolysis represents a novel form of homeostatic negative-feedback processing of VGCCs that could profoundly affect neuronal excitability, neurotransmission, neuroprotection, and calcium signaling in physiological and disease states. PMID:24908485

  7. The screw-helical voltage gating of ion channels.

    PubMed Central

    Keynes, R D; Elinder, F

    1999-01-01

    In the voltage-gated ion channels of every animal, whether they are selective for K+, Na+ or Ca2+, the voltage sensors are the S4 transmembrane segments carrying four to eight positive charges always separated by two uncharged residues. It is proposed that they move across the membrane in a screw-helical fashion in a series of three or more steps that each transfer a single electronic charge. The unit steps are stabilized by ion pairing between the mobile positive charges and fixed negative charges, of which there are invariably two located near the inner ends of segments S2 and S3 and a third near the outer end of either S2 or S3. Opening of the channel involves three such steps in each domain. PMID:10343407

  8. Molecular modeling and dynamics of the sodium channel inactivation gate.

    PubMed Central

    Sirota, Fernanda L; Pascutti, Pedro G; Anteneodo, Celia

    2002-01-01

    The intracellular linker L(III-IV) of voltage-gated sodium channels is known to be involved in their mechanism of inactivation. Its primary sequence is well conserved in sodium channels from different tissues and species. However, the role of charged residues in this region, first thought to play an important role in inactivation, has not been well identified, whereas the IFM triad (I1488-M1490) has been characterized as the crucial element for inactivation. In this work, we constructed theoretical models and performed molecular dynamics simulations, exploring the role of L(III-IV)-charged residues in the presence of a polar/nonpolar planar interface represented by a dielectric discontinuity. From structural predictions, two alpha-helical segments are proposed. Moreover, from dynamics simulations, a time-conserved motif is detected and shown to play a relevant role in guiding the inactivation particle toward its receptor site. PMID:11867438

  9. G-protein modulation of N-type calcium channel gating current in human embryonic kidney cells (HEK 293).

    PubMed Central

    Jones, L P; Patil, P G; Snutch, T P; Yue, D T

    1997-01-01

    1. Voltage-dependent inhibition of N-type calcium currents by G-proteins contributes importantly to presynaptic inhibition. To examine the effect of G-proteins on key intermediary transitions leading to channel opening, we measured both gating and ionic currents arising from recombinant N-type channels (alpha 1B, beta 1b and alpha 2) expressed in transiently transfected human embryonic kidney cells (HEK 293). Recombinant expression of a homogeneous population of channels provided a favourable system for rigorous examination of the mechanisms underlying G-protein modulation. 2. During intracellular dialysis with GTP gamma S to activate G-proteins, ionic currents demonstrated classic features of voltage-dependent inhibition, i.e. strong depolarizing prepulses increased ionic currents and produced hyperpolarizing shifts in the voltage-dependent activation of ionic current. No such effects were observed with GDP beta S present to minimize G-protein activity. 3. Gating currents were clearly resolved after ionic current blockade with 0.1 mM free La3+, enabling this first report of gating charge translocation arising exclusively from N-type channels. G-proteins decreased the amplitude of gating currents and produced depolarizing shifts in the voltage-dependent activation of gating charge movement. However, the greatest effect was to induce a approximately 20 mV separation between the voltage-dependent activation of gating charge movement and ionic current. Strong depolarizing prepulses largely reversed these effects. These modulatory features provide telling clues about the kinetic steps affected by G-proteins because gating currents arise from the movement of voltage sensors that trigger channel activation. 4. The mechanistic implications of concomitant G-protein-mediated changes in gating and ionic currents are discussed. We argue that G-proteins act to inhibit both voltage-sensor movement and the transduction of voltage-sensor activation into channel opening. Images

  10. From membrane tension to channel gating: A principal energy transfer mechanism for mechanosensitive channels.

    PubMed

    Zhang, Xuejun C; Liu, Zhenfeng; Li, Jie

    2016-11-01

    Mechanosensitive (MS) channels are evolutionarily conserved membrane proteins that play essential roles in multiple cellular processes, including sensing mechanical forces and regulating osmotic pressure. Bacterial MscL and MscS are two prototypes of MS channels. Numerous structural studies, in combination with biochemical and cellular data, provide valuable insights into the mechanism of energy transfer from membrane tension to gating of the channel. We discuss these data in a unified two-state model of thermodynamics. In addition, we propose a lipid diffusion-mediated mechanism to explain the adaptation phenomenon of MscS.

  11. Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium.

    PubMed Central

    Altenhofen, W; Ludwig, J; Eismann, E; Kraus, W; Bönigk, W; Kaupp, U B

    1991-01-01

    Cyclic nucleotide-gated ionic channels in photoreceptors and olfactory sensory neurons are activated by binding of cGMP or cAMP to a receptor site on the channel polypeptide. By site-directed mutagenesis and functional expression of bovine wild-type and mutant channels in Xenopus oocytes, we have tested the hypothesis that an alanine/threonine difference in the cyclic nucleotide-binding site determines the specificity of ligand binding, as has been proposed for cyclic nucleotide-dependent protein kinases [Weber, I.T., Shabb, J.B. & Corbin, J.D. (1989) Biochemistry 28, 6122-6127]. The wild-type olfactory channel is approximately 25-fold more sensitive to both cAMP and cGMP than the wild-type rod photoreceptor channel, and both channels are 30- to 40-fold more sensitive to cGMP than to cAMP. Substitution of the respective threonine by alanine in the rod photoreceptor and olfactory channels decreases the cGMP sensitivity of channel activation 30-fold but little affects activation by cAMP. Substitution of threonine by serine, an amino acid that also carries a hydroxyl group, even improves cGMP sensitivity of the wild-type channels 2- to 5-fold. We conclude that the hydroxyl group of Thr-560 (rod) and Thr-537 (olfactory) forms an additional hydrogen bond with cGMP, but not cAMP, and thereby provides the structural basis for ligand discrimination in cyclic nucleotide-gated channels. PMID:1719541

  12. Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium.

    PubMed

    Altenhofen, W; Ludwig, J; Eismann, E; Kraus, W; Bönigk, W; Kaupp, U B

    1991-11-01

    Cyclic nucleotide-gated ionic channels in photoreceptors and olfactory sensory neurons are activated by binding of cGMP or cAMP to a receptor site on the channel polypeptide. By site-directed mutagenesis and functional expression of bovine wild-type and mutant channels in Xenopus oocytes, we have tested the hypothesis that an alanine/threonine difference in the cyclic nucleotide-binding site determines the specificity of ligand binding, as has been proposed for cyclic nucleotide-dependent protein kinases [Weber, I.T., Shabb, J.B. & Corbin, J.D. (1989) Biochemistry 28, 6122-6127]. The wild-type olfactory channel is approximately 25-fold more sensitive to both cAMP and cGMP than the wild-type rod photoreceptor channel, and both channels are 30- to 40-fold more sensitive to cGMP than to cAMP. Substitution of the respective threonine by alanine in the rod photoreceptor and olfactory channels decreases the cGMP sensitivity of channel activation 30-fold but little affects activation by cAMP. Substitution of threonine by serine, an amino acid that also carries a hydroxyl group, even improves cGMP sensitivity of the wild-type channels 2- to 5-fold. We conclude that the hydroxyl group of Thr-560 (rod) and Thr-537 (olfactory) forms an additional hydrogen bond with cGMP, but not cAMP, and thereby provides the structural basis for ligand discrimination in cyclic nucleotide-gated channels.

  13. Spinal morphine but not ziconotide or gabapentin analgesia is affected by alternative splicing of voltage-gated calcium channel CaV2.2 pre-mRNA.

    PubMed

    Jiang, Yu-Qiu; Andrade, Arturo; Lipscombe, Diane

    2013-12-26

    Presynaptic voltage-gated calcium Ca(V)2.2 channels play a privileged role in spinal level sensitization following peripheral nerve injury. Direct and indirect inhibitors of Ca(V)2.2 channel activity in spinal dorsal horn are analgesic in chronic pain states. Ca(V)2.2 channels represent a family of splice isoforms that are expressed in different combinations according to cell-type. A pair of mutually exclusive exons in the Ca(V)2.2 encoding Cacna1b gene, e37a and e37b, differentially influence morphine analgesia. In mice that lack exon e37a, which is enriched in nociceptors, the analgesic efficacy of intrathecal morphine against noxious thermal stimuli is reduced. Here we ask if sequences unique to e37a influence: the development of abnormal thermal and mechanical sensitivity associated with peripheral nerve injury; and the actions of two other classes of analgesics that owe part or all of their efficacy to Ca(V)2.2 channel inhibition. We find that: i) the analgesic efficacy of morphine, but not ziconotide or gabapentin, is reduced in mice lacking e37a, ii) the induction and maintenance of behaviors associated with sensitization that accompany peripheral nerve injury, do not require e37a-specific sequence, iii) intrathecal morphine, but not ziconotide or gabapentin analgesia to thermal stimuli is significantly lower in wild-type mice after peripheral nerve injury, iv) the analgesic efficacy of ziconotide and gabapentin to mechanical stimuli is reduced following nerve injury, and iv) intrathecal morphine analgesia to thermal stimuli in mice lacking e37a is not further reduced by peripheral nerve injury. Our findings show that the analgesic action of morphine, but not ziconotide or gabapentin, to thermal stimuli is linked to which Cacna1b exon, e37a or e37b, is selected during alternative pre-mRNA splicing.

  14. A Point Mutation in the Pore Region Alters Gating, Ca2+Blockage, and Permeation of Olfactory Cyclic Nucleotide–Gated Channels

    PubMed Central

    Gavazzo, Paola; Picco, Cristiana; Eismann, Elisabeth; Kaupp, U. Benjamin; Menini, Anna

    2000-01-01

    Upon stimulation by odorants, Ca2+ and Na+ enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide–gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the α subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide–gated channels play a similar role. We studied the corresponding glutamate (E340) of the α subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca2+ and Mg2+. E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca2+ and Mg2+ powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca2+ relative to Na+ was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca2+ and Mg2+, and affects channel gating by cAMP more than by cGMP. PMID:10962010

  15. Voltage-dependent gating of single gap junction channels in an insect cell line.

    PubMed Central

    Bukauskas, F F; Weingart, R

    1994-01-01

    De novo formation of cell pairs was used to examine the gating properties of single gap junction channels. Two separate cells of an insect cell line (clone C6/36, derived from the mosquito Aedes albopictus) were pushed against each other to provoke formation of gap junction channels. A dual voltage-clamp method was used to control the voltage gradient between the cells (Vj) and measure the intercellular current (Ij). The first sign of channel activity was apparent 4.7 min after cell contact. Steady-state coupling reached after 30 min revealed a conductance of 8.7 nS. Channel formation involved no leak between the intra- and extracellular space. The first opening of a newly formed channel was slow (25-28 ms). Each preparation passed through a phase with only one operational gap junction channel. This period was exploited to examine the single channel properties. We found that single channels exhibit several conductance states with different conductances gamma j; a fully open state (gamma j(main state)), several substates (gamma j(substates)), a residual state (gamma j(residual)) and a closed state (gamma j(closed)). The gamma j(main state) was 375 pS, and gamma j(residual) ranged from 30 to 90 pS. The transitions between adjacent substates were 1/7-1/4 of gamma j(main state). Vj had no effect on gamma j(main state), but slightly affected gamma j (residual). The lj transitions involving gamma j(closed) were slow (15-60 ms), whereas those not involving gamma j(closed) were fast (< 2 ms). An increase in Vj led to a decrease in open channel probability. Depolarization of the membrane potential (Vm) increased the incidence of slow transitions leading to gamma j(closed). We conclude that insect gap junctions possess two gates, a fast gate controlled by Vj and giving rise to gamma j(substates) and gamma j(residual), and a slow gate sensitive to Vm and able to close the channel completely. PMID:7524710

  16. Transmembrane allosteric coupling of the gates in a potassium channel

    PubMed Central

    Wylie, Benjamin J.; Bhate, Manasi P.; McDermott, Ann E.

    2014-01-01

    It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98–103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening. PMID:24344306

  17. Transmembrane allosteric coupling of the gates in a potassium channel.

    PubMed

    Wylie, Benjamin J; Bhate, Manasi P; McDermott, Ann E

    2014-01-07

    It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98-103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.

  18. Inactivation of Gating Currents of L-Type Calcium Channels

    PubMed Central

    Shirokov, Roman; Ferreira, Gonzalo; Yi, Jianxun; Ríos, Eduardo

    1998-01-01

    In studies of gating currents of rabbit cardiac Ca channels expressed as α1C/β2a or α1C/β2a/α2δ subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489–517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863–895). Charge 1 (voltage of half-maximal transfer, V1/2 ≃ 0 mV) gates noninactivated channels, while charge 2 (V1/2 ≃ −90 mV) is generated in inactivated channels. In α1C/β2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant τ ≃ 8, while the available charge 2 decreased upon recovery from inactivation (at −200 mV) with τ ≃ 0.3 s. These processes therefore are much slower than charge movement, which takes <50 ms. This separation between the time scale of measurable charge movement and that of changes in their availability, which was even wider in the presence of α2δ, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the α2δ subunit did not change the V1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to τ ≃ 2 s), while slowing the reduction of charge 2 upon recovery (τ ≃ 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489–498) and inactivation as discrete modal change. The effects of α2δ are reproduced assuming that the subunit lowers the free energy of the inactivated mode. PMID:9607938

  19. Regulation of cough and action potentials by voltage-gated Na channels.

    PubMed

    Carr, Michael J

    2013-10-01

    The classical role ascribed to voltage-gated Na channels is the conduction of action potentials. Some excitable tissues such as cardiac muscle and skeletal muscle predominantly express a single voltage-gated Na channels isoform. Of the nine voltage-gated Na channels, seven are expressed in neurons, of these Nav 1.7, 1.8 and 1.9 are expressed in sensory neurons including vagal sensory neurons that innervate the airways and initiate cough. Nav 1.7 and Nav 1.9 are of particular interest as they represent two extremes in the functional diversity of voltage-gated Na channels. Voltage-gated Na channel isoforms expressed in airway sensory neurons produce multiple distinct Na currents that underlie distinct aspects of sensory neuron function. The interaction between voltage-gated Na currents underlies the characteristic ability of airway sensory nerves to encode encounters with irritant stimuli into action potential discharge and evoke the cough reflex.

  20. How voltage-gated calcium channels gate forms of homeostatic synaptic plasticity

    PubMed Central

    Frank, C. Andrew

    2014-01-01

    Throughout life, animals face a variety of challenges such as developmental growth, the presence of toxins, or changes in temperature. Neuronal circuits and synapses respond to challenges by executing an array of neuroplasticity paradigms. Some paradigms allow neurons to up- or downregulate activity outputs, while countervailing ones ensure that outputs remain within appropriate physiological ranges. A growing body of evidence suggests that homeostatic synaptic plasticity (HSP) is critical in the latter case. Voltage-gated calcium channels gate forms of HSP. Presynaptically, the aggregate data show that when synapse activity is weakened, homeostatic signaling systems can act to correct impairments, in part by increasing calcium influx through presynaptic CaV2-type channels. Increased calcium influx is often accompanied by parallel increases in the size of active zones and the size of the readily releasable pool of presynaptic vesicles. These changes coincide with homeostatic enhancements of neurotransmitter release. Postsynaptically, there is a great deal of evidence that reduced network activity and loss of calcium influx through CaV1-type calcium channels also results in adaptive homeostatic signaling. Some adaptations drive presynaptic enhancements of vesicle pool size and turnover rate via retrograde signaling, as well as de novo insertion of postsynaptic neurotransmitter receptors. Enhanced calcium influx through CaV1 after network activation or single cell stimulation can elicit the opposite response—homeostatic depression via removal of excitatory receptors. There exist intriguing links between HSP and calcium channelopathies—such as forms of epilepsy, migraine, ataxia, and myasthenia. The episodic nature of some of these disorders suggests alternating periods of stable and unstable function. Uncovering information about how calcium channels are regulated in the context of HSP could be relevant toward understanding these and other disorders. PMID

  1. 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.

  2. Modification of sodium and potassium channel gating kinetics by ether and halothane

    SciTech Connect

    Bean, B.P.; Shrager, P.; Goldstein, D.A.

    1981-03-01

    The effects of ether and halothane on the kinetics of sodium and potassium currents were investigated in the crayfish giant axon. Both general anesthetics produced a reversible, dose-dependent speeding up of sodium current inactivation at all membrane potentials, with no change in the rising phase of the currents. Double-pulse inactivation experiments with ether also showed faster inactivation, but the rate of recovery from inactivation at negative potentials was not affected. Ether shifted the midpoint of the steady-state fast inactivation curve in the hyperpolarizing direction and made the curve steeper. The activation of potassium currents was faster with ether present, with no change in the voltage dependence of steady-state potassium currents. Ether and halothane are known to perturb the structure of lipid bilayer membranes; the alterations in sodium and potassium channel gating kinetics are consistent with the hypothesis that the rats of the gating processes of the channels can be affected by the state of the lipids surrounding the channels, but a direct effect of ether and halothane on the protein part of the channels cannot be ruled out.

  3. Tetrodotoxin block of sodium channels in rabbit Purkinje fibers. Interactions between toxin binding and channel gating

    PubMed Central

    1981-01-01

    Tetrodotoxin (TTX) block of cardiac sodium channels was studied in rabbit Purkinje fibers using a two-microelectrode voltage clamp to measure sodium current. INa decreases with TTX as if one toxin molecule blocks one channel with a dissociation constant KD approximately equal to 1 microM. KD remains unchanged when INa is partially inactivated by steady depolarization. Thus, TTX binding and channel inactivation are independent at equilibrium. Interactions between toxin binding and gating were revealed, however, by kinetic behavior that depends on rates of equilibration. For example, frequent suprathreshold pulses produce extra use-dependent block beyond the tonic block seen with widely spaced stimuli. Such lingering aftereffects of depolarization were characterized by double-pulse experiments. The extra block decays slowly enough (tau approximately equal to 5 s) to be easily separated from normal recovery from inactivation (tau less than 0.2 s at 18 degrees C). The amount of extra block increases to a saturating level with conditioning depolarizations that produce inactivation without detectable activation. Stronger depolarizations that clearly open channels give the same final level of extra block, but its development includes a fast phase whose voltage- and time-dependence resemble channel activation. Thus, TTX block and channel gating are not independent, as believed for nerve. Kinetically, TTX resembles local anesthetics, but its affinity remains unchanged during maintained depolarization. On this last point, comparison of our INa results and earlier upstroke velocity (Vmax) measurements illustrates how much these approaches can differ. PMID:6270235

  4. ZnO-based multiple channel and multiple gate FinMOSFETs

    NASA Astrophysics Data System (ADS)

    Lee, Ching-Ting; Huang, Hung-Lin; Tseng, Chun-Yen; Lee, Hsin-Ying

    2016-02-01

    In recent years, zinc oxide (ZnO)-based metal-oxide-semiconductor field-effect transistors (MOSFETs) have attracted much attention, because ZnO-based semiconductors possess several advantages, including large exciton binding energy, nontoxicity, biocompatibility, low material cost, and wide direct bandgap. Moreover, the ZnO-based MOSFET is one of most potential devices, due to the applications in microwave power amplifiers, logic circuits, large scale integrated circuits, and logic swing. In this study, to enhance the performances of the ZnO-based MOSFETs, the ZnObased multiple channel and multiple gate structured FinMOSFETs were fabricated using the simple laser interference photolithography method and the self-aligned photolithography method. The multiple channel structure possessed the additional sidewall depletion width control ability to improve the channel controllability, because the multiple channel sidewall portions were surrounded by the gate electrode. Furthermore, the multiple gate structure had a shorter distance between source and gate and a shorter gate length between two gates to enhance the gate operating performances. Besides, the shorter distance between source and gate could enhance the electron velocity in the channel fin structure of the multiple gate structure. In this work, ninety one channels and four gates were used in the FinMOSFETs. Consequently, the drain-source saturation current (IDSS) and maximum transconductance (gm) of the ZnO-based multiple channel and multiple gate structured FinFETs operated at a drain-source voltage (VDS) of 10 V and a gate-source voltage (VGS) of 0 V were respectively improved from 11.5 mA/mm to 13.7 mA/mm and from 4.1 mS/mm to 6.9 mS/mm in comparison with that of the conventional ZnO-based single channel and single gate MOSFETs.

  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. Mechanics of Channel Gating of the Nicotinic Acetylcholine Receptor

    PubMed Central

    Liu, Xinli; Xu, Yechun; Li, Honglin; Wang, Xicheng; Jiang, Hualiang; Barrantes, Francisco J

    2008-01-01

    The nicotinic acetylcholine receptor (nAChR) is a key molecule involved in the propagation of signals in the central nervous system and peripheral synapses. Although numerous computational and experimental studies have been performed on this receptor, the structural dynamics of the receptor underlying the gating mechanism is still unclear. To address the mechanical fundamentals of nAChR gating, both conventional molecular dynamics (CMD) and steered rotation molecular dynamics (SRMD) simulations have been conducted on the cryo-electron microscopy (cryo-EM) structure of nAChR embedded in a dipalmitoylphosphatidylcholine (DPPC) bilayer and water molecules. A 30-ns CMD simulation revealed a collective motion amongst C-loops, M1, and M2 helices. The inward movement of C-loops accompanying the shrinking of acetylcholine (ACh) binding pockets induced an inward and upward motion of the outer β-sheet composed of β9 and β10 strands, which in turn causes M1 and M2 to undergo anticlockwise motions around the pore axis. Rotational motion of the entire receptor around the pore axis and twisting motions among extracellular (EC), transmembrane (TM), and intracellular MA domains were also detected by the CMD simulation. Moreover, M2 helices undergo a local twisting motion synthesized by their bending vibration and rotation. The hinge of either twisting motion or bending vibration is located at the middle of M2, possibly the gate of the receptor. A complementary twisting-to-open motion throughout the receptor was detected by a normal mode analysis (NMA). To mimic the pulsive action of ACh binding, nonequilibrium MD simulations were performed by using the SRMD method developed in one of our laboratories. The result confirmed all the motions derived from the CMD simulation and NMA. In addition, the SRMD simulation indicated that the channel may undergo an open-close (O ↔ C) motion. The present MD simulations explore the structural dynamics of the receptor under its gating process

  8. Ion effects on gating of the Ca(2+)-activated K+ channel correlate with occupancy of the pore.

    PubMed Central

    Demo, S D; Yellen, G

    1992-01-01

    We studied the effects of permeant ions on the gating of the large conductance Ca(2+)-activated K+ channel from rat skeletal muscle. Rb+ blockade of inward K+ current caused an increase in the open probability as though Rb+ occupancy of the pore interferes with channel closing. In support of this hypothesis, we directly measured the occupancy of the pore by the impermeant ion Cs+ and found that it strongly correlates with its effect on gating. This is consistent with the "foot-in-the-door" model of gating, which states that channels cannot close with an ion in the pore. However, because Rb+ and Cs+ not only slow the closing rate (as predicted by the model), but also speed the opening rate, our results are more consistent with a modified version of the model in which the channel can indeed close while occupied, but the occupancy destabilizes the closed state. Increasing the occupancy of the pore by the addition of other permeant (K+ and Tl+) and impermeant (tetraethylammonium) ions did not affect the open probability. To account for this disparity, we used a two-site permeation model in which only one of the sites influenced gating. Occupancy of this "gating site" interferes with channel closing and hastens opening. Ions that directly or indirectly increase the occupancy of this site will increase the open probability. Images FIGURE 1 FIGURE 2 PMID:1504240

  9. Screening for voltage-gated sodium channel interacting peptides.

    PubMed

    Meng, Er; Cai, Tian-Fu; Zhang, Hui; Tang, Si; Li, Meng-Jie; Li, Wen-Ying; Huang, Peng-Fei; Liu, Kai; Wu, Lei; Zhu, Ling-Yun; Liu, Long; Peng, Kuan; Dai, Xian-Dong; Jiang, Hui; Zeng, Xiong-Zhi; Liang, Song-Ping; Zhang, Dong-Yi

    2014-04-02

    The voltage-gated sodium channel (VGSC) interacting peptide is of special interest for both basic research and pharmaceutical purposes. In this study, we established a yeast-two-hybrid based strategy to detect the interaction(s) between neurotoxic peptide and the extracellular region of VGSC. Using a previously reported neurotoxin JZTX-III as a model molecule, we demonstrated that the interactions between JZTX-III and the extracellular regions of its target hNav1.5 are detectable and the detected interactions are directly related to its activity. We further applied this strategy to the screening of VGSC interacting peptides. Using the extracellular region of hNav1.5 as the bait, we identified a novel sodium channel inhibitor SSCM-1 from a random peptide library. This peptide selectively inhibits hNav1.5 currents in the whole-cell patch clamp assays. This strategy might be used for the large scale screening for target-specific interacting peptides of VGSCs or other ion channels.

  10. The myotonia congenita mutation A331T confers a novel hyperpolarization-activated gate to the muscle chloride channel ClC-1.

    PubMed

    Warnstedt, Maike; Sun, Chen; Poser, Barbara; Escriva, Maria Jose; Tranebjaerg, Lisbeth; Torbergsen, Torberg; van Ghelue, Marijke; Fahlke, Christoph

    2002-09-01

    Mutations in the muscle chloride channel gene CLCN1 cause myotonia congenita, an inherited disorder of skeletal muscle excitability leading to a delayed relaxation after muscle contraction. Here, we examine the functional consequences of a novel disease-causing mutation that predicts the substitution of alanine by threonine at position 331 (A331T) by whole-cell patch-clamp recording of recombinant mutant channels. A331T hClC-1 channels exhibit a novel slow gate that activates during membrane hyperpolarization and closes at positive potentials. This novel gate acts in series with fast opening and closing transitions that are common to wild-type (WT) and mutant channels. Under conditions at which this novel gate is not activated, i.e., a holding potential of 0 mV, the typical depolarization-induced activation gating of WT hClC-1 was only slightly affected by the mutation. In contrast, A331T hClC-1 channels with an open slow gate display an altered voltage dependence of open probability. These novel gating features of mutant channels produce a decreased open probability at -85 mV, the normal muscle resting potential, leading to a reduced resting chloride conductance of affected muscle fibers. The A331T mutation causes an unprecedented alteration of ClC-1 gating and reveals novel processes defining transitions between open and closed states in ClC chloride channels.

  11. The depressant scorpion neurotoxin LqqIT2 selectively modulates the insect voltage-gated sodium channel.

    PubMed

    Bosmans, Frank; Martin-Eauclaire, Marie-France; Tytgat, Jan

    2005-03-15

    LqqIT2 is a depressant neurotoxin present in the venom of the Leiurus quinquestriatus quinquestriatus scorpion, one of the world's most dangerous scorpions endemic to dry habitats in Africa and Asia. In order to determine its efficacy, potency and selectivity, LqqIT2 was subjected for the first time to an electrophysiological and pharmacological comparison between two different cloned sodium channels expressed in Xenopus laevis oocytes. Aside from typical beta-toxin effects, LqqIT2 also affected the inactivation process and ion selectivity of the insect voltage-gated sodium channel. The most interesting feature of LqqIT2 is its total insect-selectivity. At a concentration of 1 microM, the insect-voltage-gated sodium channel, para, was profoundly modulated while its mammalian counterpart, the rat brain Na(v)1.2 channel, was not affected. This trait offers excellent prospects for the development of novel insecticides.

  12. Effective gating charges per channel in voltage-dependent K+ and Ca2+ channels

    PubMed Central

    1996-01-01

    In voltage-dependent ion channels, the gating of the channels is determined by the movement of the voltage sensor. This movement reflects the rearrangement of the protein in response to a voltage stimulus, and it can be thought of as a net displacement of elementary charges (e0) through the membrane (z: effective number of elementary charges). In this paper, we measured z in Shaker IR (inactivation removed) K+ channels, neuronal alpha 1E and alpha 1A, and cardiac alpha 1C Ca2+ channels using two methods: (a) limiting slope analysis of the conductance-voltage relationship and (b) variance analysis, to evaluate the number of active channels in a patch, combined with the measurement of charge movement in the same patch. We found that in Shaker IR K+ channels the two methods agreed with a z congruent to 13. This suggests that all the channels that gate can open and that all the measured charge is coupled to pore opening in a strictly sequential kinetic model. For all Ca2+ channels the limiting slope method gave consistent results regardless of the presence or type of beta subunit tested (z = 8.6). However, as seen with alpha 1E, the variance analysis gave different results depending on the beta subunit used. alpha 1E and alpha 1E beta 1a gave higher z values (z = 14.77 and z = 15.13 respectively) than alpha 1E beta 2a (z = 9.50, which is similar to the limiting slope results). Both the beta 1a and beta 2a subunits, coexpressed with alpha 1E Ca2+ channels facilitated channel opening by shifting the activation curve to more negative potentials, but only the beta 2a subunit increased the maximum open probability. The higher z using variance analysis in alpha 1E and alpha 1E beta 1a can be explained by a set of charges not coupled to pore opening. This set of charges moves in transitions leading to nulls thus not contributing to the ionic current fluctuations but eliciting gating currents. Coexpression of the beta 2a subunit would minimize the fraction of nulls leading to

  13. 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

  14. N channel JFET based digital logic gate structure

    NASA Technical Reports Server (NTRS)

    Krasowski, Michael J. (Inventor)

    2010-01-01

    A circuit topography is presented which is used to create usable digital logic gates using N (negatively doped) channel Junction Field Effect Transistors (JFETs) and load resistors, level shifting resistors, and supply rails whose values are based on the direct current (DC) parametric distributions of those JFETs. This method has direct application to the current state of the art in high temperature, for example 300.degree. C. to 500.degree. C. and higher, silicon carbide (SiC) device production. The ability to produce inverting and combinatorial logic enables the production of pulse and edge triggered latches. This scale of logic synthesis would bring digital logic and state machine capabilities to devices operating in extremely hot environments, such as the surface of Venus, near hydrothermal vents, within nuclear reactors (SiC is inherently radiation hardened), and within internal combustion engines. The basic logic gate can be configured as a driver for oscillator circuits allowing for time bases and simple digitizers for resistive or reactive sensors. The basic structure of this innovation, the inverter, can be reconfigured into various analog circuit topographies through the use of feedback structures.

  15. Open channel block and alteration of N-methyl-D-aspartic acid receptor gating by an analog of phencyclidine.

    PubMed Central

    Dilmore, J G; Johnson, J W

    1998-01-01

    We investigated inhibition of the N-methyl-D-aspartic acid (NMDA) receptor-channel complex by N-ethyl-1,4,9, 9alpha-tetrahydro-4alphaR-cis-4alphaH-fluoren-++ +4alpha-amine (NEFA), a structural analog of phencyclidine (PCP). Using the whole-cell recording technique, we demonstrated that NEFA inhibits NMDA responses with an IC50 of 0.51 microM at -66 mV. We determined that NEFA binds to the open channel, and subsequently the channel can close and trap the blocker. Once the channel has closed, NEFA is unable to dissociate until the channel reopens. Single-channel recordings revealed that NEFA reduces the mean open time of single NMDA-activated channels in a concentration-dependent manner with a forward blocking rate (k+) of 39.9 microM-1 s-1. A computational model of antagonism by NEFA was developed and constrained using kinetic measurements of single-channel data. By multiple criteria, only models in which blocker binding in the channel causes a change in receptor operation adequately fit or predicted whole-cell data. By comparing model predictions and experimental measurements of NEFA action at a high NMDA concentration, we determined that NEFA affects receptor operation through an influence on channel gating. We conclude that inhibition of NMDA receptors by PCP-like blockers involves a modification of channel gating as well as block of current flow through the open channel. PMID:9746522

  16. A leucine zipper motif essential for gating of hyperpolarization-activated channels.

    PubMed

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

    2012-11-23

    It is poorly understood how hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) function. We have identified a leucine zipper in the S5 segment of HCNs, regulating hyperpolarization-activated and instantaneous current components. The leucine zipper is essential for HCN channel gating. The identification and functional characterization of the leucine zipper is an important step toward the understanding of HCN channel function. 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-P(o). 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.

  17. Changes in mechanosensitive channel gating following mechanical stimulation in skeletal muscle myotubes from the mdx mouse.

    PubMed

    Franco-Obregón, Alfredo; Lansman, Jeffry B

    2002-03-01

    We studied the effects of membrane stretch and voltage on the gating of single mechanosensitive (MS) channels in myotubes from dystrophin-deficient mdx mice. In earlier studies of MS channels in mdx myotubes, we found a novel class of stretch-inactivated channels. In the present experiments, we used a gentle suction protocol to determine whether seal formation damaged the membrane and altered MS channel gating, since dystrophin-deficiency is known to be associated with an increased susceptibility to mechanically induced damage. In some recordings from mdx myotubes, MS channel open probability gradually increased to levels approaching unity following seal formation. In these recordings, channels remained open for the duration of the recording. In other recordings, MS channel open probability remained low after seal formation and applying weak suction evoked conventional stretch-activated gating. Applying strong suction or very positive voltages, however, caused some channels to enter a high open probability gating mode. The shift to a high open probability gating mode coincided with the appearance of stretch-inactivated gating. These findings suggested that mechanical stimulation altered the mechanical properties of the patch causing some MS channels to enter a novel gating mode. In support of this idea, stretch-activated and stretch-inactivated channels were not detected in the same membrane patch and channel inactivation occurred at lower pressures than activation (P(1/2,) = -13 and -26.5 mmHg, respectively). Other experiments showed that stretch-inactivated gating was not due to a simple loss of MS channel activity from a non-random process such as vesiculation or bleb formation: channel inactivation by suction was readily reversible, stable over tens of minutes, and followed the predictions of the binomial theorem for independent, randomly gating channels. In addition, the voltage-dependent gating of stretch-inactivated channels was similar to that of stretch

  18. Biophysical Adaptations of Prokaryotic Voltage-Gated Sodium Channels.

    PubMed

    Vien, T N; DeCaen, P G

    2016-01-01

    This chapter describes the adaptive features found in voltage-gated sodium channels (NaVs) of prokaryotes and eukaryotes. These two families are distinct, having diverged early in evolutionary history but maintain a surprising degree of convergence in function. While prokaryotic NaVs are required for growth and motility, eukaryotic NaVs selectively conduct fast electrical currents for short- and long-range signaling across cell membranes in mammalian organs. Current interest in prokaryotic NaVs is stoked by their resolved high-resolution structures and functional features which are reminiscent of eukaryotic NaVs. In this chapter, comparisons between eukaryotic and prokaryotic NaVs are made to highlight the shared and unique aspects of ion selectivity, voltage sensitivity, and pharmacology. Examples of prokaryotic and eukaryotic NaV convergent evolution will be discussed within the context of their structural features. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Voltage-gated sodium channels and metastatic disease.

    PubMed

    Brackenbury, William J

    2012-01-01

    Voltage-gated Na (+) channels (VGSCs) are macromolecular protein complexes containing a pore-forming α subunit and smaller non-pore-forming β subunits. VGSCs are expressed in metastatic cells from a number of cancers. In these cells, Na (+) current carried by α subunits enhances migration, invasion and metastasis in vivo. In contrast, the β subunits mediate cellular adhesion and process extension. The prevailing hypothesis is that VGSCs are upregulated in cancer, in general favoring an invasive/metastatic phenotype, although the mechanisms are still not fully clear. Expression of the Nav 1.5 α subunit associates with poor prognosis in clinical breast cancer specimens, suggesting that VGSCs may have utility as prognostic markers for cancer progression. Furthermore, repurposing existing VGSC-blocking therapeutic drugs may provide a new strategy to improve outcomes in patients suffering from metastatic disease, which is the major cause of cancer-related deaths, and for which there is currently no cure.

  20. Shellfish toxins targeting voltage-gated sodium channels.

    PubMed

    Zhang, Fan; Xu, Xunxun; Li, Tingting; Liu, Zhonghua

    2013-11-28

    Voltage-gated sodium channels (VGSCs) play a central role in the generation and propagation of action potentials in excitable neurons and other cells and are targeted by commonly used local anesthetics, antiarrhythmics, and anticonvulsants. They are also common targets of neurotoxins including shellfish toxins. Shellfish toxins are a variety of toxic secondary metabolites produced by prokaryotic cyanobacteria and eukaryotic dinoflagellates in both marine and fresh water systems, which can accumulate in marine animals via the food chain. Consumption of shellfish toxin-contaminated seafood may result in potentially fatal human shellfish poisoning. This article provides an overview of the structure, bioactivity, and pharmacology of shellfish toxins that act on VGSCs, along with a brief discussion on their pharmaceutical potential for pain management.

  1. Shellfish Toxins Targeting Voltage-Gated Sodium Channels

    PubMed Central

    Zhang, Fan; Xu, Xunxun; Li, Tingting; Liu, Zhonghua

    2013-01-01

    Voltage-gated sodium channels (VGSCs) play a central role in the generation and propagation of action potentials in excitable neurons and other cells and are targeted by commonly used local anesthetics, antiarrhythmics, and anticonvulsants. They are also common targets of neurotoxins including shellfish toxins. Shellfish toxins are a variety of toxic secondary metabolites produced by prokaryotic cyanobacteria and eukaryotic dinoflagellates in both marine and fresh water systems, which can accumulate in marine animals via the food chain. Consumption of shellfish toxin-contaminated seafood may result in potentially fatal human shellfish poisoning. This article provides an overview of the structure, bioactivity, and pharmacology of shellfish toxins that act on VGSCs, along with a brief discussion on their pharmaceutical potential for pain management. PMID:24287955

  2. Congruent pattern of accessibility identifies minimal pore gate in a non-symmetric voltage-gated sodium channel

    PubMed Central

    Oelstrom, Kevin; Chanda, Baron

    2016-01-01

    Opening and closing of the central ion-conducting pore in voltage-dependent ion channels is gated by changes in membrane potential. Although a gate residue in the eukaryotic voltage-gated sodium channel has been identified, the minimal molecular determinants of this gate region remain unknown. Here, by measuring the closed- and open-state reactivity of MTSET to substituted cysteines in all the pore-lining helices, we show that the state-dependent accessibility is delineated by four hydrophobic residues at homologous positions in each domain. Introduced cysteines above these sites do not react with intracellular MTSET while the channels are closed and yet are rapidly modified while the channels are open. These findings, in conjunction with state-dependent metal cross-bridging, support the notion that the gate residues in each of the four S6 segments of the eukaryotic sodium channel form an occlusion for ions in the closed state and are splayed open on activation. PMID:27186888

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

    PubMed Central

    Bodhinathan, Karthik; Slesinger, Paul A.

    2013-01-01

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

  4. Role of extracellular Ca2+ in gating of CaV1.2 channels

    PubMed Central

    Babich, Olga; Isaev, Dmytro; Shirokov, Roman

    2005-01-01

    We examined changes in ionic and gating currents in CaV1.2 channels when extracellular Ca2+ was reduced from 10 mm to 0.1 μm. Saturating gating currents decreased by two-thirds (KD≈ 40 μm) and ionic currents increased 5-fold (KD≈ 0.5 μm) due to increasing Na+ conductance. A biphasic time dependence for the activation of ionic currents was observed at low [Ca2+], which appeared to reflect the rapid activation of channels that were not blocked by Ca2+ and a slower reversal of Ca2+ blockade of the remaining channels. Removal of Ca2+ following inactivation of Ca2+ currents showed that Na+ currents were not affected by Ca2+-dependent inactivation. Ca2+-dependent inactivation also induced a negative shift of the reversal potential for ionic currents suggesting that inactivation alters channel selectivity. Our findings suggest that activation of Ca2+ conductance and Ca2+-dependent inactivation depend on extracellular Ca2+ and are linked to changes in selectivity. PMID:15845581

  5. Structural basis for the coupling between activation and inactivation gates in K+ channels

    PubMed Central

    Cuello, Luis G.; Jogini, Vishwanath; Cortes, D. Marien.; Pan, Albert C; Gagnon, Dominique G.; Dalmas, Olivier; Cordero-Morales, Julio F.; Chakrapani, Sudha; Roux, Benoit; Perozo, Eduardo

    2011-01-01

    The coupled interplay between activation and inactivation gating is a functional hallmark of K+ channels1,2. This coupling has been experimentally demonstrated from ion interaction effects3,4, cysteine accessibility1 and is associated with a well-defined boundary of energetically coupled residues2. The structure of KcsA in its fully open conformation, as well as four other partial openings, richly illustrates the structural basis of activation-inactivation gating5. Here, we have identified the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that as a consequence of the hinge bending and rotation of TM2, the aromatic ring of Phe103 tilts towards residues Thr74 and Thr75 in the pore helix as well as Ile100 in the neighboring subunit. This allows the network of hydrogen bonds among residues W67, E71, and D80 to destabilize the selectivity filter6,7, facilitating entry to its non-conductive conformation. Mutations at position 103, affect gating kinetics in a size-dependent way: small side chain substitutions F103A and F103C severely impair inactivation kinetics, while larger side chains (F103W) have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open state interaction-energies between Phe103 and surrounding residues. Similar interactions were probed in the Shaker K-channel where inactivation was impaired in the mutant I470A. We propose that side chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K channels. PMID:20613845

  6. Target promiscuity and heterogeneous effects of tarantula venom peptides affecting Na+ and K+ ion channels.

    PubMed

    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

    2010-02-05

    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 IC(50) 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.

  7. 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

  8. A molecular framework for temperature-dependent gating of ion channels

    PubMed Central

    Chowdhury, Sandipan; Jarecki, Brian W.; Chanda, Baron

    2014-01-01

    Summary Perception of heat or cold in higher organisms is mediated by specialized ion channels whose gating is exquisitely sensitive to temperature. The physicochemical underpinnings of this temperature-sensitive gating have proven difficult to parse. Here, we took a bottom-up protein design approach, and rationally engineered ion channels to activate in response to thermal stimuli. By varying amino acid polarities at sites undergoing state-dependent changes in solvation, we were able to systematically confer temperature-sensitivity to a canonical voltage-gated ion channel. Our results imply that the specific heat capacity change during channel gating is a major determinant of thermo-sensitive gating. We also show that reduction of gating charges amplifies temperature-sensitivity of designer channels which accounts for low voltage-sensitivity in all known temperature-gated ion channels. These emerging principles suggest a plausible molecular mechanism for temperature-dependent gating that reconcile how ion channels with an overall conserved transmembrane architecture may exhibit a wide range of temperature-sensing phenotypes. PMID:25156949

  9. 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.

  10. Multi-site Phosphorylation of Voltage-Gated Sodium Channel α Subunits from Rat Brain

    PubMed Central

    Berendt, Frank J.; Park, Kang-Sik; Trimmer, James S.

    2010-01-01

    Reversible phosphorylation of ion channels underlies cellular plasticity in mammalian neurons. Voltage-gated sodium or Nav channels underlie action potential initiation and propagation, dendritic excitability, and many other aspects of neuronal excitability. Various protein kinases have been suggested to phosphorylate the primary α subunit of Nav channels, affecting diverse aspects of channel function. Previous studies of Nav α subunit phosphorylation have led to the identification of a small set of phosphorylation sites important in meditating aspects of Nav channel function. Here we use nanoflow liquid chromatography tandem mass spectrometry (nano-LC MS/MS) on Nav α subunits affinity-purified from rat brain with two distinct monoclonal antibodies to identify 15 phosphorylation sites on Nav1.2, 12 of which have not been previously reported. We also found 3 novel phosphorylation sites on Nav1.1. In general, commonly used phosphorylation site prediction algorithms did not accurately predict these novel in vivo phosphorylation sites. Our results demonstrate that specific Nav α subunits isolated from rat brain are highly phosphorylated, and suggest extensive modulation of Nav channel activity in mammalian brain. Identification of phosphorylation sites using monoclonal antibody-based immunopurification and mass spectrometry is an effective approach to define the phosphorylation status of Nav channels and important membrane proteins in mammalian brain. PMID:20131913

  11. Targeting the voltage sensor of Kv7.2 voltage-gated K+ channels with a new gating-modifier.

    PubMed

    Peretz, Asher; Pell, Liat; Gofman, Yana; Haitin, Yoni; Shamgar, Liora; Patrich, Eti; Kornilov, Polina; Gourgy-Hacohen, Orit; Ben-Tal, Nir; Attali, Bernard

    2010-08-31

    The pore and gate regions of voltage-gated cation channels have been often targeted with drugs acting as channel modulators. In contrast, the voltage-sensing domain (VSD) was practically not exploited for therapeutic purposes, although it is the target of various toxins. We recently designed unique diphenylamine carboxylates that are powerful Kv7.2 voltage-gated K(+) channel openers or blockers. Here we show that a unique Kv7.2 channel opener, NH29, acts as a nontoxin gating modifier. NH29 increases Kv7.2 currents, thereby producing a hyperpolarizing shift of the activation curve and slowing both activation and deactivation kinetics. In neurons, the opener depresses evoked spike discharges. NH29 dampens hippocampal glutamate and GABA release, thereby inhibiting excitatory and inhibitory postsynaptic currents. Mutagenesis and modeling data suggest that in Kv7.2, NH29 docks to the external groove formed by the interface of helices S1, S2, and S4 in a way that stabilizes the interaction between two conserved charged residues in S2 and S4, known to interact electrostatically, in the open state of Kv channels. Results indicate that NH29 may operate via a voltage-sensor trapping mechanism similar to that suggested for scorpion and sea-anemone toxins. Reflecting the promiscuous nature of the VSD, NH29 is also a potent blocker of TRPV1 channels, a feature similar to that of tarantula toxins. Our data provide a structural framework for designing unique gating-modifiers targeted to the VSD of voltage-gated cation channels and used for the treatment of hyperexcitability disorders.

  12. Targeting the voltage sensor of Kv7.2 voltage-gated K+ channels with a new gating-modifier

    PubMed Central

    Peretz, Asher; Pell, Liat; Gofman, Yana; Haitin, Yoni; Shamgar, Liora; Patrich, Eti; Kornilov, Polina; Gourgy-Hacohen, Orit; Ben-Tal, Nir; Attali, Bernard

    2010-01-01

    The pore and gate regions of voltage-gated cation channels have been often targeted with drugs acting as channel modulators. In contrast, the voltage-sensing domain (VSD) was practically not exploited for therapeutic purposes, although it is the target of various toxins. We recently designed unique diphenylamine carboxylates that are powerful Kv7.2 voltage-gated K+ channel openers or blockers. Here we show that a unique Kv7.2 channel opener, NH29, acts as a nontoxin gating modifier. NH29 increases Kv7.2 currents, thereby producing a hyperpolarizing shift of the activation curve and slowing both activation and deactivation kinetics. In neurons, the opener depresses evoked spike discharges. NH29 dampens hippocampal glutamate and GABA release, thereby inhibiting excitatory and inhibitory postsynaptic currents. Mutagenesis and modeling data suggest that in Kv7.2, NH29 docks to the external groove formed by the interface of helices S1, S2, and S4 in a way that stabilizes the interaction between two conserved charged residues in S2 and S4, known to interact electrostatically, in the open state of Kv channels. Results indicate that NH29 may operate via a voltage-sensor trapping mechanism similar to that suggested for scorpion and sea-anemone toxins. Reflecting the promiscuous nature of the VSD, NH29 is also a potent blocker of TRPV1 channels, a feature similar to that of tarantula toxins. Our data provide a structural framework for designing unique gating-modifiers targeted to the VSD of voltage-gated cation channels and used for the treatment of hyperexcitability disorders. PMID:20713704

  13. Carvacrol modulates voltage-gated sodium channels kinetics in dorsal root ganglia.

    PubMed

    Joca, Humberto Cavalcante; Vieira, Daiana Cardoso Oliveira; Vasconcelos, Aliny Perreira; Araújo, Demetrius Antônio Machado; Cruz, Jader Santos

    2015-06-05

    Recent studies have shown that many of plant-derived compounds interact with specific ion channels and thereby modulate many sensing mechanisms, such as nociception. The monoterpenoid carvacrol (5-isopropyl-2-methylphenol) has an anti-nociceptive effect related to a reduction in neuronal excitability and voltage-gated Na(+) channels (NaV) inhibition in peripheral neurons. However, the detailed mechanisms of carvacrol-induced inhibition of neuronal NaV remain elusive. This study explores the interaction between carvacrol and NaV in isolated dorsal root ganglia neurons. Carvacrol reduced the total voltage-gated Na(+) current and tetrodotoxin-resistant (TTX-R) Na(+) current component in a concentration-dependent manner. Carvacrol accelerates current inactivation and induced a negative-shift in voltage-dependence of steady-state fast inactivation in total and TTX-R Na(+) current. Furthermore, carvacrol slowed the recovery from inactivation. Carvacrol provoked a leftward shift in both the voltage-dependence of steady-state inactivation and activation of the TTX-R Na(+) current component. In addition, carvacrol-induced inhibition of TTX-R Na(+) current was enhanced by an increase in stimulation frequency and when neurons were pre-conditioned with long depolarization pulse (5s at -50 mV). Taken all results together, we herein demonstrated that carvacrol affects NaV gating properties. The present findings would help to explain the mechanisms underlying the analgesic activity of carvacrol.

  14. BK channel inactivation gates daytime excitability in the circadian clock

    PubMed Central

    Whitt, Joshua P.; Montgomery, Jenna R.; Meredith, Andrea L.

    2016-01-01

    Inactivation is an intrinsic property of several voltage-dependent ion channels, closing the conduction pathway during membrane depolarization and dynamically regulating neuronal activity. BK K+ channels undergo N-type inactivation via their β2 subunit, but the physiological significance is not clear. Here, we report that inactivating BK currents predominate during the day in the suprachiasmatic nucleus, the brain's intrinsic clock circuit, reducing steady-state current levels. At night inactivation is diminished, resulting in larger BK currents. Loss of β2 eliminates inactivation, abolishing the diurnal variation in both BK current magnitude and SCN firing, and disrupting behavioural rhythmicity. Selective restoration of inactivation via the β2 N-terminal ‘ball-and-chain' domain rescues BK current levels and firing rate, unexpectedly contributing to the subthreshold membrane properties that shift SCN neurons into the daytime ‘upstate'. Our study reveals the clock employs inactivation gating as a biophysical switch to set the diurnal variation in suprachiasmatic nucleus excitability that underlies circadian rhythm. PMID:26940770

  15. BK channel inactivation gates daytime excitability in the circadian clock.

    PubMed

    Whitt, Joshua P; Montgomery, Jenna R; Meredith, Andrea L

    2016-03-04

    Inactivation is an intrinsic property of several voltage-dependent ion channels, closing the conduction pathway during membrane depolarization and dynamically regulating neuronal activity. BK K(+) channels undergo N-type inactivation via their β2 subunit, but the physiological significance is not clear. Here, we report that inactivating BK currents predominate during the day in the suprachiasmatic nucleus, the brain's intrinsic clock circuit, reducing steady-state current levels. At night inactivation is diminished, resulting in larger BK currents. Loss of β2 eliminates inactivation, abolishing the diurnal variation in both BK current magnitude and SCN firing, and disrupting behavioural rhythmicity. Selective restoration of inactivation via the β2 N-terminal 'ball-and-chain' domain rescues BK current levels and firing rate, unexpectedly contributing to the subthreshold membrane properties that shift SCN neurons into the daytime 'upstate'. Our study reveals the clock employs inactivation gating as a biophysical switch to set the diurnal variation in suprachiasmatic nucleus excitability that underlies circadian rhythm.

  16. Tectonics of a K⁺ channel: The importance of the N-terminus for channel gating.

    PubMed

    Hoffgaard, F; Kast, S M; Moroni, A; Thiel, G; Hamacher, K

    2015-12-01

    The small K⁺ channel Kcv represents the pore module of complex potassium channels. It was found that its gating can be modified by sensor domains, which are N-terminally coupled to the pore. This implies that the short N-terminus of the channel can transmit conformational changes from upstream sensors to the channel gates. To understand the functional role of the N-terminus in the context of the entire channel protein, we apply combinatorial screening of the mechanical coupling and long-range interactions in the Kcv potassium channel by reduced molecular models. The dynamics and mechanical connections in the channel complex show that the N-terminus is indeed mechanically connected to the pore domain. This includes a long rang coupling to the pore and the inner and outer transmembrane domains. Since the latter domains host the two gates of the channel, the data support the hypothesis that mechanical perturbation of the N-terminus can be transmitted to the channel gates. This effect is solely determined by the topology of the channel; sequence details only have an implicit effect on the coarse-grained dynamics via the fold and not through biochemical details at a smaller scale. This observation has important implications for engineering of synthetic channels on the basis of a K⁺ channel pore. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Signatures of protein structure in the cooperative gating of mechanosensitive ion channels

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Klug, William S.; Haselwandter, Christoph A.

    2014-08-01

    Membrane proteins deform the surrounding lipid bilayer, which can lead to membrane-mediated interactions between neighboring proteins. Using the mechanosensitive channel of large conductance (MscL) as a model system, we demonstrate how the observed differences in protein structure can affect membrane-mediated interactions and cooperativity among membrane proteins. We find that distinct oligomeric states of MscL lead to distinct gateway states for the clustering of MscL, and predict signatures of MscL structure and spatial organization in the cooperative gating of MscL. Our modeling approach establishes a quantitative relation between the observed shapes and the cooperative function of membrane proteins.

  18. Defective cyclic guanosine monophosphate-gated calcium channels and the pathogenesis of psoriasis.

    PubMed

    McKenzie, Roddie C; Oda, Yuko; Szepietowski, Jacek C; Behne, Martin J; Mauro, Theodora

    2003-01-01

    A positive association between intake of calcium channel blockers and psoriasis has been observed recently. Intake of blockers of voltage-gated calcium ion channels is associated with outbreaks of psoriasis after a latent period in patients with and without a previous family history of psoriasis. This suggests that interfering with calcium influx may trigger psoriasis. Calcium influx also occurs via cyclic guanosine monophosphate-gated channels; human keratinocytes contain functional and non-functional (splice variants) versions of these channels. We show here that keratinocytes and skin from psoriatic individuals express higher levels of mRNA encoding a non-functional cyclic guanosine monophosphate-gated calcium channel and that high expression of the splice variant by transfection of cells in culture leads to loss of protein expression for the functional cyclic guanosine monophosphate-gated Ca2+ channels.

  19. Regulation of voltage gated calcium channels by GPCRs and post-translational modification.

    PubMed

    Huang, Junting; Zamponi, Gerald W

    2016-10-18

    Calcium entry via voltage gated calcium channels mediates a wide range of physiological functions, whereas calcium channel dysregulation has been associated with numerous pathophysiological conditions. There are myriad cell signaling pathways that act on voltage gated calcium channels to fine tune their activities and to regulate their cell surface expression. These regulatory mechanisms include the activation of G protein-coupled receptors and downstream phosphorylation events, and their control over calcium channel trafficking through direct physical interactions. Calcium channels also undergo post-translational modifications that alter both function and density of the channels in the plasma membrane. Here we focus on select aspects of these regulatory mechanisms and highlight recent developments.

  20. An evolutionarily-unique heterodimeric voltage-gated cation channel found in aphids

    PubMed Central

    Amey, Joanna S.; O’Reilly, Andrias O.; Burton, Mark J.; Puinean, Alin M.; Mellor, Ian R.; Duce, Ian R.; Field, Linda M.; Wallace, B.A.; Williamson, Martin S.; Davies, T.G. Emyr

    2015-01-01

    We describe the identification in aphids of a unique heterodimeric voltage-gated sodium channel which has an atypical ion selectivity filter and, unusually for insect channels, is highly insensitive to tetrodotoxin. We demonstrate that this channel has most likely arisen by adaptation (gene fission or duplication) of an invertebrate ancestral mono(hetero)meric channel. This is the only identifiable voltage-gated sodium channel homologue in the aphid genome(s), and the channel’s novel selectivity filter motif (DENS instead of the usual DEKA found in other eukaryotes) may result in a loss of sodium selectivity, as indicated experimentally in mutagenised Drosophila channels. PMID:25637326

  1. Bromoenol Lactone Inhibits Voltage-Gated Ca2+ and Transient Receptor Potential Canonical ChannelsS⃞

    PubMed Central

    Chakraborty, Saikat; Berwick, Zachary C.; Bartlett, Paula J.; Kumar, Sanjay; Thomas, Andrew P.; Sturek, Michael; Tune, Johnathan D.

    2011-01-01

    Circulating hormones stimulate the phospholipase Cβ (PLC)/Ca2+ influx pathway to regulate numerous cell functions, including vascular tone. It was proposed previously that Ca2+-independent phospholipase A2 (iPLA2)-dependent store-operated Ca2+ influx channels mediate hormone-induced contractions in isolated arteries, because bromoenol lactone (BEL), a potent irreversible inhibitor of iPLA2, inhibited such contractions. However, the effects of BEL on other channels implicated in mediating hormone-induced vessel contractions, specifically voltage-gated Ca2+ (CaV1.2) and transient receptor potential canonical (TRPC) channels, have not been defined clearly. Using isometric tension measurements, we found that thapsigargin-induced contractions were ∼34% of those evoked by phenylephrine or KCl. BEL completely inhibited not only thapsigargin- but also phenylephrine- and KCl-induced ring contractions, suggesting that CaV1.2 and receptor-operated TRPC channels also may be sensitive to BEL. Therefore, we investigated the effects of BEL on heterologously expressed CaV1.2 and TRPC channels in human embryonic kidney cells, a model system that allows probing of individual protein function without interference from other signaling elements of native cells. We found that low micromolar concentrations of BEL inhibited CaV1.2, TRPC5, TRPC6, and heteromeric TRPC1–TRPC5 channels in an iPLA2-independent manner. BEL also attenuated PLC activity, suggesting that the compound may inhibit TRPC channel activity in part by interfering with an initial PLC-dependent step required for TRPC channel activation. Conversely, BEL did not affect endogenous voltage-gated K+ channels in human embryonic kidney cells. Our findings support the hypothesis that iPLA2-dependent store-operated Ca2+ influx channels and iPLA2-independent hormone-operated TRPC channels can serve as smooth muscle depolarization triggers to activate CaV1.2 channels and to regulate vascular tone. PMID:21795434

  2. 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.

  3. Voltage-gated ion channels in dendrites of hippocampal pyramidal neurons.

    PubMed

    Chen, Xixi; Johnston, Daniel

    2006-12-01

    The properties and distribution of voltage-gated ion channels contribute to electrical signaling in neuronal dendrites. The apical dendrites of CA1 pyramidal neurons in hippocampus express a wide variety of sodium, calcium, potassium, and other voltage-gated channels. In this report, we provide some new evidence for the role of the delayed-rectifier K(+) channel in shaping the dendritic action potential at different membrane potentials.

  4. Molecular simulation studies of hydrophobic gating in nanopores and ion channels.

    PubMed

    Trick, Jemma L; Aryal, Prafulla; Tucker, Stephen J; Sansom, Mark S P

    2015-04-01

    Gating in channels and nanopores plays a key role in regulating flow of ions across membranes. Molecular simulations provide a 'computational microscope' which enables us to examine the physical nature of gating mechanisms at the level of the single channel molecule. Water enclosed within the confines of a nanoscale pore may exhibit unexpected behaviour. In particular, if the molecular surfaces lining the pore are hydrophobic this promotes de-wetting of the pore. De-wetting is observed as stochastic liquid-vapour transitions within a pore, and may lead to functional closure of a pore to the flow of ions and/or water. Such behaviour was first observed in simulations of simple model nanopores and referred to as 'hydrophobic gating'. Simulations of both the nicotinic acetylcholine receptor and of TWIK-1 potassium channels (the latter alongside experimental studies) suggest hydrophobic gating may occur in some biological ion channels. Current studies are focused on designing hydrophobic gates into biomimetic nanopores.

  5. Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels.

    PubMed

    Zeng, Xu-Hui; Xia, Xiao-Ming; Lingle, Christopher J

    2003-06-01

    An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.

  6. Resveratrol attenuates cortical neuron activity: roles of large conductance calcium-activated potassium channels and voltage-gated sodium channels.

    PubMed

    Wang, Ya-Jean; Chan, Ming-Huan; Chen, Linyi; Wu, Sheng-Nan; Chen, Hwei-Hisen

    2016-05-21

    Resveratrol, a phytoalexin found in grapes and red wine, exhibits diverse pharmacological activities. However, relatively little is known about whether resveratrol modulates the ion channels in cortical neurons. The large-conductance calcium-activated potassium channels (BKCa) and voltage-gated sodium channels were expressed in cortical neurons and play important roles in regulation of neuronal excitability. The present study aimed to determine the effects of resveratrol on BKCa currents and voltage-gated sodium currents in cortical neurons. Resveratrol concentration-dependently increased the current amplitude and the opening activity of BKCa channels, but suppressed the amplitude of voltage-gated sodium currents. Similar to the BKCa channel opener NS1619, resveratrol decreased the firing rate of action potentials. In addition, the enhancing effects of BKCa channel blockers tetraethylammonium (TEA) and paxilline on action potential firing were sensitive to resveratrol. Our results indicated that the attenuation of action potential firing rate by resveratrol might be mediated through opening the BKCa channels and closing the voltage-gated sodium channels. As BKCa channels and sodium channels are critical molecular determinants for seizure generation, our findings suggest that regulation of these two channels in cortical neurons probably makes a considerable contribution to the antiseizure activity of resveratrol.

  7. Computer simulation of ion channel gating: the M(2) channel of influenza A virus in a lipid bilayer

    NASA Technical Reports Server (NTRS)

    Schweighofer, K. J.; Pohorille, A.

    2000-01-01

    The transmembrane fragment of the influenza virus M(2) protein forms a homotetrameric channel that transports protons. In this paper, we use molecular dynamics simulations to help elucidate the mechanism of channel gating by four histidines that occlude the channel lumen in the closed state. We test two competing hypotheses. In the "shuttle" mechanism, the delta nitrogen atom on the extracellular side of one histidine is protonated by the incoming proton, and, subsequently, the proton on the epsilon nitrogen atom is released on the opposite side. In the "water-wire" mechanism, the gate opens because of electrostatic repulsion between four simultaneously biprotonated histidines. This allows for proton transport along the water wire that penetrates the gate. For each system, composed of the channel embedded in a hydrated phospholipid bilayer, a 1.3-ns trajectory was obtained. It is found that the states involved in the shuttle mechanism, which contain either single-protonated histidines or a mixture of single-protonated histidines plus one biprotonated residue, are stable during the simulations. Furthermore, the orientations and dynamics of water molecules near the gate are conducive to proton transfer. In contrast, the fully biprotonated state is not stable. Additional simulations show that if only two histidines are biprotonated, the channel deforms but the gate remains closed. These results support the shuttle mechanism but not the gate-opening mechanism of proton gating in M(2).

  8. Functional role of cyclic nucleotide-gated channels in rat medial vestibular nucleus neurons

    PubMed Central

    Podda, Maria Vittoria; D'Ascenzo, Marcello; Leone, Lucia; Piacentini, Roberto; Azzena, Gian Battista; Grassi, Claudio

    2008-01-01

    Although cyclic nucleotide-gated (CNG) channels are expressed in numerous brain areas, little information is available on their functions in CNS neurons. The aim of the present study was to define the distribution of CNG channels in the rat medial vestibular nucleus (MVN) and their possible involvement in regulating MVN neuron (MVNn) excitability. The majority of MVNn expressed both CNG1 and CNG2 A subunits. In whole-cell current-clamp experiments carried out on brainstem slices containing the MVNn, the membrane-permeant analogues of cyclic nucleotides, 8-Br-cGMP and 8-Br-cAMP (1 mm), induced membrane depolarizations (8.9 ± 0.8 and 9.2 ± 1.0 mV, respectively) that were protein kinase independent. The cGMP-induced depolarization was associated with a significant decrease in the membrane input resistance. The effects of cGMP on membrane potential were almost completely abolished by the CNG channel blockers, Cd2+ and l-cis-diltiazem, but they were unaffected by blockade of hyperpolarization-activated cyclic nucleotide-gated channels. In voltage-clamp experiments, 8-Br-cGMP induced non-inactivating inward currents (−22.2 ± 3.9 pA) with an estimated reversal potential near 0 mV, which were markedly inhibited by reduction of extracellular Na+ and Ca2+ concentrations. Membrane depolarization induced by CNG channel activation increased the firing rate of MVNn without changing the action potential shape. Collectively, these findings provide novel evidence that CNG channels affect membrane potential and excitability of MVNn. Such action should have a significant impact on the function of these neurons in sensory–motor integration processes. More generally, it might represent a broad mechanism for regulating the excitability of different CNS neurons. PMID:18048449

  9. Current view on regulation of voltage-gated sodium channels by calcium and auxiliary proteins.

    PubMed

    Pitt, Geoffrey S; Lee, Seok-Yong

    2016-09-01

    In cardiac and skeletal myocytes, and in most neurons, the opening of voltage-gated Na(+) channels (NaV channels) triggers action potentials, a process that is regulated via the interactions of the channels' intercellular C-termini with auxiliary proteins and/or Ca(2+) . The molecular and structural details for how Ca(2+) and/or auxiliary proteins modulate NaV channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca(2+) acts directly upon the NaV channel or through interacting proteins, such as the Ca(2+) binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of NaV intracellular C-termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca(2+) affects NaV function, and how altered Ca(2+) -dependent or FHF-mediated regulation of NaV channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.

  10. Uncoupling charge movement from channel opening in voltage-gated potassium channels by ruthenium complexes.

    PubMed

    Jara-Oseguera, Andrés; Ishida, Itzel G; Rangel-Yescas, Gisela E; Espinosa-Jalapa, Noel; Pérez-Guzmán, José A; Elías-Viñas, David; Le Lagadec, Ronan; Rosenbaum, Tamara; Islas, León D

    2011-05-06

    The Kv2.1 channel generates a delayed-rectifier current in neurons and is responsible for modulation of neuronal spike frequency and membrane repolarization in pancreatic β-cells and cardiomyocytes. As with other tetrameric voltage-activated K(+)-channels, it has been proposed that each of the four Kv2.1 voltage-sensing domains activates independently upon depolarization, leading to a final concerted transition that causes channel opening. The mechanism by which voltage-sensor activation is coupled to the gating of the pore is still not understood. Here we show that the carbon-monoxide releasing molecule 2 (CORM-2) is an allosteric inhibitor of the Kv2.1 channel and that its inhibitory properties derive from the CORM-2 ability to largely reduce the voltage dependence of the opening transition, uncoupling voltage-sensor activation from the concerted opening transition. We additionally demonstrate that CORM-2 modulates Shaker K(+)-channels in a similar manner. Our data suggest that the mechanism of inhibition by CORM-2 may be common to voltage-activated channels and that this compound should be a useful tool for understanding the mechanisms of electromechanical coupling.

  11. Uncoupling Charge Movement from Channel Opening in Voltage-gated Potassium Channels by Ruthenium Complexes*

    PubMed Central

    Jara-Oseguera, Andrés; Ishida, Itzel G.; Rangel-Yescas, Gisela E.; Espinosa-Jalapa, Noel; Pérez-Guzmán, José A.; Elías-Viñas, David; Le Lagadec, Ronan; Rosenbaum, Tamara; Islas, León D.

    2011-01-01

    The Kv2.1 channel generates a delayed-rectifier current in neurons and is responsible for modulation of neuronal spike frequency and membrane repolarization in pancreatic β-cells and cardiomyocytes. As with other tetrameric voltage-activated K+-channels, it has been proposed that each of the four Kv2.1 voltage-sensing domains activates independently upon depolarization, leading to a final concerted transition that causes channel opening. The mechanism by which voltage-sensor activation is coupled to the gating of the pore is still not understood. Here we show that the carbon-monoxide releasing molecule 2 (CORM-2) is an allosteric inhibitor of the Kv2.1 channel and that its inhibitory properties derive from the CORM-2 ability to largely reduce the voltage dependence of the opening transition, uncoupling voltage-sensor activation from the concerted opening transition. We additionally demonstrate that CORM-2 modulates Shaker K+-channels in a similar manner. Our data suggest that the mechanism of inhibition by CORM-2 may be common to voltage-activated channels and that this compound should be a useful tool for understanding the mechanisms of electromechanical coupling. PMID:21454671

  12. Release-dependent feedback inhibition by a presynaptically localized ligand-gated anion channel

    PubMed Central

    Takayanagi-Kiya, Seika; Zhou, Keming; Jin, Yishi

    2016-01-01

    Presynaptic ligand-gated ion channels (LGICs) have long been proposed to affect neurotransmitter release and to tune the neural circuit activity. However, the understanding of their in vivo physiological action remains limited, partly due to the complexity in channel types and scarcity of genetic models. Here we report that C. elegans LGC-46, a member of the Cys-loop acetylcholine (ACh)-gated chloride (ACC) channel family, localizes to presynaptic terminals of cholinergic motor neurons and regulates synaptic vesicle (SV) release kinetics upon evoked release of acetylcholine. Loss of lgc-46 prolongs evoked release, without altering spontaneous activity. Conversely, a gain-of-function mutation of lgc-46 shortens evoked release to reduce synaptic transmission. This inhibition of presynaptic release requires the anion selectivity of LGC-46, and can ameliorate cholinergic over-excitation in a C. elegans model of excitation-inhibition imbalance. These data demonstrate a novel mechanism of presynaptic negative feedback in which an anion-selective LGIC acts as an auto-receptor to inhibit SV release. DOI: http://dx.doi.org/10.7554/eLife.21734.001 PMID:27782882

  13. Mechanosensitive Ion Channels in Bacteria: Functional Domains and Mechanisms of Gating

    NASA Technical Reports Server (NTRS)

    Sukharev, Sergei

    2003-01-01

    The past funding period was productive for the group. The progress in the mechanosensitive channel field was critically affected in the end of 1998 by the solution of the crystal structure of the mycobacterial homolog of MscL by our colleagues from Caltech. Having the structure of TbMscL in the closed state, we developed a detailed homology model of EcoMscL, and related the structural model with the wealth of functional phenomenology available for the E. coli version of the channel (EcoMscL). The biophysical properties of the open MscL helped to model the open conformation and infer the pathway for the entire gating transition. The following experiments provided strong support to the atomic model of the gating process, and allowed to make further predictions. The work has advanced our understanding of tension-driven conformational transitions in membrane-embedded mechanosensory proteins, determine major energetic contributions and set the stage for further exploration of the whole family of mechanosensitive channels. The results have been published in seven experimental and theoretical papers, with three other papers currently in press or in preparation.

  14. 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.

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

    SciTech Connect

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

    2015-09-28

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

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

    DOE PAGES

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

    2015-09-28

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

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

    PubMed Central

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

    2015-01-01

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

  18. The distribution and targeting of neuronal voltage-gated ion channels.

    PubMed

    Lai, Helen C; Jan, Lily Y

    2006-07-01

    Voltage-gated ion channels have to be at the right place in the right number to endow individual neurons with their specific character. Their biophysical properties together with their spatial distribution define the signalling characteristics of a neuron. Improper channel localization could cause communication defects in a neuronal network. This review covers recent studies of mechanisms for targeting voltage-gated ion channels to axons and dendrites, including trafficking, retention and endocytosis pathways for the preferential localization of particular ion channels. We also discuss how the spatial localization of these channels might contribute to the electrical excitability of neurons, and consider the need for future work in this emerging field.

  19. Permeant ion effects on the gating kinetics of the type L potassium channel in mouse lymphocytes

    PubMed Central

    1991-01-01

    Permeant ion species was found to profoundly affect the gating kinetics of type l K+ currents in mouse T lymphocytes studied with the whole- cell or on-cell patch gigaohm-seal techniques. Replacing external K+ with Rb+ (as the sole monovalent cation, at 160 mM) shifted the peak conductance voltage (g-V) relation by approximately 20 mV to more negative potentials, while NH4+ shifted the g-V curve by 15 mV to more positive potentials. Deactivation (the tail current time constant, tau tail) was slowed by an average of 14-fold at -70 mV in external Rb+, by approximately 8-fold in Cs+, and by a factor of two to three in NH4+. Changing the external K+ concentration, [K+]o, from 4.5 to 160 mM or [Rb+]o from 10 to 160 mM had no effect on tau tail. With all the internal K+ replaced by Rb+ or Cs+ and either isotonic Rb+ or K+ in the bath, tau tail was indistinguishable from that with K+ in the cell. With the exception of NH4+, activation time constants were insensitive to permeant ion species. These results indicate that external permeant ions have stronger effects than internal permeant ions, suggesting an external modulatory site that influences K+ channel gating. However, in bi-ionic experiments with reduced external permeant ion concentrations, tau tail was sensitive to the direction of current flow, indicating that the modulatory site is either within the permeation pathway or in the outer vestibule of the channel. The latter interpretation implies that outward current through an open type l K+ channel significantly alters local ion concentrations at the modulatory site in the outer vestibule, and consequently at the mouth of the channel. Experiments with mixtures of K+ and Rb+ in the external solution reveal that deactivation kinetics are minimally affected by addition of Rb+ until the Rb+ mole fraction approaches unity. This relationship between mole fraction and tau tail, together with the concentration independence of tau tail, was hard to reconcile with simple

  20. Localisation of the mechanotransducer channels in mammalian cochlear hair cells provides clues to their gating

    PubMed Central

    Furness, David N; Hackney, Carole M; Evans, Michael G

    2010-01-01

    Our sense of hearing and balance relies on the very rapid gating of mechanotransducer channels known to be located close to the tops of the hair cell stereocilia within the stereociliary bundle. The molecular identity of the channels is unknown but functional aspects such as permeation, block and sensitivity to bundle displacement are well known. The channel has high calcium permeability and this feature has been used in conjunction with fast confocal calcium imaging to unambiguously localise the channels at the top of the two shorter rows of stereocilia in mammalian cochlear hair cells. The data suggest that they are completely absent from the tallest row. It is thought that the structures connecting stereocilia in adjacent rows, the tip links, are either directly responsible for the channel's mechanical gating, or are closely associated with the gating process. The channels must therefore be associated with the bottom part of the tip links and not the top. This feature has important implications for both the channel's gating mechanism and its regulatory adaptation mechanism. The tip link remains an attractive candidate for mechanical coupling between the bundle and the channel or an accessory protein. The localisation of the mechanotransducer channels to the lower end of the tip link represents an important milestone in the journey towards eventual identification of the channel and its gating mechanism. PMID:20026619

  1. Macroscopic kinetics of pentameric ligand gated ion channels: comparisons between two prokaryotic channels and one eukaryotic channel.

    PubMed

    Laha, Kurt T; Ghosh, Borna; Czajkowski, Cynthia

    2013-01-01

    Electrochemical signaling in the brain depends on pentameric ligand-gated ion channels (pLGICs). Recently, crystal structures of prokaryotic pLGIC homologues from Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus (GLIC) in presumed closed and open channel states have been solved, which provide insight into the structural mechanisms underlying channel activation. Although structural studies involving both ELIC and GLIC have become numerous, thorough functional characterizations of these channels are still needed to establish a reliable foundation for comparing kinetic properties. Here, we examined the kinetics of ELIC and GLIC current activation, desensitization, and deactivation and compared them to the GABAA receptor, a prototypic eukaryotic pLGIC. Outside-out patch-clamp recordings were performed with HEK-293T cells expressing ELIC, GLIC, or α1β2γ2L GABAA receptors, and ultra-fast ligand application was used. In response to saturating agonist concentrations, we found both ELIC and GLIC current activation were two to three orders of magnitude slower than GABAA receptor current activation. The prokaryotic channels also had slower current desensitization on a timescale of seconds. ELIC and GLIC current deactivation following 25 s pulses of agonist (cysteamine and pH 4.0 buffer, respectively) were relatively fast with time constants of 24.9 ± 5.1 ms and 1.2 ± 0.2 ms, respectively. Surprisingly, ELIC currents evoked by GABA activated very slowly with a time constant of 1.3 ± 0.3 s and deactivated even slower with a time constant of 4.6 ± 1.2 s. We conclude that the prokaryotic pLGICs undergo similar agonist-mediated gating transitions to open and desensitized states as eukaryotic pLGICs, supporting their use as experimental models. Their uncharacteristic slow activation, slow desensitization and rapid deactivation time courses are likely due to differences in specific structural elements, whose future identification may help uncover mechanisms underlying p

  2. Calcium permeability of ligand-gated Ca2+ channels.

    PubMed

    Pankratov, Yuriy; Lalo, Ulyana

    2014-09-15

    Many of cation-permeable ionotropic receptors to various neurotransmitters, such as glutamate, acetylcholine and ATP, are permeable to Ca(2+) ions. For some of them, in particular NMDA, nicotinic Ach and P2X receptors, permeability to Ca(2+) is higher than permeability to monovalent cations. Such receptors can be viewed as ligand-gated Ca(2+)-channels (LGCCs). This review provides an overview of past works on structure LGCCs, including structural motifs responsible for their interaction with Ca(2+) ions, and functional implications of their Ca(2+)-permeability. The NMDA, P2X and nicotinic Ach receptors are abundantly expressed in the central nervous system. They are present at the nerve terminals, postsynaptic, extrasynaptic and glial membrane and therefore can contribute to synaptic function at different levels. Their heteromeric structure leads to wide variety of LGCC subtypes and great diversity of their functional properties. The influx of Ca(2+) provided by LGCCs can activate a plethora of secondary messenger cascades, which can modulate activity, trafficking and lateral mobility of LGCCs and thereby are entangled with their physiological function. In the discussion of the physiological importance of LGCCs we are focusing on emerging evidence on their role in control of synaptic transmission, plasticity and glia-neuron interaction.

  3. Voltage-gated sodium channel blockers as immunomodulators.

    PubMed

    Roselli, Francesco; Livrea, Paolo; Jirillo, Emilio

    2006-01-01

    Several Voltage-Gated Sodium Channels (VGSC) are widely expressed on lymphocytes and macrophages but their role in immune function is still debated. Nevertheless, Na(+) influx through VGSC is required for lymphocytes activation and proliferation, since these responses are blocked by Na(+)-free medium or by VGSC blockers. These effects may be mediated by the reduced intracellular Na(+) levels, which in turn may impair the activity of Na(+)/Ca(++) exchanger resulting in reduced intracellular Ca(++) levels during lymphocyte activation. Furthermore, in Jurkat cell line VGSC appear to be involved in cell volume regulation, migration in artificial matrix and cell death by apoptosis. VGSC play a role in macrophage function as well, and VGSC blockers impair both phagocytosis and inflammatory responses. Several VGSC blockers have shown immunomodulatory properties in mice models, skewing the immune response toward a Th2-mediated response, while suppressing Th1-mediated responses, and VGSC already used in clinical practice are known to modulate immunoglobulin (Ig) levels both in mice and in humans. These effects suggest that VGSC blockers may find clinical application in the treatment of autoimmune and inflammatory disease. However, many of these drugs induce a number of severe side effects. The relevance of VGSC function in immune regulation suggest that the testing of newly patented VGSC blockers for their effect on immunity may be worthwhile.

  4. Voltage-gated calcium channel autoimmune cerebellar degeneration

    PubMed Central

    McKasson, Marilyn; Clawson, Susan A.; Hill, Kenneth E.; Wood, Blair; Carlson, Noel; Bromberg, Mark; Greenlee, John E.

    2016-01-01

    Objectives: To describe response to treatment in a patient with autoantibodies against voltage-gated calcium channels (VGCCs) who presented with autoimmune cerebellar degeneration and subsequently developed Lambert-Eaton myasthenic syndrome (LEMS), and to study the effect of the patient's autoantibodies on Purkinje cells in rat cerebellar slice cultures. Methods: Case report and study of rat cerebellar slice cultures incubated with patient VGCC autoantibodies. Results: A 53-year-old man developed progressive incoordination with ataxic speech. Laboratory evaluation revealed VGCC autoantibodies without other antineuronal autoantibodies. Whole-body PET scans 6 and 12 months after presentation detected no malignancy. The patient improved significantly with IV immunoglobulin G (IgG), prednisone, and mycophenolate mofetil, but worsened after IV IgG was halted secondary to aseptic meningitis. He subsequently developed weakness with electrodiagnostic evidence of LEMS. The patient's IgG bound to Purkinje cells in rat cerebellar slice cultures, followed by neuronal death. Reactivity of the patient's autoantibodies with VGCCs was confirmed by blocking studies with defined VGCC antibodies. Conclusions: Autoimmune cerebellar degeneration associated with VGCC autoantibodies may precede onset of LEMS and may improve with immunosuppressive treatment. Binding of anti-VGCC antibodies to Purkinje cells in cerebellar slice cultures may be followed by cell death. Patients with anti-VGCC autoantibodies may be at risk of irreversible neurologic injury over time, and treatment should be initiated early. PMID:27088118

  5. Histamine operates Cl -gated channels in crayfish neurosecretory cells.

    PubMed

    Cebada, Jorge; García, Ubaldo

    2007-11-01

    We describe a histamine-activated Cl(-) conductance in the X-organ neurons from crayfish Cherax quadricarinatus, which has comparable properties to the homomultimeric histamine-gated ion channels described in Drosophila. Topical application of histamine inhibited spontaneous neuronal firing in the X-organ sinus gland tract, concomitant with an increase in the membrane conductance. In X-organ neurons in culture and under voltage-clamp conditions, histamine evoked outward currents at -40 mV that reversed at the Cl(-) equilibrium potential. Histamine sensitivity in these neurons had a half-maximal response (EC(50))=3.3+/-1 micromol l(-1), with a Hill number of 2.6+/-0.4. The histamine-evoked current was blocked by tiotidine, cimetidine, ranitidine and 256+/-11 and 483+/-11 micromol l(-1), respectively) and d-tubocurarine (IC(50)=21+/-2 micromol l(-1)), but was insensitive to picrotoxin, bicuculline and strychnine. Neither GABA nor glutamate was capable of desensitizing the histamine response, indicating that histamine activates a particular Cl(-) conductance. The presence of immunoreactive neurons to histamine in the medulla terminalis with axonal projections to the neuropile suggests a possible histaminergic modulation of the X-organ sinus gland system.

  6. Phylogenomics of Ligand-Gated Ion Channels Predicts Monepantel Effect

    PubMed Central

    Rufener, Lucien; Keiser, Jennifer; Kaminsky, Ronald; Mäser, Pascal; Nilsson, Daniel

    2010-01-01

    The recently launched veterinary anthelmintic drench for sheep (Novartis Animal Health Inc., Switzerland) containing the nematocide monepantel represents a new class of anthelmintics: the amino-acetonitrile derivatives (AADs), much needed in view of widespread resistance to the classical drugs. Recently, it was shown that the ACR-23 protein in Caenorhabditis elegans and a homologous protein, MPTL-1 in Haemonchus contortus, are potential targets for AAD action. Both proteins belong to the DEG-3 subfamily of acetylcholine receptors, which are thought to be nematode-specific, and different from those targeted by the imidazothiazoles (e.g. levamisole). Here we provide further evidence that Cel-ACR-23 and Hco-MPTL-1-like subunits are involved in the monepantel-sensitive phenotype. We performed comparative genomics of ligand-gated ion channel genes from several nematodes and subsequently assessed their sensitivity to anthelmintics. The nematode species in the Caenorhabditis genus, equipped with ACR-23/MPTL-1-like receptor subunits, are sensitive to monepantel (EC50<1.25 µM), whereas the related nematodes Pristionchus pacificus and Strongyloides ratti, which lack an ACR-23/MPTL-1 homolog, are insensitive (EC50>43 µM). Genome sequence information has long been used to identify putative targets for therapeutic intervention. We show how comparative genomics can be applied to predict drug sensitivity when molecular targets of a compound are known or suspected. PMID:20838602

  7. Structure of the gating ring from the human large-conductance Ca[superscript 2+]-gated K[superscript +] channel

    SciTech Connect

    Wu, Yunkun; Yang, Yi; Ye, Sheng; Jiang, Youxing

    2010-11-12

    Large-conductance Ca{sup 2+}-gated K{sup +} (BK) channels are essential for many biological processes such as smooth muscle contraction and neurotransmitter release. This group of channels can be activated synergistically by both voltage and intracellular Ca{sup 2+}, with the large carboxy-terminal intracellular portion being responsible for Ca{sup 2+} sensing. Here we present the crystal structure of the entire cytoplasmic region of the human BK channel in a Ca{sup 2+}-free state. The structure reveals four intracellular subunits, each comprising two tandem RCK domains, assembled into a gating ring similar to that seen in the MthK channel and probably representing its physiological assembly. Three Ca{sup 2+} binding sites including the Ca{sup 2+} bowl are mapped onto the structure based on mutagenesis data. The Ca{sup 2+} bowl, located within the second RCK domain, forms an EF-hand-like motif and is strategically positioned close to the assembly interface between two subunits. The other two Ca{sup 2+} (or Mg{sup 2+}) binding sites, Asp367 and Glu374/Glu399, are located on the first RCK domain. The Asp367 site has high Ca{sup 2+} sensitivity and is positioned in the groove between the amino- and carboxy-terminal subdomains of RCK1, whereas the low-affinity Mg{sup 2+}-binding Glu374/Glu399 site is positioned on the upper plateau of the gating ring and close to the membrane. Our structure also contains the linker connecting the transmembrane and intracellular domains, allowing us to dock a voltage-gated K{sup +} channel pore of known structure onto the gating ring with reasonable accuracy and generate a structural model for the full BK channel.

  8. Episodic ataxia type-1 mutations in the hKv1.1 cytoplasmic pore region alter the gating properties of the channel.

    PubMed Central

    D'Adamo, M C; Liu, Z; Adelman, J P; Maylie, J; Pessia, M

    1998-01-01

    Episodic ataxia type-1 is a rare human neurological syndrome which occurs during childhood and persists through the whole life of affected patients. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene hKv1.1 of different affected families. V408A and E325D mutations are located in the cytoplasmic putative pore region of hKv1.1 channels and profoundly alter their gating properties. V408A channels showed increased kinetic rates of activation, deactivation and C-type inactivation. Expression of E325D channels in Xenopus oocytes led to an approximately 13-fold current amplitude reduction and to a 52.4 mV positive shift in the voltage dependence of activation. Moreover, the E325D mutation altered the kinetics of activation, deactivation, C-type inactivation and channel open probability. Heteromeric channels composed of two wild-type and two mutated subunits, linked as dimers, showed gating properties intermediate between channels formed from four normal or four mutated subunits. The results demonstrate that the highly conserved residues Val408 and Glu325 play a pivotal role in several gating processes of a human potassium channel, and suggest a pathogenetic mechanism by which the impairment of the delayed-rectifier function of affected neurons is related to the type and number of mutated subunits which make up the hKv1.1 channels. PMID:9482717

  9. The ladder-shaped polyether toxin gambierol anchors the gating machinery of Kv3.1 channels in the resting state.

    PubMed

    Kopljar, Ivan; Labro, Alain J; de Block, Tessa; Rainier, Jon D; Tytgat, Jan; Snyders, Dirk J

    2013-03-01

    Voltage-gated potassium (Kv) and sodium (Nav) channels are key determinants of cellular excitability and serve as targets of neurotoxins. Most marine ciguatoxins potentiate Nav channels and cause ciguatera seafood poisoning. Several ciguatoxins have also been shown to affect Kv channels, and we showed previously that the ladder-shaped polyether toxin gambierol is a potent Kv channel inhibitor. Most likely, gambierol acts via a lipid-exposed binding site, located outside the K(+) permeation pathway. However, the mechanism by which gambierol inhibits Kv channels remained unknown. Using gating and ionic current analysis to investigate how gambierol affected S6 gate opening and voltage-sensing domain (VSD) movements, we show that the resting (closed) channel conformation forms the high-affinity state for gambierol. The voltage dependence of activation was shifted by >120 mV in the depolarizing direction, precluding channel opening in the physiological voltage range. The (early) transitions between the resting and the open state were monitored with gating currents, and provided evidence that strong depolarizations allowed VSD movement up to the activated-not-open state. However, for transition to the fully open (ion-conducting) state, the toxin first needed to dissociate. These dissociation kinetics were markedly accelerated in the activated-not-open state, presumably because this state displayed a much lower affinity for gambierol. A tetrameric concatemer with only one high-affinity binding site still displayed high toxin sensitivity, suggesting that interaction with a single binding site prevented the concerted step required for channel opening. We propose a mechanism whereby gambierol anchors the channel's gating machinery in the resting state, requiring more work from the VSD to open the channel. This mechanism is quite different from the action of classical gating modifier peptides (e.g., hanatoxin). Therefore, polyether toxins open new opportunities in structure

  10. Open-channel block by internally applied amines inhibits activation gate closure in batrachotoxin-activated sodium channels.

    PubMed Central

    Zamponi, G W; French, R J

    1994-01-01

    We have studied the action of several pore-blocking amines on voltage-dependent activation gating of batrachotoxin(BTX)-activated sodium channels, from bovine heart and rat skeletal muscle, incorporated into planar lipid bilayers. Although structurally simpler, the compounds studied show general structural features and channel-inhibiting actions that resemble those of lidocaine. When applied to the cytoplasmic end of the channel, these compounds cause a rapid, voltage-dependent, open-channel block seen as a reduction in apparent single-channel amplitude (companion paper). Internal application of phenylpropanolamine, phenylethylamine, phenylmethylamine, and diethylamine, as well as causing open-channel block, reduces the probability of channel closure, producing a shift of the steady-state activation curve toward more hyperpolarizing potentials. These gating effects were observed for both cardiac and skeletal muscle channels and were not evoked by addition of equimolar N-Methyl-D-Glucamine, suggesting a specific interaction of the blockers with the channel rather than a surface charge effect. Kinetic analysis of phenylpropanolamine action on skeletal muscle channels indicated that phenylpropanolamine reduced the closed probability via two separate mechanisms. First, mean closed durations were slightly abbreviated in its presence. Second, and more important, the frequency of the gating closures was reduced. This action was correlated with the degree, and the voltage dependence, of open-channel block, suggesting that the activation gate cannot close while the pore is occluded by the blocker. Such a mechanism might underlie the previously reported immobilization of gating charge associated with local anesthetic block of unmodified sodium channels. PMID:7811914

  11. β-Scorpion Toxin Modifies Gating Transitions in All Four Voltage Sensors of the Sodium Channel

    PubMed Central

    Campos, Fabiana V.; Chanda, Baron; Beirão, Paulo S.L.; Bezanilla, Francisco

    2007-01-01

    Several naturally occurring polypeptide neurotoxins target specific sites on the voltage-gated sodium channels. Of these, the gating modifier toxins alter the behavior of the sodium channels by stabilizing transient intermediate states in the channel gating pathway. Here we have used an integrated approach that combines electrophysiological and spectroscopic measurements to determine the structural rearrangements modified by the β-scorpion toxin Ts1. Our data indicate that toxin binding to the channel is restricted to a single binding site on domain II voltage sensor. Analysis of Cole-Moore shifts suggests that the number of closed states in the activation sequence prior to channel opening is reduced in the presence of toxin. Measurements of charge–voltage relationships show that a fraction of the gating charge is immobilized in Ts1-modified channels. Interestingly, the charge–voltage relationship also shows an additional component at hyperpolarized potentials. Site-specific fluorescence measurements indicate that in presence of the toxin the voltage sensor of domain II remains trapped in the activated state. Furthermore, the binding of the toxin potentiates the activation of the other three voltage sensors of the sodium channel to more hyperpolarized potentials. These findings reveal how the binding of β-scorpion toxin modifies channel function and provides insight into early gating transitions of sodium channels. PMID:17698594

  12. An external sodium ion binding site controls allosteric gating in TRPV1 channels

    PubMed Central

    Jara-Oseguera, Andres; Bae, Chanhyung; Swartz, Kenton J

    2016-01-01

    TRPV1 channels in sensory neurons are integrators of painful stimuli and heat, yet how they integrate diverse stimuli and sense temperature remains elusive. Here, we show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation. In studying the underlying mechanism, we find that the temperature sensors in TRPV1 activate in two steps to favor opening, and that the binding of sodium to an extracellular site exerts allosteric control over temperature-sensor activation and opening of the pore. The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium. Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli. DOI: http://dx.doi.org/10.7554/eLife.13356.001 PMID:26882503

  13. Independent and cooperative motions of the Kv1.2 channel: voltage sensing and gating.

    PubMed

    Yeheskel, Adva; Haliloglu, Turkan; Ben-Tal, Nir

    2010-05-19

    Voltage-gated potassium (Kv) channels, such as Kv1.2, are involved in the generation and propagation of action potentials. The Kv channel is a homotetramer, and each monomer is composed of a voltage-sensing domain (VSD) and a pore domain (PD). We analyzed the fluctuations of a model structure of Kv1.2 using elastic network models. The analysis suggested a network of coupled fluctuations of eight rigid structural units and seven hinges that may control the transition between the active and inactive states of the channel. For the most part, the network is composed of amino acids that are known to affect channel activity. The results suggested allosteric interactions and cooperativity between the subunits in the coupling between the motion of the VSD and the selectivity filter of the PD, in accordance with recent empirical data. There are no direct contacts between the VSDs of the four subunits, and the contacts between these and the PDs are loose, suggesting that the VSDs are capable of functioning independently. Indeed, they manifest many inherent fluctuations that are decoupled from the rest of the structure. In general, the analysis suggests that the two domains contribute to the channel function both individually and cooperatively.

  14. The enemy at the gates. Ca2+ entry through TRPM7 channels and anoxic neuronal death.

    PubMed

    Nicotera, Pierluigi; Bano, Daniele

    2003-12-26

    In brain ischemia, gating of postsynaptic glutamate receptors is thought to initiate Ca2+ overload leading to excitotoxic neuronal death. In this issue, Aarts and colleagues describe a novel mechanism, whereby gating of TRPM7, a Ca2+-permeable nonselective cation channel, mediates Ca2+ overload and demise of anoxic neurons.

  15. Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers.

    PubMed

    van der Cruijsen, Elwin A W; Prokofyev, Alexander V; Pongs, Olaf; Baldus, Marc

    2017-01-10

    Ion conduction across the cellular membrane requires the simultaneous opening of activation and inactivation gates of the K(+) channel pore. The bacterial KcsA channel has served as a powerful system for dissecting the structural changes that are related to four major functional states associated with K(+) gating. Yet, the direct observation of the full gating cycle of KcsA has remained structurally elusive, and crystal structures mimicking these gating events require mutations in or stabilization of functionally relevant channel segments. Here, we found that changes in lipid composition strongly increased the KcsA open probability. This enabled us to probe all four major gating states in native-like membranes by combining electrophysiological and solid-state NMR experiments. In contrast to previous crystallographic views, we found that the selectivity filter and turret region, coupled to the surrounding bilayer, were actively involved in channel gating. The increase in overall steady-state open probability was accompanied by a reduction in activation-gate opening, underscoring the important role of the surrounding lipid bilayer in the delicate conformational coupling of the inactivation and activation gates.

  16. Molecular and biophysical basis of glutamate and trace metal modulation of voltage-gated Cav2.3 calcium channels

    PubMed Central

    Vitko, Iuliia; Lazarenko, Roman M.; Orestes, Peihan; Todorovic, Slobodan M.

    2012-01-01

    Here, we describe a new mechanism by which glutamate (Glu) and trace metals reciprocally modulate activity of the Cav2.3 channel by profoundly shifting its voltage-dependent gating. We show that zinc and copper, at physiologically relevant concentrations, occupy an extracellular binding site on the surface of Cav2.3 and hold the threshold for activation of these channels in a depolarized voltage range. Abolishing this binding by chelation or the substitution of key amino acid residues in IS1–IS2 (H111) and IS2–IS3 (H179 and H183) loops potentiates Cav2.3 by shifting the voltage dependence of activation toward more negative membrane potentials. We demonstrate that copper regulates the voltage dependence of Cav2.3 by affecting gating charge movements. Thus, in the presence of copper, gating charges transition into the “ON” position slower, delaying activation and reducing the voltage sensitivity of the channel. Overall, our results suggest a new mechanism by which Glu and trace metals transiently modulate voltage-dependent gating of Cav2.3, potentially affecting synaptic transmission and plasticity in the brain. PMID:22371363

  17. Mechanistic Insights into the Modulation of Voltage-Gated Ion Channels by Inhalational Anesthetics.

    PubMed

    Covarrubias, Manuel; Barber, Annika F; Carnevale, Vincenzo; Treptow, Werner; Eckenhoff, Roderic G

    2015-11-17

    General anesthesia is a relatively safe medical procedure, which for nearly 170 years has allowed life saving surgical interventions in animals and people. However, the molecular mechanism of general anesthesia continues to be a matter of importance and debate. A favored hypothesis proposes that general anesthesia results from direct multisite interactions with multiple and diverse ion channels in the brain. Neurotransmitter-gated ion channels and two-pore K+ channels are key players in the mechanism of anesthesia; however, new studies have also implicated voltage-gated ion channels. Recent biophysical and structural studies of Na+ and K+ channels strongly suggest that halogenated inhalational general anesthetics interact with gates and pore regions of these ion channels to modulate function. Here, we review these studies and provide a perspective to stimulate further advances.

  18. Mechanistic Insights into the Modulation of Voltage-Gated Ion Channels by Inhalational Anesthetics

    PubMed Central

    Covarrubias, Manuel; Barber, Annika F.; Carnevale, Vincenzo; Treptow, Werner; Eckenhoff, Roderic G.

    2015-01-01

    General anesthesia is a relatively safe medical procedure, which for nearly 170 years has allowed life saving surgical interventions in animals and people. However, the molecular mechanism of general anesthesia continues to be a matter of importance and debate. A favored hypothesis proposes that general anesthesia results from direct multisite interactions with multiple and diverse ion channels in the brain. Neurotransmitter-gated ion channels and two-pore K+ channels are key players in the mechanism of anesthesia; however, new studies have also implicated voltage-gated ion channels. Recent biophysical and structural studies of Na+ and K+ channels strongly suggest that halogenated inhalational general anesthetics interact with gates and pore regions of these ion channels to modulate function. Here, we review these studies and provide a perspective to stimulate further advances. PMID:26588560

  19. A dipeptidyl aminopeptidase-like protein remodels gating charge dynamics in Kv4.2 channels.

    PubMed

    Dougherty, Kevin; Covarrubias, Manuel

    2006-12-01

    Dipeptidyl aminopeptidase-like proteins (DPLPs) interact with Kv4 channels and thereby induce a profound remodeling of activation and inactivation gating. DPLPs are constitutive components of the neuronal Kv4 channel complex, and recent observations have suggested the critical functional role of the single transmembrane segment of these proteins (Zagha, E., A. Ozaita, S.Y. Chang, M.S. Nadal, U. Lin, M.J. Saganich, T. McCormack, K.O. Akinsanya, S.Y. Qi, and B. Rudy. 2005. J. Biol. Chem. 280:18853-18861). However, the underlying mechanism of action is unknown. We hypothesized that a unique interaction between the Kv4.2 channel and a DPLP found in brain (DPPX-S) may remodel the channel's voltage-sensing domain. To test this hypothesis, we implemented a robust experimental system to measure Kv4.2 gating currents and study gating charge dynamics in the absence and presence of DPPX-S. The results demonstrated that coexpression of Kv4.2 and DPPX-S causes a -26 mV parallel shift in the gating charge-voltage (Q-V) relationship. This shift is associated with faster outward movements of the gating charge over a broad range of relevant membrane potentials and accelerated gating charge return upon repolarization. In sharp contrast, DPPX-S had no effect on gating charge movements of the Shaker B Kv channel. We propose that DPPX-S destabilizes resting and intermediate states in the voltage-dependent activation pathway, which promotes the outward gating charge movement. The remodeling of gating charge dynamics may involve specific protein-protein interactions of the DPPX-S's transmembrane segment with the voltage-sensing and pore domains of the Kv4.2 channel. This mechanism may determine the characteristic fast operation of neuronal Kv4 channels in the subthreshold range of membrane potentials.

  20. Forcing Open TRP channels: mechanical gating as a unifying activation mechanism

    PubMed Central

    Liu, Chao; Montell, Craig

    2015-01-01

    Transient receptor potential (TRP) proteins are cation channels that comprise a superfamily of molecular sensors that enable animals to detect a wide variety of environmental stimuli. This versatility enables vertebrate and invertebrate TRP channels to function in a diversity of senses, ranging from vision to taste, smell, touch, hearing, proprioception and thermosensation. Moreover, many individual TRP channels are activated through a surprising range of sensory stimuli. The multitasking nature of TRP channels raises the question as to whether seemingly disparate activators gate TRPs through common strategies. In this regard, a recent major advance is the discovery that a phospholipase C (PLC)-dependent signaling cascade activates the TRP channels in Drosophila photoreceptor cells through generation of force in the lipid-bilayer. The premise of this review is that mechanical force is a unifying, common strategy for gating TRP channels. In addition to several TRP channels that function in mechanosensation and are gated by force applied to the cells, changes in temperature and in the concentration of lipophilic second messengers through stimulation of signaling cascades, cause architectural modifications of the cell membrane, which in turn activate TRP channels through mechanical force. Consequently, TRPs are capable of functioning as stretch-activated channels, even in cases in which the stimuli that initiate the signaling cascades are not mechanical. We propose that most TRPs are actually mechanosensitive channels (MSCs), which undergo conformational changes in response to tension imposed on the lipid bilayer, resulting in channel gating. PMID:25998730

  1. 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.

  2. Psychiatric presentation of voltage-gated potassium channel antibody-associated encephalopathy. Case report.

    PubMed

    Parthasarathi, U D; Harrower, T; Tempest, M; Hodges, J R; Walsh, C; McKenna, P J; Fletcher, P C

    2006-08-01

    Voltage-gated potassium channel antibody encephalopathy, a rare cause of limbic encephalopathy, typically presents with memory impairment and seizures. Psychiatric symptoms have not been emphasised in the literature. Here we describe a 58-year-old man who presented with panic attacks and psychogenic non-epileptic seizures and, later on, developed delusions and hallucinations and then confusion. He was found to have antibodies to voltage-gated potassium channels. Treatment with immuno-modulatory therapy resulted in almost complete recovery.

  3. Insight into DEG/ENaC channel gating from genetics and structure.

    PubMed

    Eastwood, Amy L; Goodman, Miriam B

    2012-10-01

    The founding members of the superfamily of DEG/ENaC ion channel proteins are C. elegans proteins that form mechanosensitive channels in touch and pain receptors. For more than a decade, the research community has used mutagenesis to identify motifs that regulate gating. This review integrates insight derived from unbiased in vivo mutagenesis screens with recent crystal structures to develop new models for activation of mechanically gated DEGs.

  4. The ladder-shaped polyether toxin gambierol anchors the gating machinery of Kv3.1 channels in the resting state

    PubMed Central

    Kopljar, Ivan; Labro, Alain J.; de Block, Tessa; Rainier, Jon D.; Tytgat, Jan

    2013-01-01

    Voltage-gated potassium (Kv) and sodium (Nav) channels are key determinants of cellular excitability and serve as targets of neurotoxins. Most marine ciguatoxins potentiate Nav channels and cause ciguatera seafood poisoning. Several ciguatoxins have also been shown to affect Kv channels, and we showed previously that the ladder-shaped polyether toxin gambierol is a potent Kv channel inhibitor. Most likely, gambierol acts via a lipid-exposed binding site, located outside the K+ permeation pathway. However, the mechanism by which gambierol inhibits Kv channels remained unknown. Using gating and ionic current analysis to investigate how gambierol affected S6 gate opening and voltage-sensing domain (VSD) movements, we show that the resting (closed) channel conformation forms the high-affinity state for gambierol. The voltage dependence of activation was shifted by >120 mV in the depolarizing direction, precluding channel opening in the physiological voltage range. The (early) transitions between the resting and the open state were monitored with gating currents, and provided evidence that strong depolarizations allowed VSD movement up to the activated-not-open state. However, for transition to the fully open (ion-conducting) state, the toxin first needed to dissociate. These dissociation kinetics were markedly accelerated in the activated-not-open state, presumably because this state displayed a much lower affinity for gambierol. A tetrameric concatemer with only one high-affinity binding site still displayed high toxin sensitivity, suggesting that interaction with a single binding site prevented the concerted step required for channel opening. We propose a mechanism whereby gambierol anchors the channel’s gating machinery in the resting state, requiring more work from the VSD to open the channel. This mechanism is quite different from the action of classical gating modifier peptides (e.g., hanatoxin). Therefore, polyether toxins open new opportunities in structure

  5. Clustering of the K+ channel GORK of Arabidopsis parallels its gating by extracellular K+.

    PubMed

    Eisenach, Cornelia; Papanatsiou, Maria; Hillert, Ellin-Kristina; Blatt, Michael R

    2014-04-01

    GORK is the only outward-rectifying Kv-like K(+) channel expressed in guard cells. Its activity is tightly regulated to facilitate K(+) efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward-rectifying K(+) channel KAT1. Whereas the population of KAT1 is subject to regulated traffic to and from the plasma membrane, nothing is known about GORK, its distribution and traffic in vivo. We have used transformations with fluorescently-tagged GORK to explore its characteristics in tobacco epidermis and Arabidopsis guard cells. These studies showed that GORK assembles in puncta that reversibly dissociated as a function of the external K(+) concentration. Puncta dissociation parallelled the gating dependence of GORK, the speed of response consistent with the rapidity of channel gating response to changes in the external ionic conditions. Dissociation was also suppressed by the K(+) channel blocker Ba(2+) . By contrast, confocal and protein biochemical analysis failed to uncover substantial exo- and endocytotic traffic of the channel. Gating of GORK is displaced to more positive voltages with external K(+) , a characteristic that ensures the channel facilitates only K(+) efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K(+) above 1 mm. We suggest that GORK clustering in puncta is related to its gating and conductance, and reflects associated conformational changes and (de)stabilisation of the channel protein, possibly as a platform for transmission and coordination of channel gating in response to external K(+) .

  6. STIM1 triggers a gating rearrangement at the extracellular mouth of the ORAI1 channel

    NASA Astrophysics Data System (ADS)

    Gudlur, Aparna; Quintana, Ariel; Zhou, Yubin; Hirve, Nupura; Mahapatra, Sahasransu; Hogan, Patrick G.

    2014-10-01

    The ER-resident regulatory protein STIM1 triggers store-operated Ca2+ entry by direct interaction with the plasma membrane Ca2+ channel ORAI1. The mechanism of channel gating remains undefined. Here we establish that STIM1 gates the purified recombinant ORAI1 channel in vitro, and use Tb3+ luminescence and, separately, disulfide crosslinking to probe movements of the pore-lining helices. We show that interaction of STIM1 with the cytoplasmic face of the human ORAI1 channel elicits a conformational change near the external entrance to the pore, detectable at the pore Ca2+-binding residue E106 and the adjacent pore-lining residue V102. We demonstrate that a short nonpolar segment of the pore including V102 forms a barrier to ion flux in the closed channel, implicating the STIM1-dependent movement in channel gating. Our data explain the close coupling between ORAI1 channel gating and ion selectivity, and open a new avenue to dissect the gating, modulation and inactivation of ORAI-family channels.

  7. Multiple mechanisms underlying rectification in retinal cyclic nucleotide-gated (CNGA1) channels

    PubMed Central

    Arcangeletti, Manuel; Marchesi, Arin; Mazzolini, Monica; Torre, Vincent

    2013-01-01

    In cyclic nucleotide-gated (CNGA1) channels, in the presence of symmetrical ionic conditions, current–voltage (I-V) relationship depends, in a complex way, on the radius of permeating ion. It has been suggested that both the pore and S4 helix contribute to the observed rectification. In the present manuscript, using tail and gating current measurements from homotetrameric CNGA1 channels expressed in Xenopus oocytes, we clarify and quantify the role of the pore and of the S4 helix. We show that in symmetrical Rb+ and Cs+ single-channel current rectification dominates macroscopic currents while voltage-dependent gating becomes larger in symmetrical ethylammonium and dimethylammonium, where the open probability strongly depends on voltage. Isochronal tail currents analysis in dimethylammonium shows that at least two voltage-dependent transitions underlie the observed rectification. Only the first voltage-dependent transition is sensible to mutation of charge residues in the S4 helix. Moreover, analysis of tail and gating currents indicates that the number of elementary charges per channel moving across the membrane is less than 2, when they are about 12 in K+ channels. These results indicate the existence of distinct mechanisms underlying rectification in CNG channels. A restricted motion of the S4 helix together with an inefficient coupling to the channel gate render CNGA1 channels poorly sensitive to voltage in the presence of physiological Na+ and K+. PMID:24400150

  8. Mechanisms of closed-state inactivation in voltage-gated ion channels.

    PubMed

    Bähring, Robert; Covarrubias, Manuel

    2011-02-01

    Inactivation of voltage-gated ion channels is an intrinsic auto-regulatory process necessary to govern the occurrence and shape of action potentials and establish firing patterns in excitable tissues. Inactivation may occur from the open state (open-state inactivation, OSI) at strongly depolarized membrane potentials, or from pre-open closed states (closed-state inactivation, CSI) at hyperpolarized and modestly depolarized membrane potentials. Voltage-gated Na(+), K(+), Ca(2+) and non-selective cationic channels utilize both OSI and CSI. Whereas there are detailed mechanistic descriptions of OSI, much less is known about the molecular basis of CSI. Here, we review evidence for CSI in voltage-gated cationic channels (VGCCs) and recent findings that shed light on the molecular mechanisms of CSI in voltage-gated K(+) (Kv) channels. Particularly, complementary observations suggest that the S4 voltage sensor, the S4S5 linker and the main S6 activation gate are instrumental in the installment of CSI in Kv4 channels. According to this hypothesis, the voltage sensor may adopt a distinct conformation to drive CSI and, depending on the stability of the interactions between the voltage sensor and the pore domain, a closed-inactivated state results from rearrangements in the selectivity filter or failure of the activation gate to open. Kv4 channel CSI may efficiently exploit the dynamics of the subthreshold membrane potential to regulate spiking properties in excitable tissues.

  9. Mechanisms of closed-state inactivation in voltage-gated ion channels

    PubMed Central

    Bähring, Robert; Covarrubias, Manuel

    2011-01-01

    Inactivation of voltage-gated ion channels is an intrinsic auto-regulatory process necessary to govern the occurrence and shape of action potentials and establish firing patterns in excitable tissues. Inactivation may occur from the open state (open-state inactivation, OSI) at strongly depolarized membrane potentials, or from pre-open closed states (closed-state inactivation, CSI) at hyperpolarized and modestly depolarized membrane potentials. Voltage-gated Na+, K+, Ca2+ and non-selective cationic channels utilize both OSI and CSI. Whereas there are detailed mechanistic descriptions of OSI, much less is known about the molecular basis of CSI. Here, we review evidence for CSI in voltage-gated cationic channels (VGCCs) and recent findings that shed light on the molecular mechanisms of CSI in voltage-gated K+ (Kv) channels. Particularly, complementary observations suggest that the S4 voltage sensor, the S4S5 linker and the main S6 activation gate are instrumental in the installment of CSI in Kv4 channels. According to this hypothesis, the voltage sensor may adopt a distinct conformation to drive CSI and, depending on the stability of the interactions between the voltage sensor and the pore domain, a closed-inactivated state results from rearrangements in the selectivity filter or failure of the activation gate to open. Kv4 channel CSI may efficiently exploit the dynamics of the subthreshold membrane potential to regulate spiking properties in excitable tissues. PMID:21098008

  10. New expression profiles of voltage-gated ion channels in arteries exposed to high blood pressure.

    PubMed

    Cox, Robert H; Rusch, Nancy J

    2002-01-01

    The diameters of small arteries and arterioles are tightly regulated by the dynamic interaction between Ca(2+) and K(+) channels in the vascular smooth muscle cells. Calcium influx through voltage-gated Ca(2+) channels induces vasoconstriction, whereas the opening of K(+) channels mediates hyperpolarization, inactivation of voltage-gated Ca(2+) channels, and vasodilation. Three types of voltage-sensitive ion channels have been highly implicated in the regulation of resting vascular tone. These include the L-type Ca(2+) (Ca(L)) channels, voltage-gated K(+) (K(V)) channels, and high-conductance voltage- and Ca(2+)-sensitive K(+) (BK(Ca)) channels. Recently, abnormal expression profiles of these ion channels have been identified as part of the pathogenesis of arterial hypertension and other vasospastic diseases. An increasing number of studies suggest that high blood pressure may trigger cellular signaling cascades that dynamically alter the expression profile of arterial ion channels to further modify vascular tone. This article will briefly review the properties of Ca(L), K(V), and BK(Ca) channels, present evidence that their expression profile is altered during systemic hypertension, and suggest potential mechanisms by which the signal of elevated blood pressure may result in altered ion channel expression. A final section will discuss emerging concepts and opportunities for the development of new vasoactive drugs, which may rely on targeting disease-specific changes in ion channel expression as a mechanism to lower vascular tone during hypertensive diseases.

  11. Allosteric effects of permeating cations on gating currents during K+ channel deactivation.

    PubMed

    Chen, F S; Steele, D; Fedida, D

    1997-08-01

    K+ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K+ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K+ or Cs+, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K+ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs+ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at ECs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K+ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.

  12. Mapping of scorpion toxin receptor sites at voltage-gated sodium channels.

    PubMed

    Gurevitz, Michael

    2012-09-15

    Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Na(v)s) at two pharmacologically distinct sites. Alpha toxins bind at receptor site-3 and inhibit channel inactivation, whereas beta toxins bind at receptor site-4 and shift the voltage-dependent activation toward more hyperpolarizing potentials. The two toxin classes are subdivided to distinct pharmacological groups according to their binding preferences and ability to compete for the receptor sites at Na(v) subtypes. To elucidate the toxin-channel surface of interaction at both receptor sites and clarify the molecular basis of varying toxin preferences, an efficient bacterial system for their expression in recombinant form was established. Mutagenesis accompanied by toxicity, binding and electrophysiological assays, in parallel to determination of the three-dimensional structure using NMR and X-ray crystallography uncovered a bipartite bioactive surface in toxin representatives of all pharmacological groups. Exchange of external loops between the mammalian brain channel rNa(v)1.2a and the insect channel DmNa(v)1 highlighted channel regions involved in the varying sensitivity to assorted toxins. In parallel, thorough mutagenesis of channel external loops illuminated points of putative interaction with the toxins. Amino acid substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain II of rNa(v)1.2a had prominent impact on the activity of the beta-toxin Css4 (from Centruroides suffusus suffusus), and substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain IV affected the activity of the alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus). Rosetta modeling of toxin-Na(v) interaction using the voltage sensor module of the potassium channel as template raises commonalities in the way alpha and beta toxins interact with the channel. Css4 interacts with rNa(v)1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain

  13. Voltage-gated sodium channel β subunits: The power outside the pore in brain development and disease.

    PubMed

    Hull, Jacob M; Isom, Lori L

    2017-09-16

    Voltage gated sodium channels (VGSCs) were first identified in terms of their role in the upstroke of the action potential. The underlying proteins were later identified as saxitoxin and scorpion toxin receptors consisting of α and β subunits. We now know that VGSCs are heterotrimeric complexes consisting of a single pore forming α subunit joined by two β subunits; a noncovalently linked β1 or β3 and a covalently linked β2 or β4 subunit. VGSC α subunits contain all the machinery necessary for channel cell surface expression, ion conduction, voltage sensing, gating, and inactivation, in one central, polytopic, transmembrane protein. VGSC β subunits are more than simple accessories to α subunits. In the more than two decades since the original cloning of β1, our knowledge of their roles in physiology and pathophysiology has expanded immensely. VGSC β subunits are multifunctional. They confer unique gating mechanisms, regulate cellular excitability, affect brain development, confer distinct channel pharmacology, and have functions that are independent of the α subunits. The vast array of functions of these proteins stems from their special station in the channelome: being the only known constituents that are cell adhesion and intra/extracellular signaling molecules in addition to being part of channel complexes. This functional trifecta and how it goes awry demonstrates the power outside the pore in ion channel signaling complexes, broadening the term channelopathy beyond defects in ion conduction. Copyright © 2017. Published by Elsevier Ltd.

  14. Stabilization of the conductive conformation of a voltage-gated K+ (Kv) channel: the lid mechanism.

    PubMed

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

    2013-06-07

    Voltage-gated K(+) (Kv) channels are molecular switches that sense membrane potential and in response open to allow K(+) ions to diffuse out of the cell. In these proteins, sensor and pore belong to two distinct structural modules. We previously showed that the pore module alone is a robust yet dynamic structural unit in lipid membranes and that it senses potential and gates open to conduct K(+) with unchanged fidelity. The implication is that the voltage sensitivity of K(+) channels is not solely encoded in the sensor. Given that the coupling between sensor and pore remains elusive, we asked whether it is then possible to convert a pore module characterized by brief openings into a conductor with a prolonged lifetime in the open state. The strategy involves selected probes targeted to the filter gate of the channel aiming to modulate the probability of the channel being open assayed by single channel recordings from the sensorless pore module reconstituted in lipid bilayers. Here we show that the premature closing of the pore is bypassed by association of the filter gate with two novel open conformation stabilizers: an antidepressant and a peptide toxin known to act selectively on Kv channels. Such stabilization of the conductive conformation of the channel is faithfully mimicked by the covalent attachment of fluorescein at a cysteine residue selectively introduced near the filter gate. This modulation prolongs the occupancy of permeant ions at the gate. It is this longer embrace between ion and gate that we conjecture underlies the observed stabilization of the conductive conformation. This study provides a new way of thinking about gating.

  15. Efficient gate control of spin-valve signals and Hanle signals in GaAs channel with p-i-n junction-type back-gate structure

    NASA Astrophysics Data System (ADS)

    Miyakawa, Takumi; Akiho, Takafumi; Ebina, Yuya; Yamamoto, Masafumi; Uemura, Tetsuya

    2016-02-01

    Efficient gate control of spin-valve signals and Hanle signals was achieved in a GaAs channel with a p-i-n back-gate structure. Experiments showed that the amplitude of the spin-valve signal (ΔVNL) under constant-injection-current conditions increased for a cross nonlocal geometry when the channel was depleted by the gate voltage (VG). In contrast, the VG dependence of ΔVNL for a nonlocal geometry was complicated. The gate modulation efficiency of spin signals was approximately 50 times that with a graphene or Si channel.

  16. Kv3 voltage-gated potassium channels regulate neurotransmitter release from mouse motor nerve terminals.

    PubMed

    Brooke, Ruth E; Moores, Thomas S; Morris, Neil P; Parson, Simon H; Deuchars, Jim

    2004-12-01

    Voltage-gated potassium (Kv) channels are critical to regulation of neurotransmitter release throughout the nervous system but the roles and identity of the subtypes involved remain unclear. Here we show that Kv3 channels regulate transmitter release at the mouse neuromuscular junction (NMJ). Light- and electron-microscopic immunohistochemistry revealed Kv3.3 and Kv3.4 subunits within all motor nerve terminals of muscles examined [transversus abdominus, lumbrical and flexor digitorum brevis (FDB)]. To determine the roles of these Kv3 subunits, intracellular recordings were made of end-plate potentials (EPPs) in FDB muscle fibres evoked by electrical stimulation of tibial nerve. Tetraethylammonium (TEA) applied at low concentrations (0.05-0.5 mM), which blocks only a few known potassium channels including Kv3 channels, did not affect muscle fibre resting potential but significantly increased the amplitude of all EPPs tested. Significantly, this effect of TEA was still observed in the presence of the large-conductance calcium-activated potassium channel blockers iberiotoxin (25-150 nM) and Penitrem A (100 nM), suggesting a selective action on Kv3 subunits. Consistent with this, 15-microM 4-aminopyridine, which blocks Kv3 but not large-conductance calcium-activated potassium channels, enhanced evoked EPP amplitude. Unexpectedly, blood-depressing substance-I, a toxin selective for Kv3.4 subunits, had no effect at 0.05-1 microM. The combined presynaptic localization of Kv3 subunits and pharmacological enhancement of EPP amplitude indicate that Kv3 channels regulate neurotransmitter release from presynaptic terminals at the NMJ.

  17. Dynamics transitions at the outer vestibule of the KcsA potassium channel during gating.

    PubMed

    Raghuraman, H; Islam, Shahidul M; Mukherjee, Soumi; Roux, Benoit; Perozo, Eduardo

    2014-02-04

    In K(+) channels, the selectivity filter, pore helix, and outer vestibule play a crucial role in gating mechanisms. The outer vestibule is an important structurally extended region of KcsA in which toxins, blockers, and metal ions bind and modulate the gating behavior of K(+) channels. Despite its functional significance, the gating-related structural dynamics at the outer vestibule are not well understood. Under steady-state conditions, inactivating WT and noninactivating E71A KcsA stabilize the nonconductive and conductive filter conformations upon opening the activation gate. Site-directed fluorescence polarization of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled outer vestibule residues shows that the outer vestibule of open/conductive conformation is highly dynamic compared with the motional restriction experienced by the outer vestibule during inactivation gating. A wavelength-selective fluorescence approach shows a change in hydration dynamics in inactivated and noninactivated conformations, and supports a possible role of restricted/bound water molecules in C-type inactivation gating. Using a unique restrained ensemble simulation method, along with distance measurements by EPR, we show that, on average, the outer vestibule undergoes a modest backbone conformational change during its transition to various functional states, although the structural dynamics of the outer vestibule are significantly altered during activation and inactivation gating. Taken together, our results support the role of a hydrogen bond network behind the selectivity filter, side-chain conformational dynamics, and water molecules in the gating mechanisms of K(+) channels.

  18. Bubble gating in biological ion channels: A density functional theory study

    NASA Astrophysics Data System (ADS)

    Gußmann, Florian; Roth, Roland

    2017-06-01

    We study the behavior of a waterlike liquid inside the gate of a biological ion channel following the basic geometry of the well studied potassium channel KcsA. We calculate the three-dimensional density distribution ρ (r ) of the liquid within the framework of classical density functional theory and observe the formation of a low density region (bubble) when the gate is narrow. This observation corresponds to a finite-size form of capillary evaporation and supports the so-called bubble-gate theory. From the density profile we also compute the energy landscape of the gate and the energy required to change the gate from a closed (narrow) to an open (wide) state and vice versa.

  19. The Interaction of Na+ and K+ in Voltage-gated Potassium Channels

    PubMed Central

    Kiss, Laszlo; Immke, David; LoTurco, Joseph; Korn, Stephen J.

    1998-01-01

    Voltage-gated potassium (K+) channels are multi-ion pores. Recent studies suggest that, similar to calcium channels, competition between ionic species for intrapore binding sites may contribute to ionic selectivity in at least some K+ channels. Molecular studies suggest that a putative constricted region of the pore, which is presumably the site of selectivity, may be as short as one ionic diameter in length. Taken together, these results suggest that selectivity may occur at just a single binding site in the pore. We are studying a chimeric K+ channel that is highly selective for K+ over Na+ in physiological solutions, but conducts Na+ in the absence of K+. Na+ and K+ currents both display slow (C-type) inactivation, but had markedly different inactivation and deactivation kinetics; Na+ currents inactivated more rapidly and deactivated more slowly than K+ currents. Currents carried by 160 mM Na+ were inhibited by external K+ with an apparent IC50 <30 μM. K+ also altered both inactivation and deactivation kinetics of Na+ currents at these low concentrations. In the complementary experiment, currents carried by 3 mM K+ were inhibited by external Na+, with an apparent IC50 of ∼100 mM. In contrast to the effects of low [K+] on Na+ current kinetics, Na+ did not affect K+ current kinetics, even at concentrations that inhibited K+ currents by 40–50%. These data suggest that Na+ block of K+ currents did not involve displacement of K+ from the high affinity site involved in gating kinetics. We present a model that describes the permeation pathway as a single high affinity, cation-selective binding site, flanked by low affinity, nonselective sites. This model quantitatively predicts the anomalous mole fraction behavior observed in two different K+ channels, differential K+ and Na+ conductance, and the concentration dependence of K+ block of Na+ currents and Na+ block of K+ currents. Based on our results, we hypothesize that the permeation pathway contains a single high

  20. Molecular mapping of general anesthetic sites in a voltage-gated ion channel.

    PubMed

    Barber, Annika F; Liang, Qiansheng; Amaral, Cristiano; Treptow, Werner; Covarrubias, Manuel

    2011-10-05

    Several voltage-gated ion channels are modulated by clinically relevant doses of general anesthetics. However, the structural basis of this modulation is not well understood. Previous work suggested that n-alcohols and inhaled anesthetics stabilize the closed state of the Shaw2 voltage-gated (Kv) channel (K-Shaw2) by directly interacting with a discrete channel site. We hypothesize that the inhibition of K-Shaw2 channels by general anesthetics is governed by interactions between binding and effector sites involving components of the channel's activation gate. To investigate this hypothesis, we applied Ala/Val scanning mutagenesis to the S4-S5 linker and the post-PVP S6 segment, and conducted electrophysiological analysis to evaluate the energetic impact of the mutations on the inhibition of the K-Shaw2 channel by 1-butanol and halothane. These analyses identified residues that determine an apparent binding cooperativity and residue pairs that act in concert to modulate gating upon anesthetic binding. In some instances, due to their critical location, key residues also influence channel gating. Complementing these results, molecular dynamics simulations and in silico docking experiments helped us visualize possible anesthetic sites and interactions. We conclude that the inhibition of K-Shaw2 by general anesthetics results from allosteric interactions between distinct but contiguous binding and effector sites involving inter- and intrasubunit interfaces.

  1. Energetic Contributions to Channel Gating of Residues in the Muscle Nicotinic Receptor β1 Subunit

    PubMed Central

    Akk, Gustav; Eaton, Megan; Li, Ping; Zheng, Steven; Lo, Joshua; Steinbach, Joe Henry

    2013-01-01

    In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit. PMID:24194945

  2. The sorting receptor Rer1 controls Purkinje cell function via voltage gated sodium channels

    PubMed Central

    Valkova, Christina; Liebmann, Lutz; Krämer, Andreas; Hübner, Christian A.; Kaether, Christoph

    2017-01-01

    Rer1 is a sorting receptor in the early secretory pathway that controls the assembly and the cell surface transport of selected multimeric membrane protein complexes. Mice with a Purkinje cell (PC) specific deletion of Rer1 showed normal polarization and differentiation of PCs and normal development of the cerebellum. However, PC-specific loss of Rer1 led to age-dependent motor deficits in beam walk, ladder climbing and gait. Analysis of brain sections revealed a specific degeneration of PCs in the anterior cerebellar lobe in old animals. Electrophysiological recordings demonstrated severe deficits in spontaneous action potential generation. Measurements of resurgent currents indicated decreased surface densities of voltage-gated sodium channels (Nav), but not changes in individual channels. Analysis of mice with a whole brain Rer1-deletion demonstrated a strong down-regulation of Nav1.6 and 1.1 in the absence of Rer1, whereas protein levels of the related Cav2.1 and of Kv3.3 and 7.2 channels were not affected. The data suggest that Rer1 controls the assembly and transport of Nav1.1 and 1.6, the principal sodium channels responsible for recurrent firing, in PCs. PMID:28117367

  3. Effects of stochastic channel gating and distribution on the cardiac action potential.

    PubMed

    Lemay, Mathieu; de Lange, Enno; Kucera, Jan P

    2011-07-21

    Ion channels exhibit stochastic conformational changes determining their gating behavior. In addition, the process of protein turnover leads to a natural variability of the number of membrane and gap junctional channels. Nevertheless, in computational models, these two aspects are scarcely considered and their impacts are largely unknown. We investigated the effects of stochastic current fluctuations and channel distributions on action potential duration (APD), intercellular conduction delays (ICDs) and conduction blocks using a modified ventricular cell model (Rudy et al.) with Markovian formulations of the principal ion currents (to simulate their stochastic open-close gating behavior) and with channel counts drawn from Poisson distributions (to simulate their natural variability). In single cells, APD variability (coefficient of variation: 1.6% at BCL=1000ms) was essentially caused by stochastic channel gating of I(Ks), persistent I(Na) and I(Ca,L). In cell strands, ICD variability induced by stochastic channel gating and Poissonian channel distributions was low under normal conditions. Nonetheless, at low intercellular coupling levels, Poissonian gap junctional channel distribution resulted in a large ICD variability (coefficient of variation >20%), highly heterogeneous conduction patterns and conduction blocks. Therefore, the stochastic behavior of current fluctuations and channel distributions can contribute to the heterogeneity of conduction patterns and to conduction block, as observed previously in experiments in cardiac tissue with altered intercellular coupling.

  4. Investigations of the contribution of a putative glycine hinge to ryanodine receptor channel gating.

    PubMed

    Euden, Joanne; Mason, Sammy A; Viero, Cedric; Thomas, N Lowri; Williams, Alan J

    2013-06-07

    Ryanodine receptor channels (RyR) are key components of striated muscle excitation-contraction coupling, and alterations in their function underlie both inherited and acquired disease. A full understanding of the disease process will require a detailed knowledge of the mechanisms and structures involved in RyR function. Unfortunately, high-resolution structural data, such as exist for K(+)-selective channels, are not available for RyR. In the absence of these data, we have used modeling to identify similarities in the structural elements of K(+) channel pore-forming regions and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining transmembrane helix of RyR (G(4864)LIIDA(4869) in RyR2) analogous to the glycine hinge motif present in many K(+) channels. Gating in these K(+) channels can be disrupted by substitution of residues for the hinge glycine. We investigated the involvement of glycine 4864 in RyR2 gating by monitoring properties of recombinant human RyR2 channels in which this glycine is replaced by residues that alter gating in K(+) channels. Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function. In contrast, function is altered when glycine 4864 is replaced by either valine or proline, the former preventing channel opening and the latter modifying both ion translocation and gating. Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K(+) channels. Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.

  5. Stochastic Ion Channel Gating in Dendritic Neurons: Morphology Dependence and Probabilistic Synaptic Activation of Dendritic Spikes

    PubMed Central

    Nolan, Matthew F.

    2010-01-01

    Neuronal activity is mediated through changes in the probability of stochastic transitions between open and closed states of ion channels. While differences in morphology define neuronal cell types and may underlie neurological disorders, very little is known about influences of stochastic ion channel gating in neurons with complex morphology. We introduce and validate new computational tools that enable efficient generation and simulation of models containing stochastic ion channels distributed across dendritic and axonal membranes. Comparison of five morphologically distinct neuronal cell types reveals that when all simulated neurons contain identical densities of stochastic ion channels, the amplitude of stochastic membrane potential fluctuations differs between cell types and depends on sub-cellular location. For typical neurons, the amplitude of membrane potential fluctuations depends on channel kinetics as well as open probability. Using a detailed model of a hippocampal CA1 pyramidal neuron, we show that when intrinsic ion channels gate stochastically, the probability of initiation of dendritic or somatic spikes by dendritic synaptic input varies continuously between zero and one, whereas when ion channels gate deterministically, the probability is either zero or one. At physiological firing rates, stochastic gating of dendritic ion channels almost completely accounts for probabilistic somatic and dendritic spikes generated by the fully stochastic model. These results suggest that the consequences of stochastic ion channel gating differ globally between neuronal cell-types and locally between neuronal compartments. Whereas dendritic neurons are often assumed to behave deterministically, our simulations suggest that a direct consequence of stochastic gating of intrinsic ion channels is that spike output may instead be a probabilistic function of patterns of synaptic input to dendrites. PMID:20711353

  6. Cav3 T-type channels: regulators for gating, membrane expression, and cation selectivity.

    PubMed

    Senatore, A; Guan, W; Spafford, J D

    2014-04-01

    Cav3 T-type channels are low-voltage-gated channels with rapid kinetics that are classified among the calcium-selective Cav1 and Cav2 type channels. Here, we outline the fundamental and unique regulators of T-type channels. An ubiquitous and proximally located "gating brake" works in concert with the voltage-sensor domain and S6 alpha-helical segment from domain II to set the canonical low-threshold and transient gating features of T-type channels. Gene splicing of optional exon 25c (and/or exon 26) in the short III-IV linker provides a developmental switch between modes of activity, such as activating in response to membrane depolarization, to channels requiring hyperpolarization input before being available to activate. Downstream of the gating brake in the I-II linker is a key region for regulating channel expression where alternative splicing patterns correlate with functional diversity of spike patterns, pacemaking rate (especially in the heart), stage of development, and animal size. A small but persistent window conductance depolarizes cells and boosts excitability at rest. T-type channels possess an ion selectivity that can resemble not only the calcium ion exclusive Cav1 and Cav2 channels but also the sodium ion selectivity of Nav1 sodium channels too. Alternative splicing in the extracellular turret of domain II generates highly sodium-permeable channels, which contribute to low-threshold sodium spikes. Cav3 channels are more ubiquitous among multicellular animals and more widespread in tissues than the more brain centric Nav1 sodium channels in invertebrates. Highly sodium-permeant Cav3 channels can functionally replace Nav1 channels in species where they are lacking, such as in Caenorhabditis elegans.

  7. H bonding at the helix-bundle crossing controls gating in Kir potassium channels.

    PubMed

    Rapedius, Markus; Fowler, Philip W; Shang, Lijun; Sansom, Mark S P; Tucker, Stephen J; Baukrowitz, Thomas

    2007-08-16

    Specific stimuli such as intracellular H+ and phosphoinositides (e.g., PIP2) gate inwardly rectifying potassium (Kir) channels by controlling the reversible transition between the closed and open states. This gating mechanism underlies many aspects of Kir channel physiology and pathophysiology; however, its structural basis is not well understood. Here, we demonstrate that H+ and PIP2 use a conserved gating mechanism defined by similar structural changes in the transmembrane (TM) helices and the selectivity filter. Our data support a model in which the gating motion of the TM helices is controlled by an intrasubunit hydrogen bond between TM1 and TM2 at the helix-bundle crossing, and we show that this defines a common gating motif in the Kir channel superfamily. Furthermore, we show that this proposed H-bonding interaction determines Kir channel pH sensitivity, pH and PIP2 gating kinetics, as well as a K+-dependent inactivation process at the selectivity filter and therefore many of the key regulatory mechanisms of Kir channel physiology.

  8. Open channel structure of MscL and the gating mechanism of mechanosensitive channels

    NASA Astrophysics Data System (ADS)

    Perozo, Eduardo; Cortes, D. Marien; Sompornpisut, Pornthep; Kloda, Anna; Martinac, Boris

    2002-08-01

    Mechanosensitive channels act as membrane-embedded mechano-electrical switches, opening a large water-filled pore in response to lipid bilayer deformations. This process is critical to the response of living organisms to direct physical stimulation, such as in touch, hearing and osmoregulation. Here, we have determined the structural rearrangements that underlie these events in the large prokaryotic mechanosensitive channel (MscL) using electron paramagnetic resonance spectroscopy and site-directed spin labelling. MscL was trapped in both the open and in an intermediate closed state by modulating bilayer morphology. Transition to the intermediate state is characterized by small movements in the first transmembrane helix (TM1). Subsequent transitions to the open state are accompanied by massive rearrangements in both TM1 and TM2, as shown by large increases in probe dynamics, solvent accessibility and the elimination of all intersubunit spin-spin interactions. The open state is highly dynamic, supporting a water-filled pore of at least 25Å, lined mostly by TM1. These structures suggest a plausible molecular mechanism of gating in mechanosensitive channels.

  9. The voltage-dependent gate in MthK potassium channels is located at the selectivity filter

    PubMed Central

    Posson, David J.; McCoy, Jason G.; Nimigean, Crina M.

    2012-01-01

    Understanding how ion channels open and close their pores is crucial for understanding their physiological roles. We used intracellular quaternary ammonium blockers to locate the voltage-dependent gate in MthK potassium channels from Methanobacterium thermoautotrophicum with electrophysiology and X-ray crystallography. Blockers bind in an aqueous cavity between two putative gates, an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location: an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding. A kinetic analysis of tetrabutylammonium block of single MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutylantimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggests that MthK inactivation leads to conformational changes within the cavity and intracellular pore entrance. PMID:23262489

  10. The voltage-dependent gate in MthK potassium channels is located at the selectivity filter.

    PubMed

    Posson, David J; McCoy, Jason G; Nimigean, Crina M

    2013-02-01

    Understanding how ion channels open and close their pores is crucial for comprehending their physiological roles. We used intracellular quaternary ammonium blockers, electrophysiology and X-ray crystallography to locate the voltage-dependent gate in MthK potassium channels from Methanobacterium thermoautotrophicum. Blockers bind in an aqueous cavity between two putative gates: an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location--an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding. Kinetic analysis of tetrabutylammonium block of single MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutyl antimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggest that MthK inactivation leads to conformational changes within the cavity and intracellular pore entrance.

  11. Further characterization of the effect of ethanol on voltage-gated Ca2+ channel function in developing CA3 hippocampal pyramidal neurons

    PubMed Central

    Morton, Russell A.; Valenzuela, C. Fernando

    2015-01-01

    Developmental ethanol exposure damages the hippocampus, a brain region involved in learning and memory. Alterations in synaptic transmission and plasticity may play a role in this effect of ethanol. We previously reported that acute and repeated exposure to ethanol during the 3rd trimester-equivalent inhibits long-term potentiation of GABAA receptor-dependent synaptic currents in CA3 pyramidal neurons through a mechanism that depends on retrograde release of brain-derived neurotrophic factor driven by activation of voltage-gated Ca2+ channels (Zucca and Valenzuela, 2010). We found evidence indicating that voltage-gated Ca2+ channels are inhibited in the presence of ethanol, an effect that may play a role in its mechanism of action. Here, we further investigated the acute effect of ethanol on the function of voltage-gated Ca2+ channels in CA3 pyramidal neurons using Ca2+ imaging techniques. These experiments revealed that acute ethanol exposure inhibits voltage-gated Ca2+ channels both in somatic and proximal dendritic compartments. To investigate the long-term consequences of ethanol on voltage-gated Ca2+ channels, we used patch-clamp electrophysiological techniques to assess the function of L-type voltage-gated Ca2+ channels during and following ten days of vapor ethanol exposure. During ethanol withdrawal periods, the function of these channels was not significantly affected by vapor chamber exposure. Taken together with our previous findings, our results suggest that 3rd trimester-equivalent ethanol exposure transiently inhibits L-type voltage-gated Ca2+ channel function in CA3 pyramidal neurons and that compensatory mechanisms restore their function during ethanol withdrawal. Transient inhibition of these channels by ethanol may be, in part, responsible for the hippocampal abnormalities associated with developmental exposure to this agent. PMID:26711851

  12. An evolutionarily-unique heterodimeric voltage-gated cation channel found in aphids.

    PubMed

    Amey, Joanna S; O'Reilly, Andrias O; Burton, Mark J; Puinean, Alin M; Mellor, Ian R; Duce, Ian R; Field, Linda M; Wallace, B A; Williamson, Martin S; Davies, T G Emyr

    2015-02-27

    We describe the identification in aphids of a unique heterodimeric voltage-gated sodium channel which has an atypical ion selectivity filter and, unusually for insect channels, is highly insensitive to tetrodotoxin. We demonstrate that this channel has most likely arisen by adaptation (gene fission or duplication) of an invertebrate ancestral mono(hetero)meric channel. This is the only identifiable voltage-gated sodium channel homologue in the aphid genome(s), and the channel's novel selectivity filter motif (DENS instead of the usual DEKA found in other eukaryotes) may result in a loss of sodium selectivity, as indicated experimentally in mutagenised Drosophila channels. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  13. The KCNQ1 channel – remarkable flexibility in gating allows for functional versatility

    PubMed Central

    Liin, Sara I; Barro-Soria, Rene; Larsson, H Peter

    2015-01-01

    The KCNQ1 channel (also called Kv7.1 or KvLQT1) belongs to the superfamily of voltage-gated K+ (Kv) channels. KCNQ1 shares several general features with other Kv channels but also displays a fascinating flexibility in terms of the mechanism of channel gating, which allows KCNQ1 to play different physiological roles in different tissues. This flexibility allows KCNQ1 channels to function as voltage-independent channels in epithelial tissues, whereas KCNQ1 function as voltage-activated channels with very slow kinetics in cardiac tissues. This flexibility is in part provided by the association of KCNQ1 with different accessory KCNE β-subunits and different modulators, but also seems like an integral part of KCNQ1 itself. The aim of this review is to describe the main mechanisms underlying KCNQ1 flexibility. PMID:25653179

  14. The role of voltage-gated potassium channels in the regulation of mouse uterine contractility.

    PubMed

    Smith, Ryan C; McClure, Marisa C; Smith, Margaret A; Abel, Peter W; Bradley, Michael E

    2007-11-02

    Uterine smooth muscle cells exhibit ionic currents that appear to be important in the control of uterine contractility, but how these currents might produce the changes in contractile activity seen in pregnant myometrium has not been established. There are conflicting reports concerning the role of voltage-gated potassium (Kv) channels and large-conductance, calcium-activated potassium (BK) channels in the regulation of uterine contractility. In this study we provide molecular and functional evidence for a role for Kv channels in the regulation of spontaneous contractile activity in mouse myometrium, and also demonstrate a change in Kv channel regulation of contractility in pregnant mouse myometrium. Functional assays which evaluated the effects of channel blockers and various contractile agonists were accomplished by quantifying contractility of isolated uterine smooth muscle obtained from nonpregnant mice as well as mice at various stages of pregnancy. Expression of Kv channel proteins in isolated uterine smooth muscle was evaluated by Western blots. The Kv channel blocker 4-aminopyridine (4-AP) caused contractions in nonpregnant mouse myometrium (EC50 = 54 micromolar, maximal effect at 300 micromolar) but this effect disappeared in pregnant mice; similarly, the Kv4.2/Kv4.3 blocker phrixotoxin-2 caused contractions in nonpregnant, but not pregnant, myometrium. Contractile responses to 4-AP were not dependent upon nerves, as neither tetrodotoxin nor storage of tissues at room temperature significantly altered these responses, nor were responses dependent upon the presence of the endometrium. Spontaneous contractions and contractions in response to 4-AP did not appear to be mediated by BK, as the BK channel-selective blockers iberiotoxin, verruculogen, or tetraethylammonium failed to affect either spontaneous contractions or 4-AP-elicited responses. A number of different Kv channel alpha subunit proteins were found in isolated myometrium from both nonpregnant and

  15. The role of voltage-gated potassium channels in the regulation of mouse uterine contractility

    PubMed Central

    Smith, Ryan C; McClure, Marisa C; Smith, Margaret A; Abel, Peter W; Bradley, Michael E

    2007-01-01

    Background Uterine smooth muscle cells exhibit ionic currents that appear to be important in the control of uterine contractility, but how these currents might produce the changes in contractile activity seen in pregnant myometrium has not been established. There are conflicting reports concerning the role of voltage-gated potassium (Kv) channels and large-conductance, calcium-activated potassium (BK) channels in the regulation of uterine contractility. In this study we provide molecular and functional evidence for a role for Kv channels in the regulation of spontaneous contractile activity in mouse myometrium, and also demonstrate a change in Kv channel regulation of contractility in pregnant mouse myometrium. Methods Functional assays which evaluated the effects of channel blockers and various contractile agonists were accomplished by quantifying contractility of isolated uterine smooth muscle obtained from nonpregnant mice as well as mice at various stages of pregnancy. Expression of Kv channel proteins in isolated uterine smooth muscle was evaluated by Western blots. Results The Kv channel blocker 4-aminopyridine (4-AP) caused contractions in nonpregnant mouse myometrium (EC50 = 54 micromolar, maximal effect at 300 micromolar) but this effect disappeared in pregnant mice; similarly, the Kv4.2/Kv4.3 blocker phrixotoxin-2 caused contractions in nonpregnant, but not pregnant, myometrium. Contractile responses to 4-AP were not dependent upon nerves, as neither tetrodotoxin nor storage of tissues at room temperature significantly altered these responses, nor were responses dependent upon the presence of the endometrium. Spontaneous contractions and contractions in response to 4-AP did not appear to be mediated by BK, as the BK channel-selective blockers iberiotoxin, verruculogen, or tetraethylammonium failed to affect either spontaneous contractions or 4-AP-elicited responses. A number of different Kv channel alpha subunit proteins were found in isolated myometrium

  16. Artificial Modulation of the Gating Behavior of a K+ Channel in a KvAP-DNA Chimera

    PubMed Central

    Wang, Andrew; Zocchi, Giovanni

    2011-01-01

    We present experiments where the gating behavior of a voltage-gated ion channel is modulated by artificial ligand binding. We construct a channel-DNA chimera with the KvAP potassium channel reconstituted in an artificial membrane. The channel is functional and the single channel ion conductivity unperturbed by the presence of the DNA. However, the channel opening probability vs. bias voltage, i.e., the gating, can be shifted considerably by the electrostatic force between the charges on the DNA and the voltage sensing domain of the protein. Different hybridization states of the chimera DNA thus lead to different response curves of the channel. PMID:21526187

  17. 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.

  18. 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

  19. The pore structure and gating mechanism of K2P channels

    PubMed Central

    Piechotta, Paula L; Rapedius, Markus; Stansfeld, Phillip J; Bollepalli, Murali K; Erhlich, Gunter; Andres-Enguix, Isabelle; Fritzenschaft, Hariolf; Decher, Niels; Sansom, Mark S P; Tucker, Stephen J; Baukrowitz, Thomas

    2011-01-01

    Two-pore domain (K2P) potassium channels are important regulators of cellular electrical excitability. However, the structure of these channels and their gating mechanism, in particular the role of the bundle-crossing gate, are not well understood. Here, we report that quaternary ammonium (QA) ions bind with high-affinity deep within the pore of TREK-1 and have free access to their binding site before channel activation by intracellular pH or pressure. This demonstrates that, unlike most other K+ channels, the bundle-crossing gate in this K2P channel is constitutively open. Furthermore, we used QA ions to probe the pore structure of TREK-1 by systematic scanning mutagenesis and comparison of these results with different possible structural models. This revealed that the TREK-1 pore most closely resembles the open-state structure of KvAP. We also found that mutations close to the selectivity filter and the nature of the permeant ion profoundly influence TREK-1 channel gating. These results demonstrate that the primary activation mechanisms in TREK-1 reside close to, or within the selectivity filter and do not involve gating at the cytoplasmic bundle crossing. PMID:21822218

  20. Kinetic effects of quaternary lidocaine block of cardiac sodium channels: a gating current study

    PubMed Central

    1994-01-01

    The interaction of antiarrhythmic drugs with ion channels is often described within the context of the modulated receptor hypothesis, which explains the action of drugs by proposing that the binding site has a variable affinity for drugs, depending upon whether the channel is closed, open, or inactivated. Lack of direct evidence for altered gating of cardiac Na channels allowed for the suggestion of an alternative model for drug interaction with cardiac channels, which postulated a fixed affinity receptor with access limited by the conformation of the channel (guarded receptor hypothesis). We report measurement of the gating currents of Na channels in canine cardiac Purkinje cells in the absence and presence of QX-222, a quaternary derivative of lidocaine, applied intracellularly, and benzocaine, a neutral local anesthetic. These data demonstrate that the cardiac Na channel behaves as a modulated rather than a guarded receptor in that drug-bound channels gate with altered kinetics. In addition, the results suggest a new interpretation of the modulated receptor hypothesis whereby drug occupancy reduces the overall voltage- dependence of gating, preventing full movement of the voltage sensor. PMID:8169596

  1. Gate-controlled conductance enhancement from quantum Hall channels along graphene p–n junctions

    PubMed Central

    Tóvári, Endre; Makk, Péter; Liu, Ming-Hao; Rickhaus, Peter; Kovács-Krausz, Zoltán; Richter, Klaus; Schönenberger, Christian

    2016-01-01

    The formation of quantum Hall channels inside the bulk of graphene is studied using various contact and gate geometries. p–n junctions are created along the longitudinal direction of samples, and enhanced conductance is observed in the case of bipolar doping due to the new conducting channels formed in the bulk, whose position, propagating direction and, in one geometry, coupling to electrodes are determined by the gate-controlled filling factor across the device. This effect could be exploited to probe the behavior and interaction of quantum Hall channels protected against uncontrolled scattering at the edges. PMID:27878177

  2. The cytoplasmic coiled-coil mediates cooperative gating temperature sensitivity in the voltage-gated H(+) channel Hv1.

    PubMed

    Fujiwara, Yuichiro; Kurokawa, Tatsuki; Takeshita, Kohei; Kobayashi, Megumi; Okochi, Yoshifumi; Nakagawa, Atsushi; Okamura, Yasushi

    2012-05-08

    Hv1/VSOP is a dimeric voltage-gated H(+) channel in which the gating of one subunit is reportedly coupled to that of the other subunit within the dimer. The molecular basis for dimer formation and intersubunit coupling, however, remains unknown. Here we show that the carboxy terminus ends downstream of the S4 voltage-sensor helix twist in a dimer coiled-coil architecture, which mediates cooperative gating. We also show that the temperature-dependent activation of H(+) current through Hv1/VSOP is regulated by thermostability of the coiled-coil domain, and that this regulation is altered by mutation of the linker between S4 and the coiled-coil. Cooperative gating within the dimer is also dependent on the linker structure, which circular dichroism spectrum analysis suggests is α-helical. Our results indicate that the cytoplasmic coiled-coil strands form continuous α-helices with S4 and mediate cooperative gating to adjust the range of temperatures over which Hv1/VSOP operates.

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

    SciTech Connect

    Du Yuzhe; Song Weizhong; Groome, James R.; Nomura, Yoshiko; Luo Ningguang; Dong Ke

    2010-08-15

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

  4. Cooperative Nature of Gating Transitions in K+ Channels as seen from Dynamic Importance Sampling Calculations

    PubMed Central

    Denning, Elizabeth J.; Woolf, Thomas B.

    2009-01-01

    The growing dataset of K+ channel x-ray structures provides an excellent opportunity to begin a detailed molecular understanding of voltage-dependent gating. These structures, while differing in sequence, represent either a stable open or closed state. However, an understanding of the molecular details of gating will require models for the transitions and experimentally testable predictions for the gating transition. To explore these ideas, we apply Dynamic Importance Sampling (DIMS) to a set of homology models for the molecular conformations of K+ channels for four different sets of sequences and eight different states. In our results, we highlight the importance of particular residues upstream from the PVP region to the gating transition. This supports growing evidence that the PVP region is important for influencing the flexibility of the S6 helix and thus the opening of the gating domain. The results further suggest how gating on the molecular level depends on intra-subunit motions to influence the cooperative behavior of all four subunits of the K+ channel. We hypothesize that the gating process occurs in steps: first sidechain movement, then inter- S5-S6 subunit motions, and lastly the large-scale domain rearrangements. PMID:19950367

  5. The voltage-sensing domain of a phosphatase gates the pore of a potassium channel.

    PubMed

    Arrigoni, Cristina; Schroeder, Indra; Romani, Giulia; Van Etten, James L; Thiel, Gerhard; Moroni, Anna

    2013-03-01

    The modular architecture of voltage-gated K(+) (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates Kv(Synth1), a functional voltage-gated, outwardly rectifying K(+) channel. Kv(Synth1) displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V(1/2) = +56 mV; z of ~1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.

  6. The hitchhiker’s guide to the voltage-gated sodium channel galaxy

    PubMed Central

    2016-01-01

    Eukaryotic voltage-gated sodium (Nav) channels contribute to the rising phase of action potentials and served as an early muse for biophysicists laying the foundation for our current understanding of electrical signaling. Given their central role in electrical excitability, it is not surprising that (a) inherited mutations in genes encoding for Nav channels and their accessory subunits have been linked to excitability disorders in brain, muscle, and heart; and (b) Nav channels are targeted by various drugs and naturally occurring toxins. Although the overall architecture and behavior of these channels are likely to be similar to the more well-studied voltage-gated potassium channels, eukaryotic Nav channels lack structural and functional symmetry, a notable difference that has implications for gating and selectivity. Activation of voltage-sensing modules of the first three domains in Nav channels is sufficient to open the channel pore, whereas movement of the domain IV voltage sensor is correlated with inactivation. Also, structure–function studies of eukaryotic Nav channels show that a set of amino acids in the selectivity filter, referred to as DEKA locus, is essential for Na+ selectivity. Structures of prokaryotic Nav channels have also shed new light on mechanisms of drug block. These structures exhibit lateral fenestrations that are large enough to allow drugs or lipophilic molecules to gain access into the inner vestibule, suggesting that this might be the passage for drug entry into a closed channel. In this Review, we will synthesize our current understanding of Nav channel gating mechanisms, ion selectivity and permeation, and modulation by therapeutics and toxins in light of the new structures of the prokaryotic Nav channels that, for the time being, serve as structural models of their eukaryotic counterparts. PMID:26712848

  7. The hitchhiker's guide to the voltage-gated sodium channel galaxy.

    PubMed

    Ahern, Christopher A; Payandeh, Jian; Bosmans, Frank; Chanda, Baron

    2016-01-01

    Eukaryotic voltage-gated sodium (Nav) channels contribute to the rising phase of action potentials and served as an early muse for biophysicists laying the foundation for our current understanding of electrical signaling. Given their central role in electrical excitability, it is not surprising that (a) inherited mutations in genes encoding for Nav channels and their accessory subunits have been linked to excitability disorders in brain, muscle, and heart; and (b) Nav channels are targeted by various drugs and naturally occurring toxins. Although the overall architecture and behavior of these channels are likely to be similar to the more well-studied voltage-gated potassium channels, eukaryotic Nav channels lack structural and functional symmetry, a notable difference that has implications for gating and selectivity. Activation of voltage-sensing modules of the first three domains in Nav channels is sufficient to open the channel pore, whereas movement of the domain IV voltage sensor is correlated with inactivation. Also, structure-function studies of eukaryotic Nav channels show that a set of amino acids in the selectivity filter, referred to as DEKA locus, is essential for Na(+) selectivity. Structures of prokaryotic Nav channels have also shed new light on mechanisms of drug block. These structures exhibit lateral fenestrations that are large enough to allow drugs or lipophilic molecules to gain access into the inner vestibule, suggesting that this might be the passage for drug entry into a closed channel. In this Review, we will synthesize our current understanding of Nav channel gating mechanisms, ion selectivity and permeation, and modulation by therapeutics and toxins in light of the new structures of the prokaryotic Nav channels that, for the time being, serve as structural models of their eukaryotic counterparts.

  8. Modulation of NMDA channel gating by Ca2+ and Cd2+ binding to the external pore mouth

    PubMed Central

    Tu, Ya-Chi; Yang, Ya-Chin; Kuo, Chung-Chin

    2016-01-01

    NMDA receptor channels are characterized by high Ca2+ permeability. It remains unclear whether extracellular Ca2+ could directly modulate channel gating and control Ca2+ influxes. We demonstrate a pore-blocking site external to the activation gate for extracellular Ca2+ and Cd2+, which has the same charge and radius as Ca2+ but is impermeable to the channel. The apparent affinity of Cd2+ or Ca2+ is higher toward the activated (a steady-state mixture of the open and desensitized, probably chiefly the latter) than the closed states. The blocking effect of Cd2+ is well correlated with the number of charges in the DRPEER motif at the external pore mouth, with coupling coefficients close to 1 in double mutant cycle analyses. The effect of Ca2+ and especially Cd2+ could be allosterically affected by T647A mutation located just inside the activation gate. A prominent “hook” also develops after wash-off of Cd2+ or Ca2+, suggesting faster unbinding rates of Cd2+ and Ca2+ with the mutation. We conclude that extracellular Ca2+ or Cd2+ directly binds to the DRPEER motif to modify NMDA channel activation (opening as well as desensitization), which seems to involve essential regional conformational changes centered at the bundle crossing point A652 (GluN1)/A651(GluN2). PMID:27848984

  9. Utrophin regulates modal gating of mechanosensitive ion channels in dystrophic skeletal muscle

    PubMed Central

    Tan, Nhi; Lansman, Jeffry B

    2014-01-01

    Dystrophin is a large, submembrane cytoskeletal protein, absence of which causes Duchenne muscular dystrophy. Utrophin is a dystrophin homologue found in both muscle and brain whose physiological function is unknown. Recordings of single-channel activity were made from membrane patches on skeletal muscle from mdx, mdx/utrn+/– heterozygotes and mdx/utrn–/– double knockout mice to investigate the role of these cytoskeletal proteins in mechanosensitive (MS) channel gating. We find complex, gene dose-dependent effects of utrophin depletion in dystrophin-deficient mdx muscle: (1) increased MS channel open probability, (2) a shift of MS channel gating to larger pressures, (3) appearance of modal gating of MS channels and small conductance channels and (4) expression of large conductance MS channels. We suggest a physical model in which utrophin acts as a scaffolding protein that stabilizes lipid microdomains and clusters MS channel subunits. Depletion of utrophin disrupts domain composition in a manner that favours open channel area expansion, as well as allowing diffusion and aggregation of additional MS channel subunits. PMID:24879867

  10. Utrophin regulates modal gating of mechanosensitive ion channels in dystrophic skeletal muscle.

    PubMed

    Tan, Nhi; Lansman, Jeffry B

    2014-08-01

    Dystrophin is a large, submembrane cytoskeletal protein, absence of which causes Duchenne muscular dystrophy. Utrophin is a dystrophin homologue found in both muscle and brain whose physiological function is unknown. Recordings of single-channel activity were made from membrane patches on skeletal muscle from mdx, mdx/utrn(+/-) heterozygotes and mdx/utrn(-/-) double knockout mice to investigate the role of these cytoskeletal proteins in mechanosensitive (MS) channel gating. We find complex, gene dose-dependent effects of utrophin depletion in dystrophin-deficient mdx muscle: (1) increased MS channel open probability, (2) a shift of MS channel gating to larger pressures, (3) appearance of modal gating of MS channels and small conductance channels and (4) expression of large conductance MS channels. We suggest a physical model in which utrophin acts as a scaffolding protein that stabilizes lipid microdomains and clusters MS channel subunits. Depletion of utrophin disrupts domain composition in a manner that favours open channel area expansion, as well as allowing diffusion and aggregation of additional MS channel subunits.

  11. Nonequilibrium gating and voltage dependence of the ClC-0 Cl- channel

    PubMed Central

    1996-01-01

    The gating of ClC-0, the voltage-dependent Cl- channel from Torpedo electric organ, is strongly influenced by Cl- ions in the external solution. Raising external Cl- over the range 1-600 mM favors the fast- gating open state and disfavors the slow-gating inactivated state. Analysis of purified single ClC-0 channels reconstituted into planar lipid bilayers was used to identify the role of Cl- ions in the channel's fast voltage-dependent gating process. External, but not internal, Cl- had a major effect on the channel's opening rate constant. The closing rate was more sensitive to internal Cl- than to external Cl-. Both opening and closing rates varied with voltage. A model was derived that postulates (a) that in the channel's closed state, Cl- is accessible to a site located at the outer end of the conduction pore, where it binds in a voltage-independent fashion, (b) that this closed conformation can open, whether liganded by Cl- or not, in a weakly voltage-dependent fashion, (c) that the Cl(-)-liganded closed channel undergoes a conformational change to a different closed state, such that concomitant with this change, Cl- ion moves inward, conferring voltage-dependence to this step, and (d) that this new Cl(-)- liganded closed state opens with a very high rate. According to this picture, Cl- movement within the pre-open channel is the major source of voltage dependence, and charge movement intrinsic to the channel protein contributes very little to voltage-dependent gating of ClC-0. Moreover, since the Cl- activation site is probably located in the ion conduction pathway, the fast gating of ClC-0 is necessarily coupled to ion conduction, a nonequilibrium process. PMID:8894974

  12. The Validation of Nematode-Specific Acetylcholine-Gated Chloride Channels as Potential Anthelmintic Drug Targets

    PubMed Central

    Wever, Claudia M.; Farrington, Danielle; Dent, Joseph A.

    2015-01-01

    New compounds are needed to treat parasitic nematode infections in humans, livestock and plants. Small molecule anthelmintics are the primary means of nematode parasite control in animals; however, widespread resistance to the currently available drug classes means control will be impossible without the introduction of new compounds. Adverse environmental effects associated with nematocides used to control plant parasitic species are also motivating the search for safer, more effective compounds. Discovery of new anthelmintic drugs in particular has been a serious challenge due to the difficulty of obtaining and culturing target parasites for high-throughput screens and the lack of functional genomic techniques to validate potential drug targets in these pathogens. We present here a novel strategy for target validation that employs the free-living nematode Caenorhabditis elegans to demonstrate the value of new ligand-gated ion channels as targets for anthelmintic discovery. Many successful anthelmintics, including ivermectin, levamisole and monepantel, are agonists of pentameric ligand-gated ion channels, suggesting that the unexploited pentameric ion channels encoded in parasite genomes may be suitable drug targets. We validated five members of the nematode-specific family of acetylcholine-gated chloride channels as targets of agonists with anthelmintic properties by ectopically expressing an ivermectin-gated chloride channel, AVR-15, in tissues that endogenously express the acetylcholine-gated chloride channels and using the effects of ivermectin to predict the effects of an acetylcholine-gated chloride channel agonist. In principle, our strategy can be applied to validate any ion channel as a putative anti-parasitic drug target. PMID:26393923

  13. Time-dependent molecular memory in single voltage-gated sodium channel.

    PubMed

    Nayak, Tapan K; Sikdar, S K

    2007-10-01

    Excitability in neurons is associated with firing of action potentials and requires the opening of voltage-gated sodium channels with membrane depolarization. Sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, can have profound implications on the biophysical properties of voltage-gated ion channels. Therefore, we sought to characterize the effect of sustained membrane depolarization on single voltage-gated Na+ channels. Single-channel activity was recorded in the cell-attached patch-clamp mode from the rNa(v)1.2 alpha channels expressed in CHO cells. Classical statistical analysis revealed complex nonlinear changes in channel dwell times and unitary conductance of single Na+ channels as a function of conditioning membrane depolarization. Signal processing tools like weighted wavelet Z (WWZ) and discrete Fourier transform analyses attributed a "pseudo-oscillatory" nature to the observed nonlinear variation in the kinetic parameters. Modeling studies using the hidden Markov model (HMM) illustrated significant changes in kinetic states and underlying state transition rate constants upon conditioning depolarization. Our results suggest that sustained membrane depolarization induces novel nonlinear properties in voltage-gated Na+ channels. Prolonged membrane depolarization also induced a "molecular memory" phenomenon, characterized by clusters of dwell time events and strong autocorrelation in the dwell time series similar to that reported recently for single enzyme molecules. The persistence of such molecular memory was found to be dependent on the duration of depolarization. Voltage-gated Na+ channel with the observed time-dependent nonlinear properties and the molecular memory phenomenon may determine the functional state of the channel and, in turn, the excitability of a neuron.

  14. Calcium Channels in Planar Lipid Bilayers: Insights into Mechanisms of Ion Permeation and Gating

    NASA Astrophysics Data System (ADS)

    Rosenberg, Robert L.; Hess, Peter; Reeves, John P.; Smilowitz, Henry; Tsien, Richard W.

    1986-03-01

    Electrophysiological recordings were used to analyze single calcium channels in planar lipid bilayers after membranes from bovine cardiac sarcolemmal vesicles had been incorporated into the bilayer. In these cell-free conditions, channels in the bilayer showed unitary barium or calcium conductances, gating kinetics, and pharmacological responses that were similar to dihydropyridine-sensitive calcium channels in intact cells. The open channel current varied in a nonlinear manner with voltage under asymmetric (that is, physiological) ionic conditions. However, with identical solutions on both sides of the bilayer, the current-voltage relation was linear. In matched experiments, calcium channels from skeletal muscle T-tubules differed significantly from cardiac calcium channels in their conductance properties and gating kinetics.

  15. 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.

  16. Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

    PubMed Central

    Abenavoli, Alessandra; DiFrancesco, Mattia Lorenzo; Schroeder, Indra; Epimashko, Svetlana; Gazzarrini, Sabrina; Hansen, Ulf Peter; Thiel, Gerhard

    2009-01-01

    Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K+ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K+ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K+, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current. PMID:19720961

  17. NMR structure of inactivation gates from mammalian voltage-dependent potassium channels.

    PubMed

    Antz, C; Geyer, M; Fakler, B; Schott, M K; Guy, H R; Frank, R; Ruppersberg, J P; Kalbitzer, H R

    1997-01-16

    The electrical signalling properties of neurons originate largely from the gating properties of their ion channels. N-type inactivation of voltage-gated potassium (Kv) channels is the best-understood gating transition in ion channels, and occurs by a 'ball-and-chain' type mechanism. In this mechanism an N-terminal domain (inactivation gate), which is tethered to the cytoplasmic side of the channel protein by a protease-cleavable chain, binds to its receptor at the inner vestibule of the channel, thereby physically blocking the pore. Even when synthesized as a peptide, ball domains restore inactivation in Kv channels whose inactivation domains have been deleted. Using high-resolution nuclear magnetic resonance (NMR) spectroscopy, we analysed the three-dimensional structure of the ball peptides from two rapidly inactivating mammalian K. channels (Raw3 (Kv3.4) and RCK4 (Kv1.4)). The inactivation peptide of Raw3 (Raw3-IP) has a compact structure that exposes two phosphorylation sites and allows the formation of an intramolecular disulphide bridge between two spatially close cysteine residues. Raw3-IP exhibits a characteristic surface charge pattern with a positively charged, a hydrophobic, and a negatively charged region. The RCK4 inactivation peptide (RCK4-IP) shows a similar spatial distribution of charged and uncharged regions, but is more flexible and less ordered in its amino-terminal part.

  18. Voltage‐gated calcium channels and their auxiliary subunits: physiology and pathophysiology and pharmacology

    PubMed Central

    2016-01-01

    Abstract Voltage‐gated calcium channels are essential players in many physiological processes in excitable cells. There are three main subdivisions of calcium channel, defined by the pore‐forming α1 subunit, the CaV1, CaV2 and CaV3 channels. For all the subtypes of voltage‐gated calcium channel, their gating properties are key for the precise control of neurotransmitter release, muscle contraction and cell excitability, among many other processes. For the CaV1 and CaV2 channels, their ability to reach their required destinations in the cell membrane, their activation and the fine tuning of their biophysical properties are all dramatically influenced by the auxiliary subunits that associate with them. Furthermore, there are many diseases, both genetic and acquired, involving voltage‐gated calcium channels. This review will provide a general introduction and then concentrate particularly on the role of auxiliary α2δ subunits in both physiological and pathological processes involving calcium channels, and as a therapeutic target. PMID:27273705

  19. A Change in the Ion Selectivity of Ligand-Gated Ion Channels Provides a Mechanism to Switch Behavior

    PubMed Central

    Alkema, Mark J.

    2015-01-01

    Behavioral output of neural networks depends on a delicate balance between excitatory and inhibitory synaptic connections. However, it is not known whether network formation and stability is constrained by the sign of synaptic connections between neurons within the network. Here we show that switching the sign of a synapse within a neural circuit can reverse the behavioral output. The inhibitory tyramine-gated chloride channel, LGC-55, induces head relaxation and inhibits forward locomotion during the Caenorhabditis elegans escape response. We switched the ion selectivity of an inhibitory LGC-55 anion channel to an excitatory LGC-55 cation channel. The engineered cation channel is properly trafficked in the native neural circuit and results in behavioral responses that are opposite to those produced by activation of the LGC-55 anion channel. Our findings indicate that switches in ion selectivity of ligand-gated ion channels (LGICs) do not affect network connectivity or stability and may provide an evolutionary and a synthetic mechanism to change behavior. PMID:26348462

  20. Gating modifier toxin interactions with ion channels and lipid bilayers: Is the trimolecular complex real?

    PubMed

    Agwa, Akello J; Henriques, Sónia T; Schroeder, Christina I

    2017-04-08

    Spider peptide toxins have attracted attention because of their ability to target voltage-gated ion channels, which are involved in several pathologies including chronic pain and some cardiovascular conditions. A class of these peptides acts by modulating the gating mechanism of voltage-gated ion channels and are thus called gating modifier toxins (GMTs). In addition to their interactions with voltage-gated ion channels, some GMTs have affinity for lipid bilayers. This review discusses the potential importance of the cell membrane on the mode of action of GMTs. We propose that peptide-membrane interactions can anchor GMTs at the cell surface, thereby increasing GMT concentration in the vicinity of the channel binding site. We also propose that modulating peptide-membrane interactions might be useful for increasing the therapeutic potential of spider toxins. Furthermore, we explore the advantages and limitations of the methodologies currently used to examine peptide-membrane interactions. Although GMT-lipid membrane binding does not appear to be a requirement for the activity of all GMTs, it is an important feature, and future studies with GMTs should consider the trimolecular peptide-lipid membrane-channel complex.

  1. Hydration properties of mechanosensitive channel pores define the energetics of gating

    NASA Astrophysics Data System (ADS)

    Anishkin, A.; Akitake, B.; Kamaraju, K.; Chiang, C.-S.; Sukharev, S.

    2010-11-01

    Opening of ion channels directly by tension in the surrounding membrane appears to be the most ancient and simple mechanism of gating. Bacterial mechanosensitive channels MscL and MscS are the best-studied tension-gated nanopores, yet the key physical factors that define their gating are still hotly debated. Here we present estimations, simulations and experimental results showing that hydration of the pore might be one of the major parameters defining the thermodynamics and kinetics of mechanosensitive channel gating. We associate closing of channel pores with complete dehydration of the hydrophobic gate (occlusion by 'vapor lock') and formation of two water-vapor interfaces above and below the constriction. The opening path is the expansion of these interfaces, ultimately leading to wetting of the hydrophobic pore, which does not appear to be the exact reverse of the closing path, thus producing hysteresis. We discuss specifically the role of polar groups (glycines) buried in narrow closed conformations but exposed in the open states that change the wetting characteristics of the pore lining and stabilize conductive states of the channels.

  2. Touch responsiveness in zebrafish requires voltage-gated calcium channel 2.1b.

    PubMed

    Low, Sean E; Woods, Ian G; Lachance, Mathieu; Ryan, Joel; Schier, Alexander F; Saint-Amant, Louis

    2012-07-01

    The molecular and physiological basis of the touch-unresponsive zebrafish mutant fakir has remained elusive. Here we report that the fakir phenotype is caused by a missense mutation in the gene encoding voltage-gated calcium channel 2.1b (CACNA1Ab). Injection of RNA encoding wild-type CaV2.1 restores touch responsiveness in fakir mutants, whereas knockdown of CACNA1Ab via morpholino oligonucleotides recapitulates the fakir mutant phenotype. Fakir mutants display normal current-evoked synaptic communication at the neuromuscular junction but have attenuated touch-evoked activation of motor neurons. NMDA-evoked fictive swimming is not affected by the loss of CaV2.1b, suggesting that this channel is not required for motor pattern generation. These results, coupled with the expression of CACNA1Ab by sensory neurons, suggest that CaV2.1b channel activity is necessary for touch-evoked activation of the locomotor network in zebrafish.

  3. Inhibitory peptidergic modulation of C. elegans serotonin neurons is gated by T-type calcium channels

    PubMed Central

    Zang, Kara E; Ho, Elver; Ringstad, Niels

    2017-01-01

    Serotonin is an evolutionarily ancient molecule that functions in generating and modulating many behavioral states. Although much is known about how serotonin acts on its cellular targets, how serotonin release is regulated in vivo remains poorly understood. In the nematode C. elegans, serotonin neurons that drive female reproductive behavior are directly modulated by inhibitory neuropeptides. Here, we report the isolation of mutants in which inhibitory neuropeptides fail to properly modulate serotonin neurons and the behavior they mediate. The corresponding mutations affect the T-type calcium channel CCA-1 and symmetrically re-tune its voltage-dependencies of activation and inactivation towards more hyperpolarized potentials. This shift in voltage dependency strongly and specifically bypasses the behavioral and cell physiological effects of peptidergic inhibition on serotonin neurons. Our results indicate that T-type calcium channels are critical regulators of a C. elegans serotonergic circuit and demonstrate a mechanism in which T-type channels functionally gate inhibitory modulation in vivo. DOI: http://dx.doi.org/10.7554/eLife.22771.001 PMID:28165324

  4. Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History.

    PubMed

    Riedelsberger, Janin; Dreyer, Ingo; Gonzalez, Wendy

    2015-01-01

    Voltage-gated potassium (K+) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K+ channel type segregates into at least two main functional categories-hyperpolarization-activated, inward-rectifying (Kin) and depolarization-activated, outward-rectifying (Kout) channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom.

  5. Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History

    PubMed Central

    Riedelsberger, Janin; Dreyer, Ingo; Gonzalez, Wendy

    2015-01-01

    Voltage-gated potassium (K+) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K+ channel type segregates into at least two main functional categories—hyperpolarization-activated, inward-rectifying (Kin) and depolarization-activated, outward-rectifying (Kout) channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom. PMID:26356684

  6. Lipid agonism: The PIP2 paradigm of ligand-gated ion channels.

    PubMed

    Hansen, Scott B

    2015-05-01

    The past decade, membrane signaling lipids emerged as major regulators of ion channel function. However, the molecular nature of lipid binding to ion channels remained poorly described due to a lack of structural information and assays to quantify and measure lipid binding in a membrane. How does a lipid-ligand bind to a membrane protein in the plasma membrane, and what does it mean for a lipid to activate or regulate an ion channel? How does lipid binding compare to activation by soluble neurotransmitter? And how does the cell control lipid agonism? This review focuses on lipids and their interactions with membrane proteins, in particular, ion channels. I discuss the intersection of membrane lipid biology and ion channel biophysics. A picture emerges of membrane lipids as bona fide agonists of ligand-gated ion channels. These freely diffusing signals reside in the plasma membrane, bind to the transmembrane domain of protein, and cause a conformational change that allosterically gates an ion channel. The system employs a catalog of diverse signaling lipids ultimately controlled by lipid enzymes and raft localization. I draw upon pharmacology, recent protein structure, and electrophysiological data to understand lipid regulation and define inward rectifying potassium channels (Kir) as a new class of PIP2 lipid-gated ion channels.

  7. High temperature sensitivity is intrinsic to voltage-gated potassium channels

    PubMed Central

    Yang, Fan; Zheng, Jie

    2014-01-01

    Temperature-sensitive transient receptor potential (TRP) ion channels are members of the large tetrameric cation channels superfamily but are considered to be uniquely sensitive to heat, which has been presumed to be due to the existence of an unidentified temperature-sensing domain. Here we report that the homologous voltage-gated potassium (Kv) channels also exhibit high temperature sensitivity comparable to that of TRPV1, which is detectable under specific conditions when the voltage sensor is functionally decoupled from the activation gate through either intrinsic mechanisms or mutations. Interestingly, mutations could tune Shaker channel to be either heat-activated or heat-deactivated. Therefore, high temperature sensitivity is intrinsic to both TRP and Kv channels. Our findings suggest important physiological roles of heat-induced variation in Kv channel activities. Mechanistically our findings indicate that temperature-sensing TRP channels may not contain a specialized heat-sensor domain; instead, non-obligatory allosteric gating permits the intrinsic heat sensitivity to drive channel activation, allowing temperature-sensitive TRP channels to function as polymodal nociceptors. DOI: http://dx.doi.org/10.7554/eLife.03255.001 PMID:25030910

  8. The α2δ subunits of voltage-gated calcium channels.

    PubMed

    Dolphin, Annette C

    2013-07-01

    Voltage-gated calcium channels consist of the main pore-forming α1 subunit, together, except in the case of the T-type channels, with β and α2δ and sometimes γ subunits, which are collectively termed auxiliary or accessory subunits. This review will concentrate on the properties and role of the α2δ subunits of these channels. These proteins are largely extracellular, membrane-associated proteins which influence the trafficking, localization, and biophysical properties of the channels. This article is part of a Special Issue entitled: Calcium channels.

  9. Mechanism of activation gating in the full-length KcsA K[superscript +] channel

    SciTech Connect

    Uysal, Serdar; Cuello, Luis G.; Cortes, D. Marien; Koide, Shohei; Kossiakoff, Anthony A.; Perozo, Eduardo

    2012-10-25

    Using a constitutively active channel mutant, we solved the structure of full-length KcsA in the open conformation at 3.9 {angstrom}. The structure reveals that the activation gate expands about 20 {angstrom}, exerting a strain on the bulge helices in the C-terminal domain and generating side windows large enough to accommodate hydrated K{sup +} ions. Functional and spectroscopic analysis of the gating transition provides direct insight into the allosteric coupling between the activation gate and the selectivity filter. We show that the movement of the inner gate helix is transmitted to the C-terminus as a straightforward expansion, leading to an upward movement and the insertion of the top third of the bulge helix into the membrane. We suggest that by limiting the extent to which the inner gate can open, the cytoplasmic domain also modulates the level of inactivation occurring at the selectivity filter.

  10. Tityustoxin-K(alpha) blockade of the voltage-gated potassium channel Kv1.3

    PubMed Central

    Rodrigues, Aldo Rogelis A; Arantes, Eliane C; Monje, Francisco; Stuhmer, Walter; Varanda, Wamberto Antonio

    2003-01-01

    We investigated the action of TsTX-Kα on cloned Kv1.3 channels of the Shaker subfamily of voltage-gated potassium channels, using the voltage–clamp technique. Highly purified TsTX-Kα was obtained from the venom of the Brazilian scorpion Tityus serrulatus using a new purification protocol. Our results show that TsTX-Kα blocks Kv1.3 with high affinity in two expression systems. TsTX-Kα blockade of Kv1.3 channels expressed in Xenopus oocytes was found to be completely reversible and to exhibit a pH dependence. The KD was 3.9 nM at pH 7.5, 9.5 nM at pH 7.0 and 94.5 nM at pH 6.5. The blocking properties of TsTX-Kα in a mammalian cell line (L929), stably transfected to express Kv1.3, were studied using the patch–clamp technique. In this preparation, the toxin had a KD of 19.8 nM at pH 7.4. TsTX-Kα was found to affect neither the voltage-dependence of activation, nor the activation and deactivation time constants. The block appeared to be independent of the transmembrane voltage and the toxin did not interfere with the C-type inactivation process. Taken as a whole, our findings indicate that TsTX-Kα acts as a simple blocker of Kv1.3 channels. It is concluded that this toxin is a useful tool for probing not only the physiological roles of Kv1.2, but also those mediated by Kv1.3 channels. PMID:12871837

  11. Materials characterization for process integration of multi-channel gate all around (GAA) devices

    NASA Astrophysics Data System (ADS)

    Muthinti, Gangadhara Raja; Loubet, Nicolas; Chao, Robin; de la Peña, Abraham A.; Li, Juntao; Guillorn, Michael A.; Yamashita, Tenko; Kanakasabapathy, Sivananda; Gaudiello, John; Cepler, Aron J.; Sendelbach, Matthew; Emans, Susan; Wolfling, Shay; Ger, Avron; Kandel, Daniel; Koret, Roy; Lee, Wei Ti; Gin, Peter; Matney, Kevin; Wormington, Matthew

    2017-03-01

    Multi-channel gate all around (GAA) semiconductor devices march closer to becoming a reality in production as their maturity in development continues. From this development, an understanding of what physical parameters affecting the device has emerged. The importance of material property characterization relative to that of other physical parameters has continued to increase for GAA architecture when compared to its relative importance in earlier architectures. Among these materials properties are the concentration of Ge in SiGe channels and the strain in these channels and related films. But because these properties can be altered by many different process steps, each one adding its own variation to these parameters, their characterization and control at multiple steps in the process flow is crucial. This paper investigates the characterization of strain and Ge concentration, and the relationships between these properties, in the PFET SiGe channel material at the earliest stages of processing for GAA devices. Grown on a bulk Si substrate, multiple pairs of thin SiGe/Si layers that eventually form the basis of the PFET channel are measured and characterized in this study. Multiple measurement techniques are used to measure the material properties. In-line X-Ray Photoelectron Spectroscopy (XPS) and Low Energy X-Ray Fluorescence (LE-XRF) are used to characterize Ge content, while in-line High Resolution X-Ray Diffraction (HRXRD) is used to characterize strain. Because both patterned and un-patterned structures were investigated, scatterometry (also called optical critical dimension, or OCD) is used to provide valuable geometrical metrology.

  12. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra.

    PubMed

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D; Williamson, Michael; Kalbacher, Hubert; Grimmelikhuijzen, Cornelis J P; Holstein, Thomas W; Gründer, Stefan

    2010-04-16

    Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na(+) channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na(+) channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamide-activated Na(+) channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore properties, like a low Na(+) selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new subunit is closely related to HyNaC2 and -3 and co-localizes with HyNaC2 and -3 to the base of the tentacles. Coexpression in Xenopus oocytes of HyNaC5 with HyNaC2 and -3 largely increases current amplitude after peptide stimulation and affinity of the channel to Hydra-RFamides I and II. Moreover, the HyNaC2/3/5 channel has altered pore properties and amiloride affinity, more similarly to other DEG/ENaC channels. Collectively, our results suggest that the three homologous subunits HyNaC2, -3, and -5 form a peptide-gated ion channel in Hydra that could contribute to fast synaptic transmission.

  13. Charybdotoxin blocks voltage-gated K+ channels in human and murine T lymphocytes

    PubMed Central

    1989-01-01

    A variety of scorpion venoms and purified toxins were tested for effects on ion channels in human T lymphocytes, a human T leukemia cell line (Jurkat), and murine thymocytes, using the whole-cell patch-clamp method. Nanomolar concentrations of charbdotoxin (CTX), a purified peptide component of Leiurus quinquestriatus venom known to block Ca2+- activated K+ channels from muscle, blocked "type n" voltage-gated K+ channels in human T lymphoid cells. The Na+ channels occasionally expressed in these cells were unaffected by the toxin. From the time course of development and removal of K+ channel block we determined the rates of CTX binding and unbinding. CTX blocks K+ channels in Jurkat cells with a Kd value between 0.5 and 1.5 nM. Of the three types of voltage-gated K+ channels present in murine thymocytes, types n and n' are blocked by CTX at nanomolar concentrations. The third variety of K+ channels, "type l," is unaffected by CTX. Noxiustoxin (NTX), a purified toxin from Centruroides noxius known to block Ca2+-activated K+ channels, also blocked type n K+ channels with a high degree of potency (Kd = 0.2 nM). In addition, several types of crude scorpion venoms from the genera Androctonus, Buthus, Centruroides, and Pandinus blocked type n channels. We conclude that CTX and NTX are not specific for Ca2+ activated K+ channels and that purified scorpion toxins will provide useful probes of voltage-gated K+ channels in T lymphocytes. The existence of high-affinity sites for scorpion toxin binding may help to classify structurally related K+ channels and provide a useful tool for their biochemical purification. PMID:2475579

  14. Mechanisms of Gain Control by Voltage-Gated Channels in Intrinsically-Firing Neurons

    PubMed Central

    Patel, Ameera X.; Burdakov, Denis

    2015-01-01

    Gain modulation is a key feature of neural information processing, but underlying mechanisms remain unclear. In single neurons, gain can be measured as the slope of the current-frequency (input-output) relationship over any given range of inputs. While much work has focused on the control of basal firing rates and spike rate adaptation, gain control has been relatively unstudied. Of the limited studies on gain control, some have examined the roles of synaptic noise and passive somatic currents, but the roles of voltage-gated channels present ubiquitously in neurons have been less explored. Here, we systematically examined the relationship between gain and voltage-gated ion channels in a conductance-based, tonically-active, model neuron. Changes in expression (conductance density) of voltage-gated channels increased (Ca2+ channel), reduced (K+ channels), or produced little effect (h-type channel) on gain. We found that the gain-controlling ability of channels increased exponentially with the steepness of their activation within the dynamic voltage window (voltage range associated with firing). For depolarization-activated channels, this produced a greater channel current per action potential at higher firing rates. This allowed these channels to modulate gain by contributing to firing preferentially at states of higher excitation. A finer analysis of the current-voltage relationship during tonic firing identified narrow voltage windows at which the gain-modulating channels exerted their effects. As a proof of concept, we show that h-type channels can be tuned to modulate gain by changing the steepness of their activation within the dynamic voltage window. These results show how the impact of an ion channel on gain can be predicted from the relationship between channel kinetics and the membrane potential during firing. This is potentially relevant to understanding input-output scaling in a wide class of neurons found throughout the brain and other nervous systems

  15. Domain and interdomain energetics underlying gating in Shaker-type Kv channels.

    PubMed

    Peyser, Alexander; Gillespie, Dirk; Roth, Roland; Nonner, Wolfgang

    2014-10-21

    To understand gating events with a time-base many orders-of-magnitude slower than that of atomic motion in voltage-gated ion channels such as the Shaker-type KV channels, a multiscale physical model is constructed from the experimentally well-characterized voltage-sensor (VS) domains coupled to a hydrophobic gate. The four VS domains are described by a continuum electrostatic model under voltage-clamp conditions, the control of ion flow by the gate domain is described by a vapor-lock mechanism, and the simple coupling principle is informed by known experimental results and trial-and-error. The configurational energy computed for each element is used to produce a total Hamiltonian that is a function of applied voltage, VS positions, and gate radius. We compute statistical-mechanical expectation values of macroscopic laboratory observables. This approach stands in contrast with molecular-dynamic models which are challenged by increasing scale, and kinetic models which assume a probability distribution rather than derive it from the underlying physics. This generic model predicts well the Shaker charge/voltage and conductance/voltage relations; the tight constraints underlying these results allow us to quantitatively assess the underlying physical mechanisms. The total electrical work picked up by the VS domains is an order-of-magnitude larger than the work required to actuate the gate itself, suggesting an energetic basis for the evolutionary flexibility of the voltage-gating mechanism. The cooperative slide-and-interlock behavior of the VS domains described by the VS-gate coupling relation leads to the experimentally observed bistable gating. This engineering approach should prove useful in the investigation of various elements underlying gating characteristics and degraded behavior due to mutation.

  16. KvDB; mining and mapping sequence variants in voltage-gated potassium channels.

    PubMed

    Stead, Lucy F; Wood, Ian C; Westhead, David R

    2010-08-01

    We have created KvDB: a voltage-gated potassium (Kv) channel-specific database that houses natural and experimental variant data and includes highly curated multiple sequence alignments and additional analytical tools, such as structural variant mapping and transmembrane segment prediction. KvDB is available at www.bioinformatics.leeds.ac.uk/KvDB. Analyzing the characterized gene variants in terms of topological location revealed the following. The S4, S4-S5, S5, S5-S6, and S6 segments are most likely to house disease-causing variants. Neurological disorders are more likely to be caused by variants affecting voltage sensing, whereas cardiac disorders are more likely to be caused by variants in the pore. Long QT Syndrome 2 (LQT2) is more often caused by N-terminus variation, a region containing a domain that affects deactivation, suggesting a potential disease mechanism. Conversely, a higher proportion of LQT1-causing variants reside in S4-S5, suggesting communication of voltage-sensing to the pore as a disease mechanism. By structurally mapping functionally characterized variants, we also provide mechanistic insight into Kv channel function; identifying an intersubunit interaction that may be partly responsible for setting activation voltage. Investigating phenotypically characterized variants that map to the same position as functionally characterized ones indicates only weak association between locations that cause disease and those that alter electrophysiological properties.

  17. Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels

    PubMed Central

    Shi, Liheng; Ko, Michael L.; Ko, Gladys Y.-P.

    2017-01-01

    Modulation of ion channels by extracellular proteins plays critical roles in shaping synaptic plasticity. Retinoschisin (RS1) is an extracellular adhesive protein secreted from photoreceptors and bipolar cells, and it plays an important role during retinal development, as well as in maintaining the stability of retinal layers. RS1 is known to form homologous octamers and interact with molecules on the plasma membrane including phosphatidylserine, sodium-potassium exchanger complex, and L-type voltage-gated calcium channels (LTCCs). However, how this physical interaction between RS1 and ion channels might affect the channel gating properties is unclear. In retinal photoreceptors, two major LTCCs are Cav1.3 (α1D) and Cav1.4 (α1F) with distinct biophysical properties, functions and distributions. Cav1.3 is distributed from the inner segment (IS) to the synaptic terminal and is responsible for calcium influx to the photoreceptors and overall calcium homeostasis. Cav1.4 is only expressed at the synaptic terminal and is responsible for neurotransmitter release. Mutations of the gene encoding Cav1.4 cause X-linked incomplete congenital stationary night blindness type 2 (CSNB2), while null mutations of Cav1.3 cause a mild decrease of retinal light responses in mice. Even though RS1 is known to maintain retinal architecture, in this study, we present that RS1 interacts with both Cav1.3 and Cav1.4 and regulates their activations. RS1 was able to co-immunoprecipitate with Cav1.3 and Cav1.4 from porcine retinas, and it increased the LTCC currents and facilitated voltage-dependent activation in HEK cells co-transfected with RS1 and Cav1.3 or Cav1.4, thus providing evidence of a functional interaction between RS1 and LTCCs. The interaction between RS1 and Cav1.3 did not change the calcium-dependent inactivation of Cav1.3. In mice lacking RS1, the expression of Cav1.3 and Cav1.4 in the retina decreased, while in mice with Cav1.4 deletion, the retinal level of RS1 decreased

  18. Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains

    NASA Astrophysics Data System (ADS)

    Lörinczi, Éva; Gómez-Posada, Juan Camilo; de La Peña, Pilar; Tomczak, Adam P.; Fernández-Trillo, Jorge; Leipscher, Ulrike; Stühmer, Walter; Barros, Francisco; Pardo, Luis A.

    2015-03-01

    Voltage-gated channels open paths for ion permeation upon changes in membrane potential, but how voltage changes are coupled to gating is not entirely understood. Two modules can be recognized in voltage-gated potassium channels, one responsible for voltage sensing (transmembrane segments S1 to S4), the other for permeation (S5 and S6). It is generally assumed that the conversion of a conformational change in the voltage sensor into channel gating occurs through the intracellular S4-S5 linker that provides physical continuity between the two regions. Using the pathophysiologically relevant KCNH family, we show that truncated proteins interrupted at, or lacking the S4-S5 linker produce voltage-gated channels in a heterologous model that recapitulate both the voltage-sensing and permeation properties of the complete protein. These observations indicate that voltage sensing by the S4 segment is transduced to the channel gate in the absence of physical continuity between the modules.

  19. Molecular interactions involved in proton-dependent gating in KcsA potassium channels

    PubMed Central

    Posson, David J.; Thompson, Ameer N.; McCoy, Jason G.

    2013-01-01

    The bacterial potassium channel KcsA is gated open by the binding of protons to amino acids on the intracellular side of the channel. We have identified, via channel mutagenesis and x-ray crystallography, two pH-sensing amino acids and a set of nearby residues involved in molecular interactions that influence gating. We found that the minimal mutation of one histidine (H25) and one glutamate (E118) near the cytoplasmic gate completely abolished pH-dependent gating. Mutation of nearby residues either alone or in pairs altered the channel’s response to pH. In addition, mutations of certain pairs of residues dramatically increased the energy barriers between the closed and open states. We proposed a Monod–Wyman–Changeux model for proton binding and pH-dependent gating in KcsA, where H25 is a “strong” sensor displaying a large shift in pKa between closed and open states, and E118 is a “weak” pH sensor. Modifying model parameters that are involved in either the intrinsic gating equilibrium or the pKa values of the pH-sensing residues was sufficient to capture the effects of all mutations. PMID:24218397

  20. Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

    PubMed Central

    Wang, Jin; Yu, Ye

    2016-01-01

    P2X receptors, as ATP-gated non-selective trimeric ion channels, are permeable to Na+, K+ and Ca2+. Comparing with other ligand-gated ion channel families, P2X receptors are distinct in their unique gating properties and pathophysiological roles, and have attracted attention as promising drug targets for a variety of diseases, such as neuropathic pain, multiple sclerosis, rheumatoid arthritis and thrombus. Several small molecule inhibitors for distinct P2X subtypes have entered into clinical trials. However, many questions regarding the gating mechanism of P2X remain unsolved. The structural determinations of P2X receptors at the resting and ATP-bound open states revealed that P2X receptor gating is a cooperative allosteric process involving multiple domains, which marks the beginning of the post-structure era of P2X research at atomic level. Here, we review the current knowledge on the structure-function relationship of P2X receptors, depict the whole picture of allosteric changes during the channel gating, and summarize the active sites that may contribute to new strategies for developing novel allosteric drugs targeting P2X receptors. PMID:26725734

  1. Regulation of N-type voltage-gated calcium channels and presynaptic function by cyclin-dependent kinase 5

    PubMed Central

    Su, Susan C.; Seo, Jinsoo; Pan, Jen Q.; Samuels, Benjamin Adam; Rudenko, Andrii; Ericsson, Maria; Neve, Rachael L.; Yue, David T.; Tsai, Li-Huei

    2012-01-01

    SUMMARY N-type voltage-gated calcium channels (CaV2.2) localize to presynaptic nerve terminals and mediate key events including synaptogenesis and neurotransmission. While several kinases have been implicated in the modulation of calcium channels, their impact on presynaptic functions remains unclear. Here we report that the N-type calcium channel is a substrate for cyclin-dependent kinase 5 (Cdk5). The pore-forming α1 subunit of the N-type calcium channel is phosphorylated in the C-terminal domain, and phosphorylation results in enhanced calcium influx due to increased channel open probability. Phosphorylation of the N-type calcium channel by Cdk5 facilitates neurotransmitter release and alters presynaptic plasticity by increasing the number of docked vesicles at the synaptic cleft. These effects are mediated by an altered interaction between N-type calcium channels and RIM1, which tethers presynaptic calcium channels to the active zone. Collectively, our results highlight a molecular mechanism by which N-type calcium channels are regulated by Cdk5 to affect presynaptic functions. PMID:22920258

  2. Structural sensitivity of a prokaryotic pentameric ligand-gated ion channel to its membrane environment.

    PubMed

    Labriola, Jonathan M; Pandhare, Akash; Jansen, Michaela; Blanton, Michael P; Corringer, Pierre-Jean; Baenziger, John E

    2013-04-19

    Although the activity of the nicotinic acetylcholine receptor (nAChR) is exquisitely sensitive to its membrane environment, the underlying mechanisms remain poorly defined. The homologous prokaryotic pentameric ligand-gated ion channel, Gloebacter ligand-gated ion channel (GLIC), represents an excellent model for probing the molecular basis of nAChR sensitivity because of its high structural homology, relative ease of expression, and amenability to crystallographic analysis. We show here that membrane-reconstituted GLIC exhibits structural and biophysical properties similar to those of the membrane-reconstituted nAChR, although GLIC is substantially more thermally stable. GLIC, however, does not possess the same exquisite lipid sensitivity. In particular, GLIC does not exhibit the same propensity to adopt an uncoupled conformation where agonist binding is uncoupled from channel gating. Structural comparisons provide insight into the chemical features that may predispose the nAChR to the formation of an uncoupled state.

  3. Structural Sensitivity of a Prokaryotic Pentameric Ligand-gated Ion Channel to Its Membrane Environment*

    PubMed Central

    Labriola, Jonathan M.; Pandhare, Akash; Jansen, Michaela; Blanton, Michael P.; Corringer, Pierre-Jean; Baenziger, John E.

    2013-01-01

    Although the activity of the nicotinic acetylcholine receptor (nAChR) is exquisitely sensitive to its membrane environment, the underlying mechanisms remain poorly defined. The homologous prokaryotic pentameric ligand-gated ion channel, Gloebacter ligand-gated ion channel (GLIC), represents an excellent model for probing the molecular basis of nAChR sensitivity because of its high structural homology, relative ease of expression, and amenability to crystallographic analysis. We show here that membrane-reconstituted GLIC exhibits structural and biophysical properties similar to those of the membrane-reconstituted nAChR, although GLIC is substantially more thermally stable. GLIC, however, does not possess the same exquisite lipid sensitivity. In particular, GLIC does not exhibit the same propensity to adopt an uncoupled conformation where agonist binding is uncoupled from channel gating. Structural comparisons provide insight into the chemical features that may predispose the nAChR to the formation of an uncoupled state. PMID:23463505

  4. Evidence for a role of GABA- and glutamate-gated chloride channels in olfactory memory.

    PubMed

    Boumghar, Katia; Couret-Fauvel, Thomas; Garcia, Mikael; Armengaud, Catherine

    2012-11-01

    In the honeybee, we investigated the role of transmissions mediated by GABA-gated chloride channels and glutamate-gated chloride channels (GluCls) of the mushroom bodies (MBs) on olfactory learning using a single-trial olfactory conditioning paradigm. The GABAergic antagonist picrotoxin (PTX) or the GluCl antagonist L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) was injected alone or in combination into the α-lobes of MBs. PTX impaired early long-term olfactory memory when injected before conditioning or before testing. L-trans-PDC alone induced no significant effect on learning and memory but induced a less specific response to the conditioned odor. When injected before PTX, L-trans-PDC was able to modulate PTX effects. These results emphasize the role of MB GABA-gated chloride channels in consolidation processes and strongly support that GluCls are involved in the perception of the conditioned stimulus.

  5. An update on transcriptional and post-translational regulation of brain voltage-gated sodium channels.

    PubMed

    Onwuli, Donatus O; Beltran-Alvarez, Pedro

    2016-03-01

    Voltage-gated sodium channels are essential proteins in brain physiology, as they generate the sodium currents that initiate neuronal action potentials. Voltage-gated sodium channels expression, localisation and function are regulated by a range of transcriptional and post-translational mechanisms. Here, we review our understanding of regulation of brain voltage-gated sodium channels, in particular SCN1A (NaV1.1), SCN2A (NaV1.2), SCN3A (NaV1.3) and SCN8A (NaV1.6), by transcription factors, by alternative splicing, and by post-translational modifications. Our focus is strongly centred on recent research lines, and newly generated knowledge.

  6. NEUROSCIENCE. Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics.

    PubMed

    Govorunova, Elena G; Sineshchekov, Oleg A; Janz, Roger; Liu, Xiaoqin; Spudich, John L

    2015-08-07

    Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization. We describe anion channel rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision.

  7. Ciguatoxins: Cyclic Polyether Modulators of Voltage-gated Iion Channel Function

    PubMed Central

    Nicholson, Graham M.; Lewis, Richard J.

    2006-01-01

    Ciguatoxins are cyclic polyether toxins, derived from marine dinoflagellates, which are responsible for the symptoms of ciguatera poisoning. Ingestion of tropical and subtropical fin fish contaminated by ciguatoxins results in an illness characterised by neurological, cardiovascular and gastrointestinal disorders. The pharmacology of ciguatoxins is characterised by their ability to cause persistent activation of voltage-gated sodium channels, to increase neuronal excitability and neurotransmitter release, to impair synaptic vesicle recycling, and to cause cell swelling. It is these effects, in combination with an action to block voltage-gated potassium channels at high doses, which are believed to underlie the complex of symptoms associated with ciguatera. This review examines the sources, structures and pharmacology of ciguatoxins. In particular, attention is placed on their cellular modes of actions to modulate voltage-gated ion channels and other Na+-dependent mechanisms in numerous cell types and to current approaches for detection and treatment of ciguatera.

  8. The voltage-gated proton channel: a riddle, wrapped in a mystery, inside an enigma

    PubMed Central

    DeCoursey, Thomas E.

    2016-01-01

    The main properties of voltage gated proton channels are described, along with what is known about how the channel protein structure accomplishes these functions. Just as protons are unique among ions, proton channels are unique among ion channels. Their four transmembrane helices sense voltage, the pH gradient, and conduct protons exclusively. Selectivity is achieved by the unique ability of H3O+ to protonate an Asp-Arg salt bridge. Pathognomonic sensitivity of gating to the pH gradient ensures channel opening only when acid extrusion will result, which is crucial to most biological functions. An exception occurs in dinoflagellates in which H+ influx through HV1 triggers the bioluminescent flash. Pharmacological interventions that promise to ameliorate cancer, asthma, brain damage in ischemic stroke, Alzheimer’s disease, autoimmune diseases, and numerous other conditions, await future progress. PMID:25964989

  9. Clustering of the K+ channel GORK of Arabidopsis parallels its gating by extracellular K+

    PubMed Central

    Eisenach, Cornelia; Papanatsiou, Maria; Hillert, Ellin-Kristina; Blatt, Michael R

    2014-01-01

    GORK is the only outward-rectifying Kv-like K+ channel expressed in guard cells. Its activity is tightly regulated to facilitate K+ efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward-rectifying K+ channel KAT1. Whereas the population of KAT1 is subject to regulated traffic to and from the plasma membrane, nothing is known about GORK, its distribution and traffic in vivo. We have used transformations with fluorescently-tagged GORK to explore its characteristics in tobacco epidermis and Arabidopsis guard cells. These studies showed that GORK assembles in puncta that reversibly dissociated as a function of the external K+ concentration. Puncta dissociation parallelled the gating dependence of GORK, the speed of response consistent with the rapidity of channel gating response to changes in the external ionic conditions. Dissociation was also suppressed by the K+ channel blocker Ba2+. By contrast, confocal and protein biochemical analysis failed to uncover substantial exo- and endocytotic traffic of the channel. Gating of GORK is displaced to more positive voltages with external K+, a characteristic that ensures the channel facilitates only K+ efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K+ above 1 mm. We suggest that GORK clustering in puncta is related to its gating and conductance, and reflects associated conformational changes and (de)stabilisation of the channel protein, possibly as a platform for transmission and coordination of channel gating in response to external K+. PMID:24517091

  10. Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating.

    PubMed

    Perozo, Eduardo; Kloda, Anna; Cortes, D Marien; Martinac, Boris

    2002-09-01

    In mechanosensitive (MS) channels, gating is initiated by changes in intra-bilayer pressure profiles originating from bilayer deformation. Here we evaluated two physical mechanisms as triggers of MS channel gating: the energetic cost of protein-bilayer hydrophobic mismatches and the geometric consequences of bilayer intrinsic curvature. Structural changes in the Escherichia coli large MS channel (MscL) were studied under nominally zero transbilayer pressures using both patch clamp and EPR spectroscopic approaches. Changes in membrane intrinsic curvature induced by the external addition of lysophosphatidylcholine (LPC) generated massive spectroscopic changes in the narrow constriction that forms the channel 'gate', trapping the channel in the fully open state. Hydrophobic mismatch alone was unable to open the channel, but decreasing bilayer thickness lowered MscL activation energy, stabilizing a structurally distinct closed channel intermediate. We propose that the mechanism of mechanotransduction in MS channels is defined by both local and global asymmetries in the transbilayer pressure profile at the lipid-protein interface.

  11. Comparison of short-circuit characteristics of trench gate and planar gate U-shaped channel SOI-LIGBTs

    NASA Astrophysics Data System (ADS)

    Zhang, Long; Zhu, Jing; Sun, Weifeng; Zhao, Minna; Huang, Xuequan; Chen, Jiajun; Shi, Longxing; Chen, Jian; Ding, Desheng

    2017-09-01

    Comparison of short-circuit (SC) characteristics of 500 V rated trench gate U-shaped channel (TGU) SOI-LIGBT and planar gate U-shaped channel (PGU) SOI-LIGBT is made for the first time in this paper. The on-state carrier profile of the TGU structure is reshaped by the dual trenches (a gate trench G1 and a hole barrier trench G2), which leads to a different conduction behavior from that of the PGU structure. The TGU structure exhibits a higher latchup immunity but a severer self-heating effect. At current density (JC) < 640 A/cm2, the SC destruction is suspected to be latchup-dependent and short-circuit withstand time (tSC) of the TGU structure is much longer than that of the PGU structure. Due to the high lattice temperature rise caused by the high current density at the emitter side in the TGU structure, the PGU exhibits a better JC-tSC trade-off at JC > 640 A/cm2. Comparison of layouts and fabrication processes are also made between the two types of devices.

  12. A gating motif in the translocation channel sets the hydrophobicity threshold for signal sequence function

    PubMed Central

    Trueman, Steven F.; Mandon, Elisabet C.

    2012-01-01

    A critical event in protein translocation across the endoplasmic reticulum is the structural transition between the closed and open conformations of Sec61, the eukaryotic translocation channel. Channel opening allows signal sequence insertion into a gap between the N- and C-terminal halves of Sec61. We have identified a gating motif that regulates the transition between the closed and open channel conformations. Polar amino acid substitutions in the gating motif cause a gain-of-function phenotype that permits translocation of precursors with marginally hydrophobic signal sequences. In contrast, hydrophobic substitutions at certain residues in the gating motif cause a protein translocation defect. We conclude that the gating motif establishes the hydrophobicity threshold for functional insertion of a signal sequence into the Sec61 complex, thereby allowing the wild-type translocation channel to discriminate between authentic signal sequences and the less hydrophobic amino acid segments in cytosolic proteins. Bioinformatic analysis indicates that the gating motif is conserved between eubacterial and archaebacterial SecY and eukaryotic Sec61. PMID:23229898

  13. Energetic role of the paddle motif in voltage gating of Shaker K(+) channels.

    PubMed

    Xu, Yanping; Ramu, Yajamana; Shin, Hyeon-Gyu; Yamakaze, Jayden; Lu, Zhe

    2013-05-01

    Voltage-gated ion channels underlie rapid electric signaling in excitable cells. Electrophysiological studies have established that the N-terminal half of the fourth transmembrane segment ((NT)S4) of these channels is the primary voltage sensor, whereas crystallographic studies have shown that (NT)S4 is not located within a proteinaceous pore. Rather, (NT)S4 and the C-terminal half of S3 ((CT)S3 or S3b) form a helix-turn-helix motif, termed the voltage-sensor paddle. This unexpected structural finding raises two fundamental questions: does the paddle motif also exist in voltage-gated channels in a biological membrane, and, if so, what is its function in voltage gating? Here, we provide evidence that the paddle motif exists in the open state of Drosophila Shaker voltage-gated K(+) channels expressed in Xenopus oocytes and that (CT)S3 acts as an extracellular hydrophobic 'stabilizer' for (NT)S4, thus biasing the gating chemical equilibrium toward the open state.

  14. Gate Structure Dependence of Variability in Polycrystalline Silicon Fin-Channel Flash Memories

    NASA Astrophysics Data System (ADS)

    Liu, Yongxun; Kamei, Takahiro; Matsukawa, Takashi; Endo, Kazuhiko; O'uchi, Shinichi; Tsukada, Junichi; Yamauchi, Hiromi; Ishikawa, Yuki; Hayashida, Tetsuro; Sakamoto, Kunihiro; Ogura, Atsushi; Masahara, Meishoku

    2013-06-01

    Polycrystalline silicon (poly-Si) fin-channel tri-gate (TG)- and double-gate (DG)-type flash memories with a thin n+-poly-Si floating gate (FG) and different control-gate (CG) lengths (LCG's) from 76 to 256 nm have been fabricated and their electrical characteristics including statistical threshold voltage (Vt) and subthreshold slope (S-slope) have been comparatively investigated before and after one program/erase (P/E) cycle. It was experimentally found that better short-channel effect (SCE) immunity, a smaller Vt variation, and a higher program speed are obtained in TG-type flash memories than in DG-type memories. The higher performance of TG-type flash memories is contributed by the additional top gate and recessed bottom silicon dioxide (SiO2) regions, which strengthen the controllability of the channel potential and increase the coupling ratio of the FG to the CG. Therefore, the developed poly-Si fin-channel TG structure is expected to be very useful for the fabrication of high-density and low-cost flash memories.

  15. Linker-gating ring complex as passive spring and Ca(2+)-dependent machine for a voltage- and Ca(2+)-activated potassium channel.

    PubMed

    Niu, Xiaowei; Qian, Xiang; Magleby, Karl L

    2004-06-10

    Ion channels are proteins that control the flux of ions across cell membranes by opening and closing (gating) their pores. It has been proposed that channels gated by internal agonists have an intracellular gating ring that extracts free energy from agonist binding to open the gates using linkers that directly connect the gating ring to the gates. Here we find for a voltage- and Ca(2+)-activated K+ (BK) channel that shortening the linkers increases channel activity and lengthening the linkers decreases channel activity, both in the presence and absence of intracellular Ca2+. These observations are consistent with a mechanical model in which the linker-gating ring complex forms a passive spring that applies force to the gates in the absence of Ca2+ to modulate the voltage-dependent gating. Adding Ca2+ then changes the force to further activate the channel. Both the passive and Ca(2+)-induced forces contribute to the gating of the channel.

  16. Clinical Features of Neuromuscular Disorders in Patients with N-Type Voltage-Gated Calcium Channel Antibodies

    PubMed Central

    Totzeck, Andreas; Mummel, Petra; Kastrup, Oliver; Hagenacker, Tim

    2016-01-01

    Neuromuscular junction disorders affect the pre- or postsynaptic nerve to muscle transmission due to autoimmune antibodies. Members of the group like myasthenia gravis and Lambert-Eaton syndrome have pathophysiologically distinct characteristics. However, in practice, distinction may be difficult. We present a series of three patients with a myasthenic syndrome, dropped-head syndrome, bulbar and respiratory muscle weakness and positive testing for anti-N-type voltage-gated calcium channel antibodies. In two cases anti-acetylcholin receptor antibodies were elevated, anti-P/Q-type voltage-gated calcium channel antibodies were negative. All patients initially responded to pyridostigmine with a non-response in the course of the disease. While one patient recovered well after treatment with intravenous immunoglobulins, 3,4-diaminopyridine, steroids and later on immunosuppression with mycophenolate mofetil, a second died after restriction of treatment due to unfavorable cancer diagnosis, the third patient declined treatment. Although new antibodies causing neuromuscular disorders were discovered, clinical distinction has not yet been made. Our patients showed features of pre- and postsynaptic myasthenic syndrome as well as severe dropped-head syndrome and bulbar and axial muscle weakness, but only anti-N-type voltage-gated calcium channel antibodies were positive. When administered, one patient benefited from 3,4-diaminopyridine. We suggest that this overlap-syndrome should be considered especially in patients with assumed seronegative myasthenia gravis and lack of improvement under standard therapy. PMID:28078065

  17. Development of multi-channel gated integrator and PXI-DAQ system for nuclear detector arrays

    NASA Astrophysics Data System (ADS)

    Kong, Jie; Su, Hong; Chen, Zhi-Qiang; Dong, Cheng-Fu; Qian, Yi; Gao, Shan-Shan; Zhou, Chao-Yang; Lu, Wan; Ye, Rui-Ping; Ma, Jun-Bing

    2010-10-01

    A multi-channel gated integrator and PXI based data acquisition system have been developed for nuclear detector arrays with hundreds of detector units. The multi-channel gated integrator can be controlled by a programmable GI controller. The PXI-DAQ system consists of NI PXI-1033 chassis with several PXI-DAQ cards. The system software has a user-friendly GUI which is written in C language using LabWindows/CVI under Windows XP operating system. The performance of the PXI-DAQ system is very reliable and capable of handling event rate up to 40 kHz.

  18. Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR

    PubMed Central

    Mukhtasimova, Nuriya; daCosta, Corrie J.B.

    2016-01-01

    constants. As observed for a full and a partial agonist, the gain-of-function mutation affects the relationship between rate and equilibrium constants for priming but not for channel gating. Thus, resolving brief single channel currents distinguishes priming from gating steps and reveals how the corresponding rate and equilibrium constants depend on agonist occupancy. PMID:27353445

  19. Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR.

    PubMed

    Mukhtasimova, Nuriya; daCosta, Corrie J B; Sine, Steven M

    2016-07-01

    constants. As observed for a full and a partial agonist, the gain-of-function mutation affects the relationship between rate and equilibrium constants for priming but not for channel gating. Thus, resolving brief single channel currents distinguishes priming from gating steps and reveals how the corresponding rate and equilibrium constants depend on agonist occupancy. © 2016 Mukhtasimova et al.

  20. Promiscuous gating modifiers target the voltage sensor of K(v)7.2, TRPV1, and H(v)1 cation channels.

    PubMed

    Kornilov, Polina; Peretz, Asher; Lee, Yoonji; Son, Karam; Lee, Jin Hee; Refaeli, Bosmat; Roz, Netta; Rehavi, Moshe; Choi, Sun; Attali, Bernard

    2014-06-01

    Some of the fascinating features of voltage-sensing domains (VSDs) in voltage-gated cation channels (VGCCs) are their modular nature and adaptability. Here we examined the VSD sensitivity of different VGCCs to 2 structurally related nontoxin gating modifiers, NH17 and NH29, which stabilize K(v)7.2 potassium channels in the closed and open states, respectively. The effects of NH17 and NH29 were examined in Chinese hamster ovary cells transfected with transient receptor potential vanilloid 1 (TRPV1) or K(v)7.2 channels, as well as in dorsal root ganglia neurons, using the whole-cell patch-clamp technique. NH17 and NH29 exert opposite effects on TRPV1 channels, operating, respectively, as an activator and a blocker of TRPV1 currents (EC50 and IC50 values ranging from 4 to 40 μM). Combined mutagenesis, electrophysiology, structural homology modeling, molecular docking, and molecular dynamics simulation indicate that both compounds target the VSDs of TRPV1 channels, which, like vanilloids, are involved in π-π stacking, H-bonding, and hydrophobic interactions. Reflecting their promiscuity, the drugs also affect the lone VSD proton channel mVSOP. Thus, the same gating modifier can promiscuously interact with different VGCCs, and subtle differences at the VSD-ligand interface will dictate whether the gating modifier stabilizes channels in either the closed or the open state.

  1. Light-induced regulation of ligand-gated channel activity.

    PubMed

    Bregestovski, Piotr; Maleeva, Galyna; Gorostiza, Pau

    2017-08-31

    The control of ligand-gated receptors with light using photochromic compounds has evolved from the first handcrafted examples to accurate, engineered receptors, whose development is supported by rational design, high-resolution protein structures, comparative pharmacology and molecular biology manipulations. Photoswitchable regulators have been designed and characterized for a large number of ligand-gated receptors in the mammalian nervous system, including nicotinic acetylcholine, glutamate and GABA receptors. They provide a well-equipped toolbox to investigate synaptic and neuronal circuits in all-optical experiments. This focused review discusses the design and properties of these photoswitches, their applications and shortcomings and future perspectives in the field. © 2017 The British Pharmacological Society.

  2. Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons

    PubMed Central

    Lazcano-Pérez, Fernando; Castro, Héctor; Arenas, Isabel; García, David E.; González-Muñoz, Ricardo; Arreguín-Espinosa, Roberto

    2016-01-01

    The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels. PMID:27164140

  3. PIP2 regulation of KCNQ channels: biophysical and molecular mechanisms for lipid modulation of voltage-dependent gating.

    PubMed

    Zaydman, Mark A; Cui, Jianmin

    2014-01-01

    Voltage-gated potassium (Kv) channels contain voltage-sensing (VSD) and pore-gate (PGD) structural domains. During voltage-dependent gating, conformational changes in the two domains are coupled giving rise to voltage-dependent opening of the channel. In addition to membrane voltage, KCNQ (Kv7) channel opening requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Recent studies suggest that PIP2 serves as a cofactor to mediate VSD-PGD coupling in KCNQ1 channels. In this review, we put these findings in the context of the current understanding of voltage-dependent gating, lipid modulation of Kv channel activation, and PIP2-regulation of KCNQ channels. We suggest that lipid-mediated coupling of functional domains is a common mechanism among KCNQ channels that may be applicable to other Kv channels and membrane proteins.

  4. PIP2 regulation of KCNQ channels: biophysical and molecular mechanisms for lipid modulation of voltage-dependent gating

    PubMed Central

    Zaydman, Mark A.; Cui, Jianmin

    2014-01-01

    Voltage-gated potassium (Kv) channels contain voltage-sensing (VSD) and pore-gate (PGD) structural domains. During voltage-dependent gating, conformational changes in the two domains are coupled giving rise to voltage-dependent opening of the channel. In addition to membrane voltage, KCNQ (Kv7) channel opening requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Recent studies suggest that PIP2 serves as a cofactor to mediate VSD-PGD coupling in KCNQ1 channels. In this review, we put these findings in the context of the current understanding of voltage-dependent gating, lipid modulation of Kv channel activation, and PIP2-regulation of KCNQ channels. We suggest that lipid-mediated coupling of functional domains is a common mechanism among KCNQ channels that may be applicable to other Kv channels and membrane proteins. PMID:24904429

  5. Gate-controlled topological conducting channels in bilayer graphene.

    PubMed

    Li, Jing; Wang, Ke; McFaul, Kenton J; Zern, Zachary; Ren, Yafei; Watanabe, Kenji; Taniguchi, Takashi; Qiao, Zhenhua; Zhu, Jun

    2016-12-01

    The existence of inequivalent valleys K and K' in the momentum space of 2D hexagonal lattices provides a new electronic degree of freedom, the manipulation of which can potentially lead to new types of electronics, analogous to the role played by electron spin. In materials with broken inversion symmetry, such as an electrically gated bilayer graphene (BLG), the momentum-space Berry curvature Ω carries opposite sign in the K and K' valleys. A sign reversal of Ω along an internal boundary of the sheet gives rise to counterpropagating 1D conducting modes encoded with opposite-valley indices. These metallic states are topologically protected against backscattering in the absence of valley-mixing scattering, and thus can carry current ballistically. In BLG, the reversal of Ω can occur at the domain wall of AB- and BA-stacked domains, or at the line junction of two oppositely gated regions. The latter approach can provide a scalable platform to implement valleytronic operations, such as valves and waveguides, but it is technically challenging to realize. Here, we fabricate a dual-split-gate structure in BLG and present evidence of the predicted metallic states in electrical transport. The metallic states possess a mean free path (MFP) of up to a few hundred nanometres in the absence of a magnetic field. The application of a perpendicular magnetic field suppresses the backscattering significantly and enables a junction 400 nm in length to exhibit conductance close to the ballistic limit of 4e(2)/h at 8 T. Our experiment paves the way to the realization of gate-controlled ballistic valley transport and the development of valleytronic applications in atomically thin materials.

  6. Gate-controlled topological conducting channels in bilayer graphene

    NASA Astrophysics Data System (ADS)

    Li, Jing; Wang, Ke; McFaul, Kenton J.; Zern, Zachary; Ren, Yafei; Watanabe, Kenji; Taniguchi, Takashi; Qiao, Zhenhua; Zhu, Jun

    2016-12-01

    The existence of inequivalent valleys K and K‧ in the momentum space of 2D hexagonal lattices provides a new electronic degree of freedom, the manipulation of which can potentially lead to new types of electronics, analogous to the role played by electron spin. In materials with broken inversion symmetry, such as an electrically gated bilayer graphene (BLG), the momentum-space Berry curvature Ω carries opposite sign in the K and K‧ valleys. A sign reversal of Ω along an internal boundary of the sheet gives rise to counterpropagating 1D conducting modes encoded with opposite-valley indices. These metallic states are topologically protected against backscattering in the absence of valley-mixing scattering, and thus can carry current ballistically. In BLG, the reversal of Ω can occur at the domain wall of AB- and BA-stacked domains, or at the line junction of two oppositely gated regions. The latter approach can provide a scalable platform to implement valleytronic operations, such as valves and waveguides, but it is technically challenging to realize. Here, we fabricate a dual-split-gate structure in BLG and present evidence of the predicted metallic states in electrical transport. The metallic states possess a mean free path (MFP) of up to a few hundred nanometres in the absence of a magnetic field. The application of a perpendicular magnetic field suppresses the backscattering significantly and enables a junction 400 nm in length to exhibit conductance close to the ballistic limit of 4e2/h at 8 T. Our experiment paves the way to the realization of gate-controlled ballistic valley transport and the development of valleytronic applications in atomically thin materials.

  7. Calmodulin and Ca(2+) control of voltage gated Na(+) channels.

    PubMed

    Gabelli, Sandra B; Yoder, Jesse B; Tomaselli, Gordon F; Amzel, L Mario

    2016-01-01

    The structures of the cytosolic portion of voltage activated sodium channels (CTNav) in complexes with calmodulin and other effectors in the presence and the absence of calcium provide information about the mechanisms by which these effectors regulate channel activity. The most studied of these complexes, those of Nav1.2 and Nav1.5, show details of the conformations and the specific contacts that are involved in channel regulation. Another voltage activated sodium channel, Nav1.4, shows significant calcium dependent inactivation, while its homolog Nav1.5 does not. The available structures shed light on the possible localization of the elements responsible for this effect. Mutations in the genes of these 3 Nav channels are associated with several disease conditions: Nav1.2, neurological conditions; Nav1.4, syndromes involving skeletal muscle; and Nav1.5, cardiac arrhythmias. Many of these disease-specific mutations are located at the interfaces involving CTNav and its effectors.

  8. Surface dynamics of voltage-gated ion channels

    PubMed Central

    Heine, Martin; Ciuraszkiewicz, Anna; Voigt, Andreas; Heck, Jennifer; Bikbaev, Arthur

    2016-01-01

    ABSTRACT Neurons encode information in fast changes of the membrane potential, and thus electrical membrane properties are critically important for the integration and processing of synaptic inputs by a neuron. These electrical properties are largely determined by ion channels embedded in the membrane. The distribution of most ion channels in the membrane is not spatially uniform: they undergo activity-driven changes in the range of minutes to days. Even in the range of milliseconds, the composition and topology of ion channels are not static but engage in highly dynamic processes including stochastic or activity-dependent transient association of the pore-forming and auxiliary subunits, lateral diffusion, as well as clustering of different channels. In this review we briefly discuss the potential impact of mobile sodium, calcium and potassium ion channels and the functional significance of this for individual neurons and neuronal networks. PMID:26891382

  9. Modulation of BKCa channel gating by endogenous signaling molecules.

    PubMed

    Hou, Shangwei; Heinemann, Stefan H; Hoshi, Toshinori

    2009-02-01

    Large-conductance Ca(2+)- and voltage-activated K(+) (BK(Ca), MaxiK, or Slo1) channels are expressed in almost every tissue in our body and participate in many critical functions such as neuronal excitability, vascular tone regulation, and neurotransmitter release. The functional versatility of BK(Ca) channels owes in part to the availability of a spectacularly wide array of biological modulators of the channel function. In this review, we focus on modulation of BK(Ca) channels by small endogenous molecules, emphasizing their molecular mechanisms. The mechanistic information available from studies on the small naturally occurring modulators is expected to contribute to our understanding of the physiological and pathophysiological roles of BK(Ca) channels.

  10. Different effects on gating of three myotonia-causing mutations in the inactivation gate of the human muscle sodium channel.

    PubMed Central

    Mitrović, N; George, A L; Lerche, H; Wagner, S; Fahlke, C; Lehmann-Horn, F

    1995-01-01

    1. Three mutations at the same site in the inactivation gate of the alpha-subunit of the human muscle Na+ channel, G1306E, G1306V and G1306A, cause three phenotypes of K(+)-aggravated myotonia: G1306E as the most severe and G1306A as the most benign form. 2. Recombinant wildtype (WT) and mutant (G1306E, G1306V and G1306A) human Na+ channels were expressed in human embryonic kidney cells (HEK293). G1306E and G1306V channels showed a distinct increase in the time constants of inactivation (tau h1 and tau h2) and in the ratios of steady-state to peak currents (Iss/Ipeak) (e.g. at 0 mV, G1306E vs. WT; tau h1, 1.29 +/- 0.10 vs. 0.52 +/- 0.01 ms; Iss/Ipeak, 2.90 +/- 0.40 vs. 0.93 +/- 0.19%). G1306A channels showed only an increase in tau h1 (0.74 +/- 0.07 ms). For G1306E and G1306V channels, the steady-state inactivation curves, as well as the voltage dependence of the rate of recovery from inactivation, were shifted by +15 mV. For G1306A the h infinity curve was shifted by only +5 mV. 3. G1306E and G1306V channels showed prolonged current rise times and later first openings suggesting slowing of activation. For G1306E channels only, the steady-state activation curve was shifted by -7 mV. For all mutants the deactivation time constants were increased. 4. We conclude that (i) the combination of alterations in inactivation and activation produces the slowing of the current decay, (ii) the slowed inactivation is most responsible for myotonia, and (iii) the shift of the steady-state activation curve, seen only with G1306E channels, may explain the severity of this phenotype. 5. The results suggest that two of the mutations in the Na+ channel inactivation gate also alter channel activation and deactivation. PMID:7473241

  11. State-dependent blocker interactions with the CFTR chloride channel: implications for gating the pore.

    PubMed

    Linsdell, Paul

    2014-12-01

    Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is subject to voltage-dependent open-channel block by a diverse range of cytoplasmic anions. However, in most cases the ability of these blocking substances to influence the pore opening and closing process has not been reported. In the present work, patch clamp recording was used to investigate the state-dependent block of CFTR by cytoplasmic Pt(NO2)4(2-) ions. Two major effects of Pt(NO2)4(2-) were identified. First, this anion caused fast, voltage-dependent block of open channels, leading to an apparent decrease in single-channel current amplitude. Secondly, Pt(NO2)4(2-) also decreased channel open probability due to an increase in interburst closed times. Interestingly, mutations in the pore that weakened (K95Q) or strengthened (I344K, V345K) interactions with Pt(NO2)4(2-) altered blocker effects both on Cl(-) permeation and on channel gating, suggesting that both these effects are a consequence of Pt(NO2)4(2-) interaction with a single site within the pore. Experiments at reduced extracellular Cl(-) concentration hinted that Pt(NO2)4(2-) may have a third effect, possibly increasing channel activity by interfering with channel closure. These results suggest that Pt(NO2)4(2-) can enter from the cytoplasm into the pore inner vestibule of both open and closed CFTR channels, and that Pt(NO2)4(2-) bound in the inner vestibule blocks Cl(-) permeation as well as interfering with channel opening and, perhaps, channel closure. Implications for the location of the channel gate in the pore, and the operation of this gate, are discussed.

  12. Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus.

    PubMed

    DeCaen, Paul G; Takahashi, Yuka; Krulwich, Terry A; Ito, Masahiro; Clapham, David E

    2014-11-11

    Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na(+) to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na(+) channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na(+), Ca(2+) and K(+) ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na(+) or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

  13. Voltage-gated sodium channel modulation by scorpion α-toxins

    PubMed Central

    Bosmans, Frank; Tytgat, Jan

    2007-01-01

    Voltage-gated Na+ channels are integral membrane proteins that function as a gateway for a selective permeation of sodium ions across biological membranes. In this way, they are crucial players for the generation of action potentials in excitable cells. Voltage-gated Na+ channels are encoded by at least nine genes in mammals. The different isoforms have remarkably similar functional properties, but small changes in function and pharmacology are biologically well-defined, as underscored by mutations that cause several diseases and by modulation of a myriad of compounds respectively. This review will stress on the modulation of voltage-gated Na+ channels by scorpion alpha-toxins. Nature has designed these two classes of molecules as if they were predestined to each other: an inevitable ‘encounter’ between a voltage-gated Na+ channel isoform and an alpha-toxin from scorpion venom indeed results in a dramatically changed Na+ current phenotype with clear-cut consequences on electrical excitability and sometimes life or death. This fascinating aspect justifies an overview on scorpion venoms, their alpha-toxins and the Na+ channel targets they are built for, as well as on the molecular determinants that govern the selectivity and affinity of this ‘inseparable duo’. PMID:17087986

  14. FPL 64176 Modification of CaV1.2 L-Type Calcium Channels: Dissociation of Effects on Ionic Current and Gating Current

    PubMed Central

    McDonough, Stefan I.; Mori, Yasuo; Bean, Bruce P.

    2005-01-01

    FPL 64176 (FPL) is a nondihydropyridine compound that dramatically increases macroscopic inward current through L-type calcium channels and slows activation and deactivation. To understand the mechanism by which channel behavior is altered, we compared the effects of the drug on the kinetics and voltage dependence of ionic currents and gating currents. Currents from a homogeneous population of channels were obtained using cloned rabbit CaV1.2 (α1C, cardiac L-type) channels stably expressed in baby hamster kidney cells together with β1a and α2δ1 subunits. We found a striking dissociation between effects of FPL on ionic currents, which were modified strongly, and on gating currents, which were not detectably altered. Inward ionic currents were enhanced ∼5-fold for a voltage step from −90 mV to +10 mV. Kinetics of activation and deactivation were slowed dramatically at most voltages. Curiously, however, at very hyperpolarized voltages (<−250 mV), deactivation was actually faster in FPL than in control. Gating currents were measured using a variety of inorganic ions to block ionic current and also without blockers, by recording gating current at the reversal potential for ionic current (+50 mV). Despite the slowed kinetics of ionic currents, FPL had no discernible effect on the fundamental movements of gating charge that drive channel gating. Instead, FPL somehow affects the coupling of charge movement to opening and closing of the pore. An intriguing possibility is that the drug causes an inactivated state to become conducting without otherwise affecting gating transitions. PMID:15501945

  15. Cloning and functional expression of a voltage-gated calcium channel alpha1 subunit from jellyfish.

    PubMed

    Jeziorski, M C; Greenberg, R M; Clark, K S; Anderson, P A

    1998-08-28

    Voltage-gated Ca2+ channels in vertebrates comprise at least seven molecular subtypes, each of which produces a current with distinct kinetics and pharmacology. Although several invertebrate Ca2+ channel alpha1 subunits have also been cloned, their functional characteristics remain unclear, as heterologous expression of a full-length invertebrate channel has not previously been reported. We have cloned a cDNA encoding the alpha1 subunit of a voltage-gated Ca2+ channel from the scyphozoan jellyfish Cyanea capillata, one of the earliest existing organisms to possess neural and muscle tissue. The deduced amino acid sequence of this subunit, named CyCaalpha1, is more similar to vertebrate L-type channels (alpha1S, alpha1C, and alpha1D) than to non-L-type channels (alpha1A, alpha1B, and alpha1E) or low voltage-activated channels (alpha1G). Expression of CyCaalpha1 in Xenopus oocytes produces a high voltage-activated Ca2+ current that, unlike vertebrate L-type currents, is only weakly sensitive to 1,4-dihydropyridine or phenylalkylamine Ca2+ channel blockers and is not potentiated by the agonist S(-)-BayK 8644. In addition, the channel is less permeable to Ba2+ than to Ca2+ and is more permeable to Sr2+. CyCaalpha1 thus represents an ancestral L-type alpha1 subunit with significant functional differences from mammalian L-type channels.

  16. Spatial Distribution of Calcium-Gated Chloride Channels in Olfactory Cilia

    PubMed Central

    French, Donald A.; Badamdorj, Dorjsuren; Kleene, Steven J.

    2010-01-01

    Background In vertebrate olfactory receptor neurons, sensory cilia transduce odor stimuli into changes in neuronal membrane potential. The voltage changes are primarily caused by the sequential openings of two types of channel: a cyclic-nucleotide-gated (CNG) cationic channel and a calcium-gated chloride channel. In frog, the cilia are 25 to 200 µm in length, so the spatial distributions of the channels may be an important determinant of odor sensitivity. Principal Findings To determine the spatial distribution of the chloride channels, we recorded from single cilia as calcium was allowed to diffuse down the length of the cilium and activate the channels. A computational model of this experiment allowed an estimate of the spatial distribution of the chloride channels. On average, the channels were concentrated in a narrow band centered at a distance of 29% of the ciliary length, measured from the base of the cilium. This matches the location of the CNG channels determined previously. This non-uniform distribution of transduction proteins is consistent with similar findings in other cilia. Conclusions On average, the two types of olfactory transduction channel are concentrated in the same region of the cilium. This may contribute to the efficient detection of weak stimuli. PMID:21209888

  17. 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

  18. How and why are calcium currents curtailed in the skeletal muscle voltage‐gated calcium channels?

    PubMed Central

    Tuluc, Petronel

    2017-01-01

    Abstract Voltage‐gated calcium channels represent the sole mechanism converting electrical signals of excitable cells into cellular functions such as contraction, secretion and gene regulation. Specific voltage‐sensing domains detect changes in membrane potential and control channel gating. Calcium ions entering through the channel function as second messengers regulating cell functions, with the exception of skeletal muscle, where CaV1.1 essentially does not function as a channel but activates calcium release from intracellular stores. It has long been known that calcium currents are dispensable for skeletal muscle contraction. However, the questions as to how and why the channel function of CaV1.1 is curtailed remained obscure until the recent discovery of a developmental CaV1.1 splice variant with normal channel functions. This discovery provided new means to study the molecular mechanisms regulating the channel gating and led to the understanding that in skeletal muscle, calcium currents need to be restricted to allow proper regulation of fibre type specification and to prevent mitochondrial damage. PMID:27896815

  19. Optical electrophysiology for probing function and pharmacology of voltage-gated ion channels

    PubMed Central

    Zhang, Hongkang; Reichert, Elaine; Cohen, Adam E

    2016-01-01

    Voltage-gated ion channels mediate electrical dynamics in excitable tissues and are an important class of drug targets. Channels can gate in sub-millisecond timescales, show complex manifolds of conformational states, and often show state-dependent pharmacology. Mechanistic studies of ion channels typically involve sophisticated voltage-clamp protocols applied through manual or automated electrophysiology. Here, we develop all-optical electrophysiology techniques to study activity-dependent modulation of ion channels, in a format compatible with high-throughput screening. Using optical electrophysiology, we recapitulate many voltage-clamp protocols and apply to Nav1.7, a channel implicated in pain. Optical measurements reveal that a sustained depolarization strongly potentiates the inhibitory effect of PF-04856264, a Nav1.7-specific blocker. In a pilot screen, we stratify a library of 320 FDA-approved compounds by binding mechanism and kinetics, and find close concordance with patch clamp measurements. Optical electrophysiology provides a favorable tradeoff between throughput and information content for studies of NaV channels, and possibly other voltage-gated channels. DOI: http://dx.doi.org/10.7554/eLife.15202.001 PMID:27215841

  20. Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

    PubMed

    Coskun, Cagil; Purali, Nuhan

    2016-06-01

    Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.

  1. Recessed source concept in nanoscale vertical surrounding gate (VSG) MOSFETs for controlling short-channel effects

    NASA Astrophysics Data System (ADS)

    Subrahmanyam, B.; Jagadesh Kumar, M.

    2009-02-01

    In the recent past, vertical surrounding gate (VSG) MOSFETs have gained importance since defining their nanoscale channel length no longer depends on lithographic limitations and since they can lead to high packing densities. However, as the channel lengths decrease below 100 nm, VSG MOSFETs too suffer from short-channel effects due to the coupling between the drain and source side charges. In this paper, we demonstrate that using a recessed source, the short-channel effects in nanoscale VSG MOSFETs can be effectively controlled.

  2. 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

  3. A reduced mechanical model for cAMP-modulated gating in HCN channels

    PubMed Central

    Weißgraeber, Stephanie; Saponaro, Andrea; Thiel, Gerhard; Hamacher, Kay

    2017-01-01

    We developed an in silico mechanical model to analyze the process of cAMP-induced conformational modulations in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which conduct cations across the membrane of mammalian heart and brain cells. The structural analysis reveals a quaternary twist in the cytosolic parts of the four subunits in the channel tetramer. This motion augments the intrinsic dynamics of the very same protein structure. The pronounced differences between the cAMP bound and unbound form include a mutual interaction between the C-linker of the cyclic nucleotide binding domain (CNBD) and the linker between the S4 and S5 transmembrane domain of the channel. This allows a mechanistic annotation of the twisting motion in relation to the allosteric modulation of voltage-dependent gating of this channel by cAMP. PMID:28074902

  4. A reduced mechanical model for cAMP-modulated gating in HCN channels

    NASA Astrophysics Data System (ADS)

    Weißgraeber, Stephanie; Saponaro, Andrea; Thiel, Gerhard; Hamacher, Kay

    2017-01-01

    We developed an in silico mechanical model to analyze the process of cAMP-induced conformational modulations in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which conduct cations across the membrane of mammalian heart and brain cells. The structural analysis reveals a quaternary twist in the cytosolic parts of the four subunits in the channel tetramer. This motion augments the intrinsic dynamics of the very same protein structure. The pronounced differences between the cAMP bound and unbound form include a mutual interaction between the C-linker of the cyclic nucleotide binding domain (CNBD) and the linker between the S4 and S5 transmembrane domain of the channel. This allows a mechanistic annotation of the twisting motion in relation to the allosteric modulation of voltage-dependent gating of this channel by cAMP.

  5. Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel

    SciTech Connect

    Leng, Q.; Mercier, R.W.; Yao, W.; Berkowitz, G.A.

    1999-11-01

    Cyclic nucleotide-gated (cng) non-selective cation channels have been cloned from a number of animal systems. These channels are characterized by direct gating upon cAMO or cGMO binding to the intracellular portion of the channel protein, which leads to an increase in channel conductance. Animal cng channels are involved in signal transduction systems; they translate stimulus-induced changes in cytosolic cyclic nucleotide into altered cell membrane potential and/or cation flux as part of a signal cascade pathway. Putative plant homologs of animal cng channels have been identified. However, functional characterization (i.e., demonstration of cyclic-nucleotide-dependent ion currents) of a plant cng channel has not yet been accomplished. The authors report the cloning and first functional characterization of a plant member of this family of ion channels. The Arabidopsis cDNA AtCNGC2 encodes a polypeptide with deduced homology to the {alpha}-subunit of animal channels, and facilitates cyclic nucleotide-dependent cation currents upon expression in a number of heterologous systems. AtCNGC2 expression in a yeast mutant lacking a low-affinity K{sup +} uptake system complements growth inhibition only when lipophilic nucleotides are present in the culture medium. Voltage clamp analysis indicates that Xenopus lawvis oocytes injected with AtCNGC2 cRNA demonstrate cyclic-nucleotide-dependent, inward-rectifying K{sup +} currents. Human embryonic kidney cells (HEK293) transfected with AtCNGC2 cDNA demonstrate increased permeability to Ca{sup 2+} only in the presence of lipophilic cyclic nucleotides. The evidence presented here supports the functional classification of AtCNGC2 as a cyclic-nucleotide-gated cation channel, and presents the first direct evidence identifying a plant member of this ion channel family.

  6. The STIM1-binding site nexus remotely controls Orai1 channel gating

    PubMed Central

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

    2016-01-01

    The ubiquitously expressed Orai Ca2+ channels are gated through a unique process of intermembrane coupling with the Ca2+-sensing STIM proteins. Despite the significance of Orai1-mediated Ca2+ signals, how gating of Orai1 is triggered by STIM1 remains unknown. A widely held gating model invokes STIM1 binding directly to Orai1 pore-forming helix. Here we report that an Orai1 C-terminal STIM1-binding site, situated far from the N-terminal pore helix, alone provides the trigger that is necessary and sufficient for channel gating. We identify a critical ‘nexus' within Orai1 connecting the peripheral C-terminal STIM1-binding site to the Orai1 core helices. Mutation of the nexus transforms Orai1 into a persistently open state exactly mimicking the action of STIM1. We suggest that the Orai1 nexus transduces the STIM1-binding signal through a conformational change in the inner core helices, and that STIM1 remotely gates the Orai1 channel without the necessity for direct STIM1 contact with the pore-forming helix. PMID:27929067

  7. Kramers' diffusion theory applied to gating kinetics of voltage-dependent ion channels.

    PubMed Central

    Sigg, D; Qian, H; Bezanilla, F

    1999-01-01

    Kramers' diffusion theory of reaction rates in the condensed phase is considered as an alternative to the traditional discrete-state Markov (DSM) model in describing ion channel gating current kinetics. Diffusion theory can be expected to be particularly relevant in describing high-frequency (>100 kHz) events in channel activation. The generalized voltage sensor of a voltage-dependent ion channel is treated as a Brownian motion particle undergoing spatial diffusion along a one-dimensional energy landscape. Two classes of energy landscapes are considered. The first class contains large barriers, which give rise to gating currents with two distinct time scales: the usual low-frequency decay, which can modeled with a DSM scheme, and a high-frequency component arising from intrastate relaxation. Large depolarizations reduce potential barriers to such a degree that activation rates are diffusion limited, causing the two time scales to merge. Landscapes of the second class are either featureless or contain barriers that are small compared to kT; these are termed "drift landscapes." These landscapes require a larger friction coefficient to generate slow gating kinetics. The high-frequency component that appears with barrier models is not present in pure drift motion. The presence of a high-frequency component can be tested experimentally with large-bandwidth recordings of gating currents. Topics such as frequency domain analysis, spatial dependence of the friction coefficient, methods for determining the adequacy of a DSM model, and the development of physical models of gating are explored. PMID:9929481

  8. Evolution of voltage-gated ion channels at the emergence of Metazoa.

    PubMed

    Moran, Yehu; Barzilai, Maya Gur; Liebeskind, Benjamin J; Zakon, Harold H

    2015-02-15

    Voltage-gated ion channels are large transmembrane proteins that enable the passage of ions through their pore across the cell membrane. These channels belong to one superfamily and carry pivotal roles such as the propagation of neuronal and muscular action potentials and the promotion of neurotransmitter secretion in synapses. In this review, we describe in detail the current state of knowledge regarding the evolution of these channels with a special emphasis on the metazoan lineage. We highlight the contribution of the genomic revolution to the understanding of ion channel evolution and for revealing that these channels appeared long before the appearance of the first animal. We also explain how the elucidation of channel selectivity properties and function in non-bilaterian animals such as cnidarians (sea anemones, corals, jellyfish and hydroids) can contribute to the study of channel evolution. Finally, we point to open questions and future directions in this field of research. © 2015. Published by The Company of Biologists Ltd.

  9. Angular Sensitivity of Gated Micro-Channel Plate Framing Cameras

    SciTech Connect

    Landen, O L; Lobban, A; Tutt, T; Bell, P M; Costa, R; Ze, F

    2000-07-24

    Gated, microchannel-plate-based (MCP) framing cameras have been deployed worldwide for 0.2 - 9 keV x-ray imaging and spectroscopy of transient plasma phenomena. For a variety of spectroscopic and imaging applications, the angular sensitivity of MCPs must be known for correctly interpreting the data. We present systematic measurements of angular sensitivity at discrete relevant photon energies and arbitrary MCP gain. The results can been accurately predicted by using a simple 2D approximation to the 3D MCP geometry and by averaging over all possible photon ray paths.

  10. N Channel JFET Based Digital Logic Gate Structure

    NASA Technical Reports Server (NTRS)

    Krasowski, Michael J (Inventor)

    2013-01-01

    An apparatus is provided that includes a first field effect transistor with a source tied to zero volts and a drain tied to voltage drain drain (Vdd) through a first resistor. The apparatus also includes a first node configured to tie a second resistor to a third resistor and connect to an input of a gate of the first field effect transistor in order for the first field effect transistor to receive a signal. The apparatus also includes a second field effect transistor configured as a unity gain buffer having a drain tied to Vdd and an uncommitted source.

  11. Proline-induced hinges in transmembrane helices: possible roles in ion channel gating.

    PubMed

    Tieleman, D P; Shrivastava, I H; Ulmschneider, M R; Sansom, M S

    2001-08-01

    A number of ion channels contain transmembrane (TM) alpha-helices that contain proline-induced molecular hinges. These TM helices include the channel-forming peptide alamethicin (Alm), the S6 helix from voltage-gated potassium (Kv) channels, and the D5 helix from voltage-gated chloride (CLC) channels. For both Alm and KvS6, experimental data implicate hinge-bending motions of the helix in an aspect of channel gating. We have compared the hinge-bending motions of these TM helices in bilayer-like environments by multi-nanosecond MD simulations in an attempt to describe motions of these helices that may underlie possible modes of channel gating. Alm is an alpha-helical channel-forming peptide, which contains a central kink associated with a Gly-x-x-Pro motif in its sequence. Simulations of Alm in a TM orientation for 10 ns in an octane slab indicate that the Gly-x-x-Pro motif acts as a molecular hinge. The S6 helix from Shaker Kv channels contains a Pro-Val-Pro motif. Modeling studies and recent experimental data suggest that the KvS6 helix may be kinked in the vicinity of this motif. Simulations (10 ns) of an isolated KvS6 helix in an octane slab and in a POPC bilayer reveal hinge-bending motions. A pattern-matching approach was used to search for possible hinge-bending motifs in the TM helices of other ion channel proteins. This uncovered a conserved Gly-x-Pro motif in TM helix D5 of CLC channels. MD simulations of a model of hCLC1-D5 spanning an octane slab suggest that this channel also contains a TM helix that undergoes hinge-bending motion. In conclusion, our simulations suggest a model in which hinge-bending motions of TM helices may play a functional role in the gating mechanisms of several different families of ion channels.

  12. Modulation of BK channel voltage gating by different auxiliary β subunits

    PubMed Central

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

    2012-01-01

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

  13. Voltage-gated channel properties of epithelial cells in porcine vomeronasal organ.

    PubMed

    Ishida, Tetsuya; Takei, Ryo; Gautam, Shree Hari; Otsuguro, Ken-Ichi; Ohta, Toshio; Ito, Shigeo; Habara, Yoshiaki; Saito, Toshiyuki

    2008-08-29

    Bipolar vomeronasal sensory neurons (VSNs) in the vomeronasal organ (VNO) are believed to detect pheromones in most mammals. The vomeronasal sensory epithelium (VSE) is composed of VSNs and supporting cells. There are morphological differences in VNOs between species. Many electrophysiological experiments have been performed on rodent VSEs but few on other mammals. We therefore investigated voltage-gated channel properties of cells in the porcine VSE using slice whole-cell voltage-clamp techniques. In immunohistochemical study of the porcine VSE, most PGP9.5-immunoreactive cells were found between the middle and basal region, and negative cells were distributed in the apical to middle region. Depolarizing pulses to epithelial cells from -90mV produced transient inward Na+ channel currents and sustained outward K+ channel currents with various amplitudes. The distribution of cells having high and low Na+ current densities was mostly consistent with the histological distribution of VSNs and supporting cells, respectively. The half-inactivation voltage of voltage-gated Na+ channels in supporting cells was 26mV more negative than that in VSNs. Voltage-gated K+ channel currents in both cell types were suppressed by tetraethylammonium to the same extent. VSNs possessed TTX-sensitive voltage-gated Na+ channels and Ni2+ -sensitive T-type Ca2+ channels. These results suggest that the histological distribution of porcine vomeronasal epithelial cells is more similar to the dog and goat than to rodents, and that the electrophysiological characteristics of porcine vomeronasal epithelial cells are similar to those of rodents. It is also suggested that porcine VSNs detecting pheromones generate action potentials through these channels.

  14. Gating characteristics of a steeply voltage-dependent gap junction channel in rat Schwann cells

    PubMed Central

    1993-01-01

    The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, Gss was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary junctional currents was detected corresponding to an unitary channel conductance of approximately 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic Gss voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states. PMID:8301264

  15. Differential distribution of voltage-gated calcium channels in dopaminergic neurons of the rat retina.

    PubMed

    Witkovsky, Paul; Shen, Changpeng; McRory, John

    2006-07-20

    We studied by immunocytochemistry and Western blots the identity and cellular distribution of voltage-gated calcium channels within dopaminergic neurons of the rat retina. The aim was to associate particular calcium channel subtypes with known activities of the neuron (e.g., transmitter release from axon terminals). Five voltage-gated calcium channels were identified: alpha1A, alpha1B, alpha1E, alpha1F, and alpha1H. All of these, except the alpha1B subtype, were found within dopaminergic perikarya. The alpha1B channels were concentrated at axon terminal rings, together with alpha1A calcium channels. In contrast, alpha1H calcium channels were most abundant in the dendrites, and alpha1F calcium channels were restricted to the perikaryon. The alpha1E calcium channel was present at such a low density that its cellular distribution beyond the perikaryon could not be determined. Our findings are consistent with the available pharmacological data indicating that alpha1A and alpha1B calcium channels control the major fraction of dopamine release in the rat retina. (c) 2006 Wiley-Liss, Inc.

  16. The Link between Ion Permeation and Inactivation Gating of Kv4 Potassium Channels

    PubMed Central

    Shahidullah, Mohammad; Covarrubias, Manuel

    2003-01-01

    Kv4 potassium channels undergo rapid inactivation but do not seem to exhibit the classical N-type and C-type mechanisms present in other Kv channels. We have previously hypothesized that Kv4 channels preferentially inactivate from the preopen closed state, which involves regions of the channel that contribute to the internal vestibule of the pore. To further test this hypothesis, we have examined the effects of permeant ions on gating of three Kv4 channels (Kv4.1, Kv4.2, and Kv4.3) expressed in Xenopus oocytes. Rb+ is an excellent tool for this purpose because its prolonged residency time in the pore delays K+ channel closing. The data showed that, only when Rb+ carried the current, both channel closing and the development of macroscopic inactivation are slowed (1.5- to 4-fold, relative to the K+ current). Furthermore, macroscopic Rb+ currents were larger than K+ currents (1.2- to 3-fold) as the result of a more stable open state, which increases the maximum open probability. These results demonstrate that pore occupancy can influence inactivation gating in a manner that depends on how channel closing impacts inactivation from the preopen closed state. By examining possible changes in ionic selectivity and the influence of elevating the external K+ concentration, additional experiments did not support the presence of C-type inactivation in Kv4 channels. PMID:12547775

  17. Utrophin suppresses low frequency oscillations and coupled gating of mechanosensitive ion channels in dystrophic skeletal muscle.

    PubMed

    Lansman, Jeffry B

    2015-01-01

    An absence of utrophin in muscle from mdx mice prolongs the open time of single mechanosensitive channels. On a time scale much longer than the duration of individual channel activations, genetic depletion of utrophin produces low frequency oscillations of channel open probability. Oscillatory channel opening occurred in the dystrophin/utrophin mutants, but was absent in wild-type and mdx fibers. By contrast, small conductance channels showed random gating behavior when present in the same patch. Applying a negative pressure to a patch on a DKO fiber produced a burst of mode II activity, but channels subsequently closed and remained silent for tens of seconds during the maintained pressure stimulus. In addition, simultaneous opening of multiple MS channels could be frequently observed in recordings from patches on DKO fibers, but only rarely in wild-type and mdx muscle. A model which accounts for the single-channel data is proposed in which utrophin acts as gating spring which maintains the mechanical stability a caveolar-like compartment. The state of this compartment is suggested to be dynamic; its continuity with the extracellular surface varying over seconds to minutes. Loss of the mechanical stability of this compartment contributes to pathogenic Ca(2+) entry through MS channels in Duchenne dystrophy.

  18. Fast and Slow Gating Relaxations in the Muscle Chloride Channel Clc-1

    PubMed Central

    Accardi, Alessio; Pusch, Michael

    2000-01-01

    Gating of the muscle chloride channel CLC-1 involves at least two processes evidenced by double-exponential current relaxations when stepping the voltage to negative values. However, there is little information about the gating of CLC-1 at positive voltages. Here, we analyzed macroscopic gating of CLC-1 over a large voltage range (from −160 to +200 mV). Activation was fast at positive voltages but could be easily followed using envelope protocols that employed a tail pulse to −140 mV after stepping the voltage to a certain test potential for increasing durations. Activation was biexponential, demonstrating the presence of two gating processes. Both time constants became exponentially faster at positive voltages. A similar voltage dependence was also seen for the fast gate time constant of CLC-0. The voltage dependence of the time constant of the fast process of CLC-1, τf, was steeper than that of the slow one, τs (apparent activation valences were zf ∼ −0.79 and zs ∼ −0.42) such that at +200 mV the two processes became kinetically distinct by almost two orders of magnitude (τf ∼ 16 μs, τs ∼ 1 ms). This voltage dependence is inconsistent with a previously published gating model for CLC-1 (Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695–706). The kinetic difference at 200 mV allowed us to separate the steady state open probabilities of the two processes assuming that they reflect two parallel (not necessarily independent) gates that have to be open simultaneously to allow ion conduction. Both open probabilities could be described by Boltzmann functions with gating valences around one and with nonzero “offsets” at negative voltages, indicating that the two “gates” never close completely. For comparison with single channel data and to correlate the two gating processes with the two gates of CLC-0, we characterized their voltage, pHint, and [Cl]ext dependence, and the dominant myotonia inducing

  19. Voltage-sensing domain of voltage-gated proton channel Hv1 shares mechanism of block with pore domains

    PubMed Central

    Hong, Liang; Pathak, Medha M.; Kim, Iris H.; Ta, Dennis; Tombola, Francesco

    2012-01-01

    SUMMARY Voltage-gated sodium, potassium, and calcium channels are made of a pore domain (PD) controlled by four voltage-sensing domains (VSDs). The PD contains the ion permeation pathway and the activation gate located on the intracellular side of the membrane. A large number of small molecules are known to inhibit the PD by acting as open channel blockers. The voltage-gated proton channel Hv1 is made of two VSDs and lacks the PD. The location of the activation gate in the VSD is unknown and open channel blockers for VSDs have not yet been identified. Here we describe a class of small molecules which act as open channel blockers on the Hv1 VSD and find that a highly conserved phenylalanine in the charge transfer center of the VSD plays a key role in blocker binding. We then use one of the blockers to show that Hv1 contains two intracellular and allosterically-coupled gates. PMID:23352164

  20. Four-mode gating model of fast inactivation of sodium channel Nav1.2a.

    PubMed

    Huth, Tobias; Schmidtmayer, Johann; Alzheimer, Christian; Hansen, Ulf-Peter

    2008-10-01

    Basic principles of the gating mechanisms of neuronal sodium channels, especially the fast inactivation process, were revealed by a quantitative analysis of the effects of the chemically irreversible modifying agent chloramine T. The compound is known to enhance the open probability of sodium channels by interfering with the inactivation process. The key for the deduction of structure-function relationships was obtained from the analysis of single-channel patch-clamp data, especially the finding that chloramine T-induced modification of inactivation occurred in four steps. These steps were termed modes 1-4 (four-mode gating model), and their temporal sequence was always the same. The kinetic analysis of single-channel traces with an improved two-dimensional dwell-time fit revealed the possible mechanism related to each mode. Similarities to the kinetics of the sodium channel mutant F1489Q led to the assignment of modes 1 and 2 to transient defects in the locking of the inactivation particle (hinged lid). In the third mode, the hinged lid was unable to lock permanently. Finally, in mode 4, the apparent single-channel current was reduced, which could be explained by fast gating, presumably related to the selectivity filter.

  1. Voltage-Gated Proton Channels: Molecular Biology, Physiology, and Pathophysiology of the HV Family

    PubMed Central

    2013-01-01

    Voltage-gated proton channels (HV) are unique, in part because the ion they conduct is unique. HV channels are perfectly selective for protons and have a very small unitary conductance, both arguably manifestations of the extremely low H+ concentration in physiological solutions. They open with membrane depolarization, but their voltage dependence is strongly regulated by the pH gradient across the membrane (ΔpH), with the result that in most species they normally conduct only outward current. The HV channel protein is strikingly similar to the voltage-sensing domain (VSD, the first four membrane-spanning segments) of voltage-gated K+ and Na+ channels. In higher species, HV channels exist as dimers in which each protomer has its own conduction pathway, yet gating is cooperative. HV channels are phylogenetically diverse, distributed from humans to unicellular marine life, and perhaps even plants. Correspondingly, HV functions vary widely as well, from promoting calcification in coccolithophores and triggering bioluminescent flashes in dinoflagellates to facilitating killing bacteria, airway pH regulation, basophil histamine release, sperm maturation, and B lymphocyte responses in humans. Recent evidence that hHV1 may exacerbate breast cancer metastasis and cerebral damage from ischemic stroke highlights the rapidly expanding recognition of the clinical importance of hHV1. PMID:23589829

  2. Voltage-gated proton channels: molecular biology, physiology, and pathophysiology of the H(V) family.

    PubMed

    DeCoursey, Thomas E

    2013-04-01

    Voltage-gated proton channels (H(V)) are unique, in part because the ion they conduct is unique. H(V) channels are perfectly selective for protons and have a very small unitary conductance, both arguably manifestations of the extremely low H(+) concentration in physiological solutions. They open with membrane depolarization, but their voltage dependence is strongly regulated by the pH gradient across the membrane (ΔpH), with the result that in most species they normally conduct only outward current. The H(V) channel protein is strikingly similar to the voltage-sensing domain (VSD, the first four membrane-spanning segments) of voltage-gated K(+) and Na(+) channels. In higher species, H(V) channels exist as dimers in which each protomer has its own conduction pathway, yet gating is cooperative. H(V) channels are phylogenetically diverse, distributed from humans to unicellular marine life, and perhaps even plants. Correspondingly, H(V) functions vary widely as well, from promoting calcification in coccolithophores and triggering bioluminescent flashes in dinoflagellates to facilitating killing bacteria, airway pH regulation, basophil histamine release, sperm maturation, and B lymphocyte responses in humans. Recent evidence that hH(V)1 may exacerbate breast cancer metastasis and cerebral damage from ischemic stroke highlights the rapidly expanding recognition of the clinical importance of hH(V)1.

  3. Local anesthetic and antiepileptic drug access and binding to a bacterial voltage-gated sodium channel.

    PubMed

    Boiteux, Céline; Vorobyov, Igor; French, Robert J; French, Christopher; Yarov-Yarovoy, Vladimir; Allen, Toby W

    2014-09-09

    Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the "hinged lid"-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water-protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors.

  4. Novel nucleotide-binding sites in ATP-sensitive potassium channels formed at gating interfaces.

    PubMed

    Dong, Ke; Tang, Lie-Qi; MacGregor, Gordon G; Leng, Qiang; Hebert, Steven C

    2005-04-06

    The coupling of cell metabolism to membrane electrical activity is a vital process that regulates insulin secretion, cardiac and neuronal excitability and the responses of cells to ischemia. ATP-sensitive potassium channels (K(ATP); Kir6.x) are a major part of this metabolic-electrical coupling system and translate metabolic signals such as the ATP:ADP ratio to changes in the open or closed state (gate) of the channel. The localization of the nucleotide-binding site (NBS) on Kir6.x channels and how nucleotide binding gates these K(ATP) channels remain unclear. Here, we use fluorescent nucleotide binding to purified