Science.gov

Sample records for receptor channel gating

  1. Kinetics of unliganded acetylcholine receptor channel gating.

    PubMed Central

    Jackson, M B

    1986-01-01

    Open- and closed-state lifetimes of unliganded acetylcholine receptor channel activity were analyzed by the method of likelihood maximazation. For both open times and closed times, the best-fitting density is most often a sum of two exponentials. These multiple open states cannot depend on the number of receptor binding sites occupied since they are observed in the absence of ligand. The rate of spontaneous opening and the faster decay constant of closing increased as the membrane was hyperpolarized. The voltage dependence of the rate of spontaneous opening is stronger than that for curare-liganded channels. Evidence that the acetylcholine receptor channel can open spontaneously in the absence of ligand has been presented previously (Sanchez et al, 1983; Brehm et al, 1984; Jackson, 1984). To add to this evidence, alpha-bungarotoxin was added to the patch electrode, causing the frequency of openings to decay with time. The rate constant determined from this decay is similar to rate constants reported for the binding of iodinated alpha-bungarotoxin to the acetylcholine receptor. The frequency of unliganded channel opening has been estimated as 2 X 10(-3) s-1 per receptor. A comparison of carbamylcholine-liganded and spontaneous gating transition rates suggests that ligand binding increases the rate of opening by a factor of 1.4 X 10(7). Carbamylcholine binding increases the mean open time by a factor of 5. Thus, a cholinergic agonist activates the acetylcholine receptor by destabilizing the closed state. The liganded and unliganded channel gating rates were used to analyze the energetics of ligand activation of the acetylcholine receptor channel, and to relate the open channel dissociation constant to the closed channel dissociation constant. PMID:2421793

  2. Coupled gating between cardiac calcium release channels (ryanodine receptors).

    PubMed

    Marx, S O; Gaburjakova, J; Gaburjakova, M; Henrikson, C; Ondrias, K; Marks, A R

    2001-06-01

    Excitation-contraction coupling in heart muscle requires the activation of Ca(2+)-release channels/type 2 ryanodine receptors (RyR2s) by Ca(2+) influx. RyR2s are arranged on the sarcoplasmic reticular membrane in closely packed arrays such that their large cytoplasmic domains contact one another. We now show that multiple RyR2s can be isolated under conditions such that they remain physically coupled to one another. When these coupled channels are examined in planar lipid bilayers, multiple channels exhibit simultaneous gating, termed "coupled gating." Removal of the regulatory subunit, the FK506 binding protein (FKBP12.6), functionally but not physically uncouples multiple RyR2 channels. Coupled gating between RyR2 channels may be an important regulatory mechanism in excitation-contraction coupling as well as in other signaling pathways involving intracellular Ca(2+) release. PMID:11397781

  3. Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors

    NASA Astrophysics Data System (ADS)

    Zhou, Ming; Morais-Cabral, João H.; Mann, Sabine; MacKinnon, Roderick

    2001-06-01

    Many voltage-dependent K+ channels open when the membrane is depolarized and then rapidly close by a process called inactivation. Neurons use inactivating K+ channels to modulate their firing frequency. In Shaker-type K+ channels, the inactivation gate, which is responsible for the closing of the channel, is formed by the channel's cytoplasmic amino terminus. Here we show that the central cavity and inner pore of the K+ channel form the receptor site for both the inactivation gate and small-molecule inhibitors. We propose that inactivation occurs by a sequential reaction in which the gate binds initially to the cytoplasmic channel surface and then enters the pore as an extended peptide. This mechanism accounts for the functional properties of K+ channel inactivation and indicates that the cavity may be the site of action for certain drugs that alter cation channel function.

  4. 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

  5. Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels.

    PubMed

    Kasuya, Go; Fujiwara, Yuichiro; Takemoto, Mizuki; Dohmae, Naoshi; Nakada-Nakura, Yoshiko; Ishitani, Ryuichiro; Hattori, Motoyuki; Nureki, Osamu

    2016-02-01

    P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn(2+) ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn(2+) potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg(2+). Overall, our work provides structural insights into the divalent cation modulations of P2X receptors. PMID:26804916

  6. Statistical methods for model discrimination. Applications to gating kinetics and permeation of the acetylcholine receptor channel.

    PubMed Central

    Horn, R

    1987-01-01

    Methods are described for discrimination of models of the gating kinetics and permeation of single ionic channels. Both maximum likelihood and regression procedures are discussed. In simple situations, where models are nested, standard hypothesis tests can be used. More commonly, however, non-nested models are of interest, and several procedures are described for model discrimination in these cases, including Monte Carlo methods, which allow the comparison of models at significance levels of choice. As an illustration, the methods are applied to single-channel data from acetylcholine receptor channels. PMID:2435330

  7. G-protein–gated TRP-like Cationic Channel Activated by Muscarinic Receptors

    PubMed Central

    Zholos, Alexander V.; Zholos, Andrey A.; Bolton, Thomas B.

    2004-01-01

    There is little information about the mechanisms by which G-protein–coupled receptors gate ion channels although many ionotropic receptors are well studied. We have investigated gating of the muscarinic cationic channel, which mediates the excitatory effect of acetylcholine in smooth muscles, and proposed a scheme consisting of four pairs of closed and open states. Channel kinetics appeared to be the same in cell-attached or outside-out patches whether the channel was activated by carbachol application or by intracellular dialysis with GTPγS. Since in the latter case G-proteins are permanently active, it is concluded that the cationic channel is the major determinant of its own gating, similarly to the KACh channel (Ivanova-Nikolova, T.T., and G.E. Breitwieser. 1997. J. Gen. Physiol. 109:245–253). Analysis of adjacent-state dwell times revealed connections between the states that showed features conserved among many other ligand-gated ion channels (e.g., nAChR, BKCa channel). Open probability (PO) of the cationic channel was increased by membrane depolarization consistent with the prominent U-shaped I-V relationship of the muscarinic whole-cell current at negative potentials. Membrane potential affected transitions within each closed-open state pair but had little effect on transitions between pairs; thus, the latter are likely to be caused by interactions of the channel with its ligands, e.g., Ca2+ and Gαo-GTP. Channel activity was highly heterogeneous, as was evident from the prominent cycling behavior when PO was measured over 5-s intervals. This was related to the variable frequency of openings (as in the KACh channel) and, especially, to the number of long openings between consecutive long shuttings. Analysis of the underlying Markov chain in terms of probabilities allowed us to evaluate the contribution of each open state to the integral current (from shortest to longest open state: 0.1, 3, 24, and 73%) as PO increased 525-fold in three stages. PMID

  8. A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP.

    PubMed

    Valera, S; Hussy, N; Evans, R J; Adami, N; North, R A; Surprenant, A; Buell, G

    1994-10-01

    Extracellular ATP exerts its effects through P2 purinoceptors: these are ligand-gated ion channels (P2x) or G-protein-coupled receptors (P2Y, P2U). ATP at P2x receptors mediates synaptic transmission between neurons and from neurons to smooth muscle, being responsible, for example, for sympathetic vasoconstriction in small arteries and arterioles. We have now cloned a complementary DNA encoding the P2x receptor from rat vas deferens and expressed it in Xenopus oocytes and mammalian cells. ATP activates a cation-selective ion channel with relatively high calcium permeability. Structural predictions suggest that the protein (399 amino acids long) is mostly extracellular and contains only two transmembrane domains plus a pore-forming motif which resembles that of potassium channels. The P2x receptor thus defines a new family of ligand-gated ion channels. PMID:7523951

  9. Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor.

    PubMed

    Mei, Yingwu; Xu, Le; Mowrey, David D; Mendez Giraldez, Raul; Wang, Ying; Pasek, Daniel A; Dokholyan, Nikolay V; Meissner, Gerhard

    2015-07-10

    Type 1 ryanodine receptors (RyR1s) release Ca(2+) from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca(2+) release response in HEK293 cells and bound the RyR-specific ligand [(3)H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K(+) conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca(2+) release in HEK293 cells, low [(3)H]ryanodine binding levels, and channels that were not regulated by Ca(2+) and did not conduct Ca(2+) in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes. PMID:25998124

  10. Evolution of Pentameric Ligand-Gated Ion Channels: Pro-Loop Receptors

    PubMed Central

    Jaiteh, Mariama; Taly, Antoine; Hénin, Jérôme

    2016-01-01

    Pentameric ligand-gated ion channels (pLGICs) are ubiquitous neurotransmitter receptors in Bilateria, with a small number of known prokaryotic homologues. Here we describe a new inventory and phylogenetic analysis of pLGIC genes across all kingdoms of life. Our main finding is a set of pLGIC genes in unicellular eukaryotes, some of which are metazoan-like Cys-loop receptors, and others devoid of Cys-loop cysteines, like their prokaryotic relatives. A number of such “Cys-less” receptors also appears in invertebrate metazoans. Together, those findings draw a new distribution of pLGICs in eukaryotes. A broader distribution of prokaryotic channels also emerges, including a major new archaeal taxon, Thaumarchaeota. More generally, pLGICs now appear nearly ubiquitous in major taxonomic groups except multicellular plants and fungi. However, pLGICs are sparsely present in unicellular taxa, suggesting a high rate of gene loss and a non-essential character, contrasting with their essential role as synaptic receptors of the bilaterian nervous system. Multiple alignments of these highly divergent sequences reveal a small number of conserved residues clustered at the interface between the extracellular and transmembrane domains. Only the “Cys-loop” proline is absolutely conserved, suggesting the more fitting name “Pro loop” for that motif, and “Pro-loop receptors” for the superfamily. The infered molecular phylogeny shows a Cys-loop and a Cys-less clade in eukaryotes, both containing metazoans and unicellular members. This suggests new hypotheses on the evolutionary history of the superfamily, such as a possible origin of the Cys-loop cysteines in an ancient unicellular eukaryote. Deeper phylogenetic relationships remain uncertain, particularly around the split between bacteria, archaea, and eukaryotes. PMID:26986966

  11. Structural Studies of Inositol 1,4,5-Trisphosphate Receptor COUPLING LIGAND BINDING TO CHANNEL GATING

    SciTech Connect

    Chan, Jenny; Yamazaki, Haruka; Ishiyama, Noboru; Seo, Min-Duk; Mal, Tapas K.; Michikawa, Takayuki; Mikoshiba, Katsuhiko; Ikura, Mitsuhiko

    2010-11-22

    The three isoforms of the inositol 1,4,5-trisphosphate receptor (IP{sub 3}R) exhibit distinct IP{sub 3} sensitivities and cooperativities in calcium (Ca{sup 2+}) channel function. The determinants underlying this isoform-specific channel gating mechanism have been localized to the N-terminal suppressor region of IP3R. We determined the 1.9 {angstrom} crystal structure of the suppressor domain from type 3 IP{sub 3}R (IP{sub 3}R3{sub SUP}, amino acids 1-224) and revealed structural features contributing to isoform-specific functionality of IP{sub 3}R by comparing it with our previously determined structure of the type 1 suppressor domain (IP{sub 3}R1{sub SUP}). The molecular surface known to associate with the ligand binding domain (amino acids 224-604) showed marked differences between IP{sub 3}R3{sub SUP} and IP{sub 3}R1{sub SUP}. Our NMR and biochemical studies showed that three spatially clustered residues (Glu-20, Tyr-167, and Ser-217 in IP{sub 3}R1 and Glu-19, Trp-168, and Ser-218 in IP{sub 3}R3) within the N-terminal suppressor domains of IP{sub 3}R1{sub SUP} and IP{sub 3}R3{sub SUP} interact directly with their respective C-terminal fragments. Together with the accompanying paper (Yamazaki, H., Chan, J., Ikura, M., Michikawa, T., and Mikoshiba, K. (2010) J. Biol. Chem. 285, 36081-36091), we demonstrate that the single aromatic residue in this region (Tyr-167 in IP{sub 3}R1 and Trp-168 in IP{sub 3}R3) plays a critical role in the coupling between ligand binding and channel gating.

  12. Energy for Wild-Type Acetylcholine Receptor Channel Gating from Different Choline Derivatives

    PubMed Central

    Bruhova, Iva; Gregg, Timothy; Auerbach, Anthony

    2013-01-01

    Agonists, including the neurotransmitter acetylcholine (ACh), bind at two sites in the neuromuscular ACh receptor channel (AChR) to promote a reversible, global change in protein conformation that regulates the flow of ions across the muscle cell membrane. In the synaptic cleft, ACh is hydrolyzed to acetate and choline. Replacement of the transmitter’s ester acetyl group with a hydroxyl (ACh→choline) results in a +1.8 kcal/mol reduction in the energy for gating generated by each agonist molecule from a low- to high-affinity change of the transmitter binding site (ΔGB). To understand the distinct actions of structurally related agonist molecules, we measured ΔGB for 10 related choline derivatives. Replacing the hydroxyl group of choline with different substituents, such as hydrogen, chloride, methyl, or amine, increased the energy for gating (i.e., it made ΔGB more negative relative to choline). Extending the ethyl hydroxide tail of choline to propyl and butyl hydroxide also increased this energy. Our findings reveal the amount of energy that is available for the AChR conformational change provided by different, structurally related agonists. We speculate that a hydrogen bond between the choline hydroxyl and the backbone carbonyl of αW149 positions this agonist’s quaternary ammonium group so as to reduce the cation-π interaction between this moiety and the aromatic groups at the binding site. PMID:23442907

  13. Glutamate-gated Chloride Channels*

    PubMed Central

    Wolstenholme, Adrian J.

    2012-01-01

    Glutamate-gated chloride channels (GluCls) are found only in protostome invertebrate phyla but are closely related to mammalian glycine receptors. They have a number of roles in these animals, controlling locomotion and feeding and mediating sensory inputs into behavior. In nematodes and arthropods, they are targeted by the macrocyclic lactone family of anthelmintics and pesticides, making the GluCls of considerable medical and economic importance. Recently, the three-dimensional structure of a GluCl was solved, the first for any eukaryotic ligand-gated anion channel, revealing a macrocyclic lactone-binding site between the channel domains of adjacent subunits. This minireview will highlight some unique features of the GluCls and illustrate their contribution to our knowledge of the entire Cys loop ligand-gated ion channel superfamily. PMID:23038250

  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. Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors*

    PubMed Central

    Rienzo, Matthew; Rocchi, Angela R.; Threatt, Stephanie D.; Dougherty, Dennis A.; Lummis, Sarah C. R.

    2016-01-01

    Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC. PMID:26668320

  16. Molecular determinants mediating gating of Transient Receptor Potential Canonical (TRPC) channels by stromal interaction molecule 1 (STIM1).

    PubMed

    Lee, Kyu Pil; Choi, Seok; Hong, Jeong Hee; Ahuja, Malini; Graham, Sarabeth; Ma, Rong; So, Insuk; Shin, Dong Min; Muallem, Shmuel; Yuan, Joseph P

    2014-03-01

    Transient receptor potential canonical (TRPC) channels mediate a critical part of the receptor-evoked Ca(2+) influx. TRPCs are gated open by the endoplasmic reticulum Ca(2+) sensor STIM1. Here we asked which stromal interaction molecule 1 (STIM1) and TRPC domains mediate the interaction between them and how this interaction is used to open the channels. We report that the STIM1 Orai1-activating region domain of STIM1 interacts with the TRPC channel coiled coil domains (CCDs) and that this interaction is essential for opening the channels by STIM1. Thus, disruption of the N-terminal (NT) CCDs by triple mutations eliminated TRPC surface localization and reduced binding of STIM1 to TRPC1 and TRPC5 while increasing binding to TRPC3 and TRPC6. Single mutations in TRPC1 NT or C-terminal (CT) CCDs reduced interaction and activation of TRPC1 by STIM1. Remarkably, single mutations in the TRPC3 NT CCD enhanced interaction and regulation by STIM1. Disruption in the TRPC3 CT CCD eliminated regulation by STIM1 and the enhanced interaction caused by NT CCD mutations. The NT CCD mutations converted TRPC3 from a TRPC1-dependent to a TRPC1-independent, STIM1-regulated channel. TRPC1 reduced the FRET between BFP-TRPC3 and TRPC3-YFP and between CFP-TRPC3-YFP upon stimulation. Accordingly, knockdown of TRPC1 made TRPC3 STIM1-independent. STIM1 dependence of TRPC3 was reconstituted by the TRPC1 CT CCD alone. Knockout of Trpc1 and Trpc3 similarly inhibited Ca(2+) influx, and inhibition of Trpc3 had no further effect on Ca(2+) influx in Trpc1(-/-) cells. Cell stimulation enhanced the formation of Trpc1-Stim1-Trpc3 complexes. These findings support a model in which the TRPC3 NT and CT CCDs interact to shield the CT CCD from interaction with STIM1. The TRPC1 CT CCD dissociates this interaction to allow the STIM1 Orai1-activating region within STIM1 access to the TRPC3 CT CCD and regulation of TRPC3 by STIM1. These studies provide evidence that the TRPC channel CCDs participate in channel

  17. Molecular Determinants Mediating Gating of Transient Receptor Potential Canonical (TRPC) Channels by Stromal Interaction Molecule 1 (STIM1)*

    PubMed Central

    Lee, Kyu Pil; Choi, Seok; Hong, Jeong Hee; Ahuja, Malini; Graham, Sarabeth; Ma, Rong; So, Insuk; Shin, Dong Min; Muallem, Shmuel; Yuan, Joseph P.

    2014-01-01

    Transient receptor potential canonical (TRPC) channels mediate a critical part of the receptor-evoked Ca2+ influx. TRPCs are gated open by the endoplasmic reticulum Ca2+ sensor STIM1. Here we asked which stromal interaction molecule 1 (STIM1) and TRPC domains mediate the interaction between them and how this interaction is used to open the channels. We report that the STIM1 Orai1-activating region domain of STIM1 interacts with the TRPC channel coiled coil domains (CCDs) and that this interaction is essential for opening the channels by STIM1. Thus, disruption of the N-terminal (NT) CCDs by triple mutations eliminated TRPC surface localization and reduced binding of STIM1 to TRPC1 and TRPC5 while increasing binding to TRPC3 and TRPC6. Single mutations in TRPC1 NT or C-terminal (CT) CCDs reduced interaction and activation of TRPC1 by STIM1. Remarkably, single mutations in the TRPC3 NT CCD enhanced interaction and regulation by STIM1. Disruption in the TRPC3 CT CCD eliminated regulation by STIM1 and the enhanced interaction caused by NT CCD mutations. The NT CCD mutations converted TRPC3 from a TRPC1-dependent to a TRPC1-independent, STIM1-regulated channel. TRPC1 reduced the FRET between BFP-TRPC3 and TRPC3-YFP and between CFP-TRPC3-YFP upon stimulation. Accordingly, knockdown of TRPC1 made TRPC3 STIM1-independent. STIM1 dependence of TRPC3 was reconstituted by the TRPC1 CT CCD alone. Knockout of Trpc1 and Trpc3 similarly inhibited Ca2+ influx, and inhibition of Trpc3 had no further effect on Ca2+ influx in Trpc1−/− cells. Cell stimulation enhanced the formation of Trpc1-Stim1-Trpc3 complexes. These findings support a model in which the TRPC3 NT and CT CCDs interact to shield the CT CCD from interaction with STIM1. The TRPC1 CT CCD dissociates this interaction to allow the STIM1 Orai1-activating region within STIM1 access to the TRPC3 CT CCD and regulation of TRPC3 by STIM1. These studies provide evidence that the TRPC channel CCDs participate in channel gating

  18. Implications of Human Transient Receptor Potential Melastatin 8 (TRPM8) Channel Gating from Menthol Binding Studies of the Sensing Domain.

    PubMed

    Rath, Parthasarathi; Hilton, Jacob K; Sisco, Nicholas J; Van Horn, Wade D

    2016-01-12

    The transient receptor potential melastatin 8 (TRPM8) ion channel is the primary cold sensor in humans. TRPM8 is gated by physiologically relevant cold temperatures and chemical ligands that induce cold sensations, such as the analgesic compound menthol. Characterization of TRPM8 ligand-gated channel activation will lead to a better understanding of the fundamental mechanisms that underlie TRPM8 function. Here, the direct binding of menthol to the isolated hTRPM8 sensing domain (transmembrane helices S1-S4) is investigated. These data are compared with two mutant sensing domain proteins, Y745H (S2 helix) and R842H (S4 helix), which have been previously identified in full length TRPM8 to be menthol insensitive. The data presented herein show that menthol specifically binds to the wild type, Y745H, and R842H TRPM8 sensing domain proteins. These results are the first to show that menthol directly binds to the TRPM8 sensing domain and indicates that Y745 and R842 residues, previously identified in functional studies as crucial to menthol sensitivity, do not affect menthol binding but instead alter coupling between the sensing domain and the pore domain. PMID:26653082

  19. Modes of glutamate receptor gating

    PubMed Central

    Popescu, Gabriela K

    2012-01-01

    Abstract The time course of excitatory synaptic currents, the major means of fast communication between neurons of the central nervous system, is encoded in the dynamic behaviour of post-synaptic glutamate-activated channels. First-pass attempts to explain the glutamate-elicited currents with mathematical models produced reaction mechanisms that included only the most basic functionally defined states: resting vs. liganded, closed vs. open, responsive vs. desensitized. In contrast, single-molecule observations afforded by the patch-clamp technique revealed an unanticipated kinetic multiplicity of transitions: from microseconds-lasting flickers to minutes-long modes. How these kinetically defined events impact the shape of the synaptic response, how they relate to rearrangements in receptor structure, and whether and how they are physiologically controlled represent currently active research directions. Modal gating, which refers to the slowest, least frequently observed ion-channel transitions, has been demonstrated for representatives of all ion channel families. However, reaction schemes have been largely confined to the short- and medium-range time scales. For glutamate receptors as well, modal gating has only recently come under rigorous scrutiny. This article reviews the evidence for modal gating of glutamate receptors and the still developing hypotheses about the mechanism(s) by which modal shifts occur and the ways in which they may impact the time course of synaptic transmission. PMID:22106181

  20. Developmental regulation of voltage-gated K+ channel and GABAA receptor expression in Bergmann glial cells.

    PubMed

    Müller, T; Fritschy, J M; Grosche, J; Pratt, G D; Möhler, H; Kettenmann, H

    1994-05-01

    Bergmann glial cells are closely associated with neurons: during development they provide guiding structures for migrating granule cells and in the adult cerebellum they display intimate interactions with Purkinje cells. In this study, we have addressed the question of whether such changes in neuronal-glial interactions during development are accompanied by variations in the membrane properties of Bergmann glial cells. We used a mouse cerebellum slice preparation to study membrane currents of the Bergmann glial cells at various stages of development in situ using the patch-clamp technique. The distinct morphology of Bergmann glial cells was revealed by Lucifer yellow injections during recording. While Bergmann glial cells in mice of postnatal day 20 (P20) to P30 have thick processes with arborized, irregularly shaped leaf-like appendages, the processes of cells from younger mice (P5-P7) are thinner and smoother. This morphological maturation is accompanied by a variation in voltage-gated currents. In cells from P5 to P7, delayed outward- and inward-rectifying K+ currents were recorded, while older Bergmann glial cells were characterized by, large, voltage- and time-independent K+ currents. In addition, application of GABA induces two effects, a rapid activation of a Cl- conductance and a longer-lasting decrease in the (resting) K+ conductance. Both effects were mediated by benzodiazepine-insensitive GABAA receptors. Responses in cells of P5-P7 mice were large as compared to the small or even undetectable responses in P20-P30 cells. These GABAA receptors were characterized immunohistochemically in mice and rat brain sections with five subunit-specific antibodies. Bergmann glial cells exhibit a distinct but transient immunoreactivity for the GABAA receptor alpha 2-, alpha 3-, and delta-subunits. Staining is maximal between P7 and P10 and decreases gradually thereafter. In contrast, antibodies to the alpha 1- and beta 2,3-subunits fail to decorate Bergmann glial cells

  1. Molecular Insights into the Local Anesthetic Receptor within Voltage-Gated Sodium Channels Using Hydroxylated Analogs of Mexiletine

    PubMed Central

    Desaphy, Jean-François; Dipalma, Antonella; Costanza, Teresa; Carbonara, Roberta; Dinardo, Maria Maddalena; Catalano, Alessia; Carocci, Alessia; Lentini, Giovanni; Franchini, Carlo; Camerino, Diana Conte

    2011-01-01

    We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, directly blocked voltage-gated sodium channels, whereas salbutamol and nadolol did not (Desaphy et al., 2003), suggesting the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. To verify such an hypothesis, we synthesized five new mexiletine analogs by adding one or two hydroxyl groups to the aryloxy moiety of the sodium channel blocker and tested these compounds on hNav1.4 channels expressed in HEK293 cells. Concentration–response relationships were constructed using 25-ms-long depolarizing pulses at −30 mV applied from an holding potential of −120 mV at 0.1 Hz (tonic block) and 10 Hz (use-dependent block) stimulation frequencies. The half-maximum inhibitory concentrations (IC50) were linearly correlated to drug lipophilicity: the less lipophilic the drug, minor was the block. The same compounds were also tested on F1586C and Y1593C hNav1.4 channel mutants, to gain further information on the molecular interactions of mexiletine with its receptor within the sodium channel pore. In particular, replacement of Phe1586 and Tyr1593 by non-aromatic cysteine residues may help in the understanding of the role of π–π or π–cation interactions in mexiletine binding. Alteration of tonic block suggests that the aryloxy moiety of mexiletine may interact either directly or indirectly with Phe1586 in the closed sodium channel to produce low-affinity binding block, and that this interaction depends on the electrostatic potential of the drug aromatic tail. Alteration of use-dependent block suggests that addition of hydroxyl groups to the aryloxy moiety may modify high-affinity binding of the drug amine terminal to Phe1586 through cooperativity between the two pharmacophores, this effect being mainly related to drug lipophilicity. Mutation of Tyr1593 further impaired such cooperativity. In conclusion

  2. Single channel kinetics of a glutamate receptor.

    PubMed Central

    Kerry, C J; Kits, K S; Ramsey, R L; Sansom, M S; Usherwood, P N

    1987-01-01

    The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating. PMID:2436676

  3. Single Channel Kinetics of a Glutamate Receptor

    PubMed Central

    Kerry, Cathryn J.; Kits, Karel S.; Ramsey, Robert L.; Sansom, Mark S. P.; Usherwood, Peter N. R.

    1986-01-01

    The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the presence of 10-4 M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating. PMID:19431683

  4. Ectodomain movements of an ATP-gated ion channel (P2X2 receptor) probed by disulfide locking.

    PubMed

    Stelmashenko, Olga; Compan, Vincent; Browne, Liam E; North, R Alan

    2014-04-01

    The ectodomain of the P2X receptor is formed mainly from two- or three-stranded β-sheets provided symmetrically by each of the three subunits. These enclose a central cavity that is closed off furthest from the plasma membrane (the turret) and that joins with the transmembrane helices to form the ion permeation pathway. Comparison of closed and open crystal structures indicates that ATP binds in a pocket positioned between strands provided by different subunits and that this flexes the β-sheets of the lower body and enlarges the central cavity: this pulls apart the outer ends of the transmembrane helices and thereby opens an aperture, or gate, where they intersect within the membrane bilayer. In the present work, we examined this opening model by introducing pairs of cysteines into the rat P2X2 receptor that might form disulfide bonds within or between subunits. Receptors were expressed in human embryonic kidney cells, and disulfide formation was assessed by observing the effect of dithiothreitol on currents evoked by ATP. Substitutions in the turret (P90C, P89C/S97C), body wall (S65C/S190C, S65C/D315C) and the transmembrane domains (V48C/I328C, V51C/I328C, S54C/I328C) strongly inhibited ATP-evoked currents prior to reduction with dithiothreitol. Western blotting showed that these channels also formed predominately as dimers and/or trimers rather than monomers. The results strongly support the channel opening mechanism proposed on the basis of available crystal structures. PMID:24515105

  5. Characterization of a ligand-gated cation channel based on an inositol receptor in the silkworm, Bombyx mori.

    PubMed

    Kikuta, Shingo; Endo, Haruka; Tomita, Natsuo; Takada, Tomoyuki; Morita, Chiharu; Asaoka, Kiyoshi; Sato, Ryoichi

    2016-07-01

    Insect herbivores recognize non-volatile compounds in plants to direct their feeding behavior. Gustatory receptors (Gr) appear to be required for nutrient recognition by gustatory organs in the mouthparts of insects. Gr10 is expressed in Bombyx mori (BmGr10) mouthparts such as maxillary galea, maxillary palp, and labrum. BmGr10 is predicted to function in sugar recognition; however, the precise biochemical function remains obscure. Larvae of B. mori are monophagous feeders able to find and feed on mulberry leaves. Soluble mulberry leaf extract contains sucrose, glucose, fructose, and myo-inositol. In this study, we identified BmGr10 as an inositol receptor using electrophysiological analysis with the Xenopus oocyte expression system and Ca(2+) imaging techniques using mammalian cells. These results demonstrated that Xenopus oocytes or HEK293T cells expressing BmGr10 specifically respond to myo-inositol and epi-inositol but do not respond to any mono-, di-, or tri-saccharides or to some sugar alcohols. These inositols caused Ca(2+) and Na(+) influxes into the cytoplasm independently of a G protein-mediated signaling cascade, indicating that BmGr10 is a ligand-gated cation channel. Overall, BmGr10 plays an important role in the myo-inositol recognition required for B. mori larval feeding behavior. PMID:27132146

  6. Biophysics of BK Channel Gating.

    PubMed

    Pantazis, A; Olcese, R

    2016-01-01

    BK channels are universal regulators of cell excitability, given their exceptional unitary conductance selective for K(+), joint activation mechanism by membrane depolarization and intracellular [Ca(2+)] elevation, and broad expression pattern. In this chapter, we discuss the structural basis and operational principles of their activation, or gating, by membrane potential and calcium. We also discuss how the two activation mechanisms interact to culminate in channel opening. As members of the voltage-gated potassium channel superfamily, BK channels are discussed in the context of archetypal family members, in terms of similarities that help us understand their function, but also seminal structural and biophysical differences that confer unique functional properties. PMID:27238260

  7. Mapping the Interaction Sites between AMPA Receptors and TARPs Reveals a Role for the Receptor N-Terminal Domain in Channel Gating

    PubMed Central

    Cais, Ondrej; Herguedas, Beatriz; Krol, Karolina; Cull-Candy, Stuart G.; Farrant, Mark; Greger, Ingo H.

    2014-01-01

    Summary AMPA-type glutamate receptors (AMPARs) mediate fast neurotransmission at excitatory synapses. The extent and fidelity of postsynaptic depolarization triggered by AMPAR activation are shaped by AMPAR auxiliary subunits, including the transmembrane AMPAR regulatory proteins (TARPs). TARPs profoundly influence gating, an effect thought to be mediated by an interaction with the AMPAR ion channel and ligand binding domain (LBD). Here, we show that the distal N-terminal domain (NTD) contributes to TARP modulation. Alterations in the NTD-LBD linker result in TARP-dependent and TARP-selective changes in AMPAR gating. Using peptide arrays, we identify a TARP interaction region on the NTD and define the path of TARP contacts along the LBD surface. Moreover, we map key binding sites on the TARP itself and show that mutation of these residues mediates gating modulation. Our data reveal a TARP-dependent allosteric role for the AMPAR NTD and suggest that TARP binding triggers a drastic reorganization of the AMPAR complex. PMID:25373908

  8. Regulation of cyclic nucleotide-gated channels and membrane excitability in olfactory receptor cells by carbon monoxide

    NASA Technical Reports Server (NTRS)

    Leinders-Zufall, T.; Shepherd, G. M.; Zufall, F.

    1995-01-01

    1. The effect of the putative neural messenger carbon monoxide (CO) and the role of the cGMP second-messenger system for olfactory signal generation was examined in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. With the use of whole cell voltage-clamp recordings in combination with a series of ionic and pharmological tests, it is demonstrated that exogenously applied CO is a potent activator (K1/2 = 2.9 microM) of cyclic nucleotide-gated (CNG) channels previously described to mediate odor transduction. 3. Several lines of evidence suggest that CO mediates its effect through stimulation of a soluble guanylyl cyclase (sGC) leading to formation of the second-messenger cGMP. This conclusion is based on the findings that CO responses show an absolute requirement for guanosine 5'-triphosphate (GTP) in the internal solution, that no direct effect of CO on CNG currents in the absence of GTP is detectable, and that a blocker of sGC activation, LY85383 (10 microM), completely inhibits the CO response. 4. The dose-response curve for cGMP at CNG channels is used as a calibration to provide a quantitative estimate of the CO-stimulated cGMP formation. This analysis implies that CO is a potent activator of olfactory sGC. 5. Perforated patch recordings using amphotericin B demonstrate that low micromolar doses of CO effectively depolarize the membrane potential of ORNs through tonic activation of CNG channels. This effect in turn regulates excitable and adaptive properties of ORNs and modulates neuronal responsiveness. 6. These data argue for an important role of the cGMP pathway in olfactory signaling and support the idea that CO may function as a diffusible messenger in the olfactory system.

  9. Structural and Single-Channel Results Indicate that the Rates of Ligand Binding Domain Closing and Opening Directly Impact AMPA Receptor Gating

    SciTech Connect

    Zhang,W.; Cho, Y.; Lolis, E.; Howe, J.

    2008-01-01

    At most excitatory central synapses, glutamate is released from presynaptic terminals and binds to postsynaptic AMPA receptors, initiating a series of conformational changes that result in ion channel opening. Efficient transmission at these synapses requires that glutamate binding to AMPA receptors results in rapid and near-synchronous opening of postsynaptic receptor channels. In addition, if the information encoded in the frequency of action potential discharge is to be transmitted faithfully, glutamate must dissociate from the receptor quickly, enabling the synapse to discriminate presynaptic action potentials that are spaced closely in time. The current view is that the efficacy of agonists is directly related to the extent to which ligand binding results in closure of the binding domain. For glutamate to dissociate from the receptor, however, the binding domain must open. Previously, we showed that mutations in glutamate receptor subunit 2 that should destabilize the closed conformation not only sped deactivation but also altered the relative efficacy of glutamate and quisqualate. Here we present x-ray crystallographic and single-channel data that support the conclusions that binding domain closing necessarily precedes channel opening and that the kinetics of conformational changes at the level of the binding domain importantly influence ion channel gating. Our findings suggest that the stability of the closed-cleft conformation has been tuned during evolution so that glutamate dissociates from the receptor as rapidly as possible but remains an efficacious agonist.

  10. Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system

    SciTech Connect

    Houtani, Takeshi; Munemoto, Yumi; Kase, Masahiko; Sakuma, Satoru; Tsutsumi, Toshiyuki; Sugimoto, Tetsuo . E-mail: sugimoto@takii.kmu.ac.jp

    2005-09-23

    An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the

  11. 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.

  12. Modulation of Voltage-Gated Sodium Channels by Activation of Tumor Necrosis Factor Receptor-1 and Receptor-2 in Small DRG Neurons of Rats

    PubMed Central

    Leo, M.; Argalski, S.; Schäfers, M.; Hagenacker, T.

    2015-01-01

    Tumor necrosis factor- (TNF-) α is a proinflammatory cytokine involved in the development and maintenance of inflammatory and neuropathic pain. Its effects are mediated by two receptors, TNF receptor-1 (TNFR-1) and TNF receptor-2 (TNFR-2). These receptors play a crucial role in the sensitization of voltage-gated sodium channels (VGSCs), a key mechanism in the pathogenesis of chronic pain. Using the whole-cell patch-clamp technique, we examined the influence of TNFR-1 and TNFR-2 on VGSCs and TTX-resistant NaV1.8 channels in isolated rat dorsal root ganglion neurons by using selective TNFR agonists. The TNFR-1 agonist R32W (10 pg/mL) caused an increase in the VGSC current (INa(V)) by 27.2 ± 5.1%, while the TNFR-2 agonist D145 (10 pg/mL) increased the current by 44.9 ± 2.6%. This effect was dose dependent. Treating isolated NaV1.8 with R32W (100 pg/mL) resulted in an increase in INaV(1.8) by 18.9 ± 1.6%, while treatment with D145 (100 pg/mL) increased the current by 14.5 ± 3.7%. Based on the current-voltage relationship, 10 pg of R32W or D145 led to an increase in INa(V) in a bell-shaped, voltage-dependent manner with a maximum effect at −30 mV. The effects of TNFR activation on VGSCs promote excitation in primary afferent neurons and this might explain the sensitization mechanisms associated with neuropathic and inflammatory pain. PMID:26504355

  13. Neurotoxic phospholipase A2 from rattlesnake as a new ligand and new regulator of prokaryotic receptor GLIC (proton-gated ion channel from G. violaceus).

    PubMed

    Ostrowski, Maciej; Porowinska, Dorota; Prochnicki, Tomasz; Prevost, Marie; Raynal, Bertrand; Baron, Bruno; Sauguet, Ludovic; Corringer, Pierre-Jean; Faure, Grazyna

    2016-06-15

    Neurotoxic phospholipases A2 (sPLA2) from snake venoms interact with various protein targets with high specificity and potency. They regulate function of multiple receptors or channels essential to life processes including neuronal or neuromuscular chemoelectric signal transduction. These toxic sPLA2 exhibit high pharmacological potential and determination of PLA2-receptor binding sites represents challenging part in the receptor-channel biochemistry and pharmacology. To investigate the mechanism of interaction of neurotoxic PLA2 with its neuronal receptor at the molecular level, we used as a model crotoxin, a heterodimeric sPLA2 from rattlesnake venom and proton-gated ion channel GLIC, a bacterial homolog of pentameric ligand-gated ion channels. The three-dimensional structures of both partners, crotoxin and GLIC have been solved by X-ray crystallography and production of full-length pentameric GLIC (with ECD and TM domains) is well established. In the present study, for the first time, we demonstrated physical and functional interaction of full-length purified and solubilized GLIC with CB, (PLA2 subunit of crotoxin). We identified GLIC as a new protein target of CB and CB as a new ligand of GLIC, and showed that this non covalent interaction (PLA2-GLIC) involves the extracellular domain of GLIC. We also determined a novel function of CB as an inhibitor of proton-gated ion channel activity. In agreement with conformational changes observed upon formation of the complex, CB appears to be negative allosteric modulator (NAM) of GLIC. Finally, we proposed a possible stoichiometric model for CB - GLIC interaction based on analytical ultracentrifugation. PMID:26854368

  14. Electrophysiological characterization, solubilization and purification of the Tityus gamma toxin receptor associated with the gating component of the Na+ channel from rat brain.

    PubMed

    Barhanin, J; Pauron, D; Lombet, A; Norman, R I; Vijverberg, H P; Giglio, J R; Lazdunski, M

    1983-01-01

    Electrophysiological studies with neuroblastoma cells have shown that toxin gamma from the venom of the scorpion Tityus serrulatus is a new toxin specific for the gating system of the Na+ channel. The procedure which solubilizes the tetrodotoxin receptor from rat brain also solubilizes the Tityus gamma toxin receptor. Binding experiments on the solubilized receptor with a radioiodinated derivative of Tityus gamma toxin have shown: (i) that the TiTx gamma-receptor complex is very stable with a dissociation constant of 8.6 X 10(-12) M and a very slow dissociation (T 1/2 = 15 h); (ii) that the toxin recognizes a class of sites with a 1:1 stoichiometry with those for tetrodotoxin (Bmax = 1.3 pmol/mg protein). The radioiodinated Tityus gamma-receptor complex has been substantially purified by ion-exchange chromatography, lectin affinity chromatography and sucrose gradient sedimentation. A ratio of one Tityus gamma toxin binding site per tetrodotoxin binding site was found throughout the purification. The purified material exhibited a sedimentation coefficient of 10.4S and had an apparent mol. wt. of 270 000 on SDS-gel electrophoresis. No other polypeptide chains were demonstrated to be associated with this large protein in the Tityus gamma receptor. The main conclusion is that the tetrodotoxin binding site associated with the selectivity filter of the Na+ channel and the Tityus gamma toxin binding site associated with the gating component are probably carried by the same polypeptide chain. PMID:6315420

  15. The region adjacent to the C-end of the inner gate in transient receptor potential melastatin 8 (TRPM8) channels plays a central role in allosteric channel activation.

    PubMed

    Taberner, Francisco José; López-Córdoba, Ainara; Fernández-Ballester, Gregorio; Korchev, Yuri; Ferrer-Montiel, Antonio

    2014-10-10

    The ability of transient receptor potential (TRP) channels to sense and respond to environmental and endogenous cues is crucial in animal sensory physiology. The molecular mechanism of channel gating is yet elusive. The TRP box, a conserved region in the N-end of the C terminus domain, has been signaled as pivotal for allosteric activation in TRP channels. Here, we have examined the role of the linker region between the TRPM8 inner gate and the TRP box (referred to as the S6-TRP box linker) to identify structural determinants of channel gating. Stepwise substitutions of segments in the S6-TRP box linker of TRPM8 channel with the cognate TRPV1 channel sequences produced functional chimeric channels, and identified Tyr(981) as a central molecular determinant of channel function. Additionally, mutations in the 986-990 region had a profound impact on channel gating by voltage and menthol, as evidenced by the modulation of the conductance-to-voltage (G-V) relationships. Simulation of G-V curves using an allosteric model for channel activation revealed that these mutations altered the allosteric constants that couple stimuli sensing to pore opening. A molecular model of TRPM8, based on the recently reported TRPV1 structural model, showed that Tyr(981) may lie in a hydrophobic pocket at the end of the S6 transmembrane segment and is involved in inter-subunit interactions with residues from neighbor subunits. The 986-990 region holds intrasubunit interactions between the TRP domain and the S4-S5 linker. These findings substantiate a gating mechanism whereby the TRP domain acts as a coupling domain for efficient channel opening. Furthermore, they imply that protein-protein interactions of the TRP domain may be targets for channel modulation and drug intervention. PMID:25157108

  16. Neonatal Diabetes Caused by Mutations in Sulfonylurea Receptor 1: Interplay between Expression and Mg-Nucleotide Gating Defects of ATP-Sensitive Potassium Channels

    PubMed Central

    Zhou, Qing; Garin, Intza; Castaño, Luis; Argente, Jesús; Muñoz-Calvo, Ma. Teresa; Perez de Nanclares, Guiomar; Shyng, Show-Ling

    2010-01-01

    Context: ATP-sensitive potassium (KATP) channels regulate insulin secretion by coupling glucose metabolism to β-cell membrane potential. Gain-of-function mutations in the sulfonylurea receptor 1 (SUR1) or Kir6.2 channel subunit underlie neonatal diabetes. Objective: The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect KATP channel function. Design: E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gating properties of the resulting channels were assessed biochemically and electrophysiologically. Results: Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP4− or sulfonylureas. Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on β-cells. When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone. Conclusion: The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Furthermore, they suggest that diabetes severity is determined by interplay between effects of a mutation on channel expression and channel gating. PMID:20810569

  17. Optical control of an ion channel gate.

    PubMed

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

    2013-12-17

    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

  18. Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily.

    PubMed

    Gunthorpe, M J; Lummis, S C

    2001-06-15

    The 5-hydroxytryptamine(3) (5-HT(3)) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, gamma-aminobutyric acid, and glycine receptors. The receptors are either cation or anion selective, leading to their distinctive involvement in either excitatory or inhibitory neurotransmission. Using a combination of site-directed mutagenesis and electrophysiological characterization of homomeric 5-HT(3A) receptors expressed in HEK293 cells, we have identified a set of mutations that convert the ion selectivity of the 5-HT(3A) receptor from cationic to anionic; these were substitution of V13'T in M2 together with neutralization of glutamate residues (E-1'A) and the adjacent insertion of a proline residue (P-1') in the M1-M2 loop. Mutant receptors showed significant chloride permeability (P(Cl)/P(Na) = 12.3, P(Na)/P(Cl) = 0.08), whereas WT receptors are predominantly permeable to sodium (P(Na)/P(Cl) > 20, P(Cl)/P(Na) < 0.05). Since the equivalent mutations have previously been shown to convert alpha7 nicotinic acetylcholine receptors from cationic to anionic (Galzi J.-L., Devillers-Thiery, A, Hussy, N., Bertrand, S. Changeux, J. P., and Bertrand, D. (1992) Nature 359, 500-505) and, recently, the converse mutations have allowed the construction of a cation selective glycine receptor (Keramidas, A., Moorhouse, A. J., French, C. R., Schofield, P. R., and Barry, P. H. (2000) Biophys. J. 78, 247-259), it appears that the determinants of ion selectivity represent a conserved feature of the ligand-gated ion channel superfamily. PMID:11439930

  19. Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily.

    PubMed

    Gunthorpe, M J; Lummis, S C

    2001-04-01

    The 5-hydroxytryptamine(3) (5-HT(3)) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, gamma-aminobutyric acid, and glycine receptors. The receptors are either cation or anion selective, leading to their distinctive involvement in either excitatory or inhibitory neurotransmission. Using a combination of site-directed mutagenesis and electrophysiological characterization of homomeric 5-HT(3A) receptors expressed in HEK293 cells, we have identified a set of mutations that convert the ion selectivity of the 5-HT(3A) receptor from cationic to anionic; these were substitution of V13'T in M2 together with neutralization of glutamate residues (E-1'A) and the adjacent insertion of a proline residue (P-1') in the M1-M2 loop. Mutant receptors showed significant chloride permeability (P(Cl)/P(Na) = 12.3, P(Na)/P(Cl) = 0.08), whereas WT receptors are predominantly permeable to sodium (P(Na)/P(Cl) > 20, P(Cl)/P(Na) < 0.05). Since the equivalent mutations have previously been shown to convert alpha7 nicotinic acetylcholine receptors from cationic to anionic (Galzi J.-L., Devillers-Thiery, A, Hussy, N., Bertrand, S. Changeux, J. P., and Bertrand, D. (1992) Nature 359, 500-505) and, recently, the converse mutations have allowed the construction of a cation selective glycine receptor (Keramidas, A., Moorhouse, A. J., French, C. R., Schofield, P. R., and Barry, P. H. (2000) Biophys. J. 78, 247-259), it appears that the determinants of ion selectivity represent a conserved feature of the ligand-gated ion channel superfamily. PMID:11139582

  20. Single-Channel Recording of Ligand-Gated Ion Channels.

    PubMed

    Plested, Andrew J R

    2016-01-01

    Single-channel recordings reveal the microscopic properties of individual ligand-gated ion channels. Such recordings contain much more information than measurements of ensemble behavior and can yield structural and functional information about the receptors that participate in fast synaptic transmission in the brain. With a little care, a standard patch-clamp electrophysiology setup can be adapted for single-channel recording in a matter of hours. Thenceforth, it is a realistic aim to record single-molecule activity with microsecond resolution from arbitrary cell types, including cell lines and neurons. PMID:27480725

  1. Gγ7 proteins contribute to coupling of nociceptin/orphanin FQ peptide (NOP) opioid receptors and voltage-gated Ca(2+) channels in rat stellate ganglion neurons.

    PubMed

    Mahmoud, Saifeldin; Farrag, Mohamed; Ruiz-Velasco, Victor

    2016-08-01

    The nociceptin/orphanin FQ peptide (NOP) opioid receptors regulate neurotransmitter release via inhibition of voltage-gated Ca(2+) channels (CaV2.2) in sympathetic and sensory neurons. Stimulation of NOP receptors by its endogenous agonist, nociception (Noc), leads to membrane-delimited, voltage-dependent (VD) block of CaV2.2 channel currents mediated by Gβγ protein subunits. Previously we reported that the pertussis toxin-sensitive Gαi1 and Gβ2/β4 isoforms mediate the functional coupling of NOP opioid receptors with CaV channels in rat stellate ganglion (SG) sympathetic neurons. In the present report we extended our studies by identifying the Gγ subunit that forms the heterotrimer within this signaling pathway. Small interference RNA (or siRNA) was employed to silence the expression of the natively expressed Gγ subunits. Initial PCR assays indicated that SG neurons expressed seven Gγ subunits. Silencing Gγ3 subunits did not alter signaling between NOP receptors and Ca(2+) channels. However, after Gγ7 isoforms were silenced, the Noc-mediated inhibition of CaV channels was significantly decreased when compared to SG neurons transfected with scrambled siRNA. We observed that Gγ10 and Gγ11 mRNA levels increased 2.5- and 2.7-fold, respectively, after Gγ7 subunits were silenced. However, this compensatory increase in mRNA expression did not appear to fully rescue the NOP receptor coupling efficiency. Additionally, both Gγ2 and Gγ5 levels increased 50 and 75%, respectively, while Gγ3 and Gγ4 expression levels remained relatively unchanged. Taken together, our findings suggest that the Gαi1/Gβ2(β4)/Gγ7 heterotrimeric G protein complex determines the NOP receptor-mediated modulation of CaV channels in SG neurons. PMID:27238748

  2. Biphasic regulation of InsP3 receptor gating by dual Ca2+ release channel BH3-like domains mediates Bcl-xL control of cell viability.

    PubMed

    Yang, Jun; Vais, Horia; Gu, Wenen; Foskett, J Kevin

    2016-03-29

    Antiapoptotic Bcl-2 family members interact with inositol trisphosphate receptor (InsP3R) Ca(2+)release channels in the endoplasmic reticulum to modulate Ca(2+)signals that affect cell viability. However, the molecular details and consequences of their interactions are unclear. Here, we found that Bcl-xLactivates single InsP3R channels with a biphasic concentration dependence. The Bcl-xLBcl-2 homology 3 (BH3) domain-binding pocket mediates both high-affinity channel activation and low-affinity inhibition. Bcl-xLactivates channel gating by binding to two BH3 domain-like helices in the channel carboxyl terminus, whereas inhibition requires binding to one of them and to a previously identified Bcl-2 interaction site in the channel-coupling domain. Disruption of these interactions diminishes cell viability and sensitizes cells to apoptotic stimuli. Our results identify BH3-like domains in an ion channel and they provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP3R that play critical roles in Ca(2+)signaling and cell viability. PMID:26976600

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

    PubMed

    Bicknell, Brendan A; Goodhill, Geoffrey J

    2016-09-01

    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 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. PMID:27551100

  4. Opioid receptors from a lower vertebrate (Catostomus commersoni): Sequence, pharmacology, coupling to a G-protein-gated inward-rectifying potassium channel (GIRK1), and evolution

    PubMed Central

    Darlison, Mark G.; Greten, Florian R.; Harvey, Robert J.; Kreienkamp, Hans-Jürgen; Stühmer, Thorsten; Zwiers, Henk; Lederis, Karl; Richter, Dietmar

    1997-01-01

    The molecular evolution of the opioid receptor family has been studied by isolating cDNAs that encode six distinct opioid receptor-like proteins from a lower vertebrate, the teleost fish Catostomus commersoni. One of these, which has been obtained in full-length form, encodes a 383-amino acid protein that exhibits greatest sequence similarity to mammalian μ-opioid receptors; the corresponding gene is expressed predominantly in brain and pituitary. Transfection of the teleost cDNA into HEK 293 cells resulted in the appearance of a receptor having high affinity for the μ-selective agonist [d-Ala2, MePhe4-Gly-ol5]enkephalin (DAMGO) (Kd = 0.63 ± 0.15 nM) and for the nonselective antagonist naloxone (Kd = 3.1 ± 1.3 nM). The receptor had negligible affinity for U50488 and [d-Pen2, d-Pen5]enkephalin (DPDPE), which are κ- and δ-opioid receptor selective agonists, respectively. Stimulation of transfected cells with 1 μM DAMGO lowered forskolin-induced cAMP levels, an effect that could be reversed by naloxone. Experiments in Xenopus oocytes have demonstrated that the fish opioid receptor can, in an agonist-dependent fashion, activate a coexpressed mouse G-protein-gated inward-rectifying potassium channel (GIRK1). The identification of six distinct fish opioid receptor-like proteins suggests that additional mammalian opioid receptors remain to be identified at the molecular level. Furthermore, our data indicate that the μ-opioid receptor arose very early in evolution, perhaps before the appearance of vertebrates, and that the pharmacological and functional properties of this receptor have been conserved over a period of ≈400 million years implying that it fulfills an important physiological role. PMID:9223341

  5. Characterization of a Multiple Ligand-Gated Ion Channel Cellular Membrane Affinity Chromatography Column and Identification of Endogenously Expressed Receptors in Astrocytoma Cell Lines

    PubMed Central

    Kitabatake, T.; Moaddel, R.; Cole, R.; Gandhari, M.; Frazier, C.; Hartenstein, J.; Rosenberg, A.; Bernier, M.; Wainer, I. W.

    2008-01-01

    Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [3H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric α7 nicotinic acetylcholine receptors (α7 nAChR) and heteromeric nicotinic acetylcholine receptors (αxβy nAChRs), which was confirmed by the addition of subtype-specific inhibitors, κ-bungarotoxin (α7 nAChR) and K-bungarotoxin (αxβy nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), γ-aminobutyric acid (GABAA) and N-methyl-d-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABAA receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors. PMID:18847217

  6. Characterization of a multiple ligand-gated ion channel cellular membrane affinity chromatography column and identification of endogenously expressed receptors in astrocytoma cell lines.

    PubMed

    Kitabatake, T; Moaddel, R; Cole, R; Gandhari, M; Frazier, C; Hartenstein, J; Rosenberg, A; Bernier, M; Wainer, I W

    2008-11-15

    Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [(3)H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) and heteromeric nicotinic acetylcholine receptors (alpha(x)beta(y) nAChRs), which was confirmed by the addition of subtype-specific inhibitors, alpha-bungarotoxin (alpha7 nAChR) and kappa-bungarotoxin (alpha(x)beta(y) nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), gamma-aminobutyric acid (GABA(A)) and N-methyl-D-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABA(A) receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors. PMID:18847217

  7. The gating isomerization of neuromuscular acetylcholine receptors

    PubMed Central

    Auerbach, Anthony

    2010-01-01

    Acetylcholine receptor-channels are allosteric proteins that isomerize (‘gate’) between conformations that have a low vs. high affinity for the transmitter and conductance for ions. In order to comprehend the mechanism by which the affinity and conductance changes are linked it is of value to know the magnitude, timing and distribution of energy flowing through the system. Knowing both the di- and unliganded gating equilibrium constants (E2 and E0) is a foundation for understanding the AChR gating mechanism and for engineering both the ligand and the protein to operate in predictable ways. In adult mouse neuromuscular receptors activated by acetylcholine, E2= 28 and E0≈ 6.5 × 10−7. At each (equivalent) transmitter binding site acetylcholine provides ∼5.2 kcal mol−1 to motivate the isomerization. The partial agonist choline provides ∼3.3 kcal mol−1. The relative time of a residue's gating energy change is revealed by the slope of its rate–equilibrium constant relationship. A map of this parameter suggests that energy propagates as a conformational cascade between the transmitter binding sites and the gate region. Although gating energy changes are widespread throughout the protein, some residues are particularly sensitive to perturbations. Several specific proposals for the structural events that comprise the gating conformational cascade are discussed. PMID:19933754

  8. Gramicidin Channels Are Internally Gated

    PubMed Central

    Jones, Tyson L.; Fu, Riqiang; Nielson, Frederick; Cross, Timothy A.; Busath, David D.

    2010-01-01

    Abstract Gramicidin channels are archetypal molecular subjects for solid-state NMR studies and investigations of single-channel or cation conductance. Until now, the transitions between on and off conductance states have been thought, based on multichannel studies, to represent monomer ↔ dimer reactions. Here we use a single-molecule deposition method (vesicle fusion to a planar bilayer) to show that gramicidin dimer channels do not normally dissociate when conductance terminates. Furthermore, the observation of two 13C peaks in solid-state NMR indicates very stable dichotomous conformations for both the first and second peptide bonds in the monomers, and a two-dimensional chemical exchange spectrum with a 12-s mixing time demonstrates that the Val1 carbonyl conformations exchange slowly, with lifetimes of several seconds. It is proposed that gramicidin channels are gated by small conformational changes in the channel near the permeation pathway. These studies demonstrate how regulation of conformations governing closed ↔ open transitions may be achieved and studied at the molecular level. PMID:20409467

  9. Regulation of CFTR channel gating.

    PubMed

    Gadsby, D C; Hwang, T C; Baukrowitz, T; Nagel, G; Horie, M; Nairn, A C

    1994-01-01

    Findings outlined here support a complex model for the regulation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel gating that incorporates incremental protein kinase A (PKA) phosphorylation of CFTR at multiple sites which, in turn, differentially control the activity of CFTR's two nucleotide-binding domains (NBDs). The NBDs are functionally distinct: only one can respond to the non-hydrolyzable ATP analogue AMP-PNP, and then only after ATP has acted at the other. Moreover, the nature of the responses to AMP-PNP, and to the inorganic phosphate analogue orthovanadate, argues that ATP hydrolysis normally occurs at both NBDs, at one to initiate channel opening and at the other to initiate closing. PMID:7752525

  10. 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

  11. 5. GATE 5, INTAKE CHANNEL LOOKING SOUTH; WATER FROM GATE ...

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

    5. GATE 5, INTAKE CHANNEL LOOKING SOUTH; WATER FROM GATE 5 ENTERED DITCH AND IRRIGATED HONDIUS' FIELDS. - Hondius Water Line, 1.6 miles Northwest of Park headquarters building & 1 mile Northwest of Beaver Meadows entrance station, Estes Park, Larimer County, CO

  12. 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

  13. Salmon lice (Lepeophtheirus salmonis) showing varying emamectin benzoate susceptibilities differ in neuronal acetylcholine receptor and GABA-gated chloride channel mRNA expression

    PubMed Central

    2013-01-01

    Background Caligid copepods, also called sea lice, are fish ectoparasites, some species of which cause significant problems in the mariculture of salmon, where the annual cost of infection is in excess of €300 million globally. At present, caligid control on farms is mainly achieved using medicinal treatments. However, the continued use of a restricted number of medicine actives potentially favours the development of drug resistance. Here, we report transcriptional changes in a laboratory strain of the caligid Lepeophtheirus salmonis (Krøyer, 1837) that is moderately (~7-fold) resistant to the avermectin compound emamectin benzoate (EMB), a component of the anti-salmon louse agent SLICE® (Merck Animal Health). Results Suppression subtractive hybridisation (SSH) was used to enrich transcripts differentially expressed between EMB-resistant (PT) and drug-susceptible (S) laboratory strains of L. salmonis. SSH libraries were subjected to 454 sequencing. Further L. salmonis transcript sequences were available as expressed sequence tags (EST) from GenBank. Contiguous sequences were generated from both SSH and EST sequences and annotated. Transcriptional responses in PT and S salmon lice were investigated using custom 15 K oligonucleotide microarrays designed using the above sequence resources. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains, these genes being enriched for functions such as calcium ion binding, chitin metabolism and muscle structure. γ-aminobutyric acid (GABA)-gated chloride channel (GABA-Cl) and neuronal acetylcholine receptor (nAChR) subunits showed significantly lower transcript levels in PT lice compared to S lice. Using RT-qPCR, the decrease in mRNA levels was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR. Salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 μg L-1 of EMB, a drug concentration tolerated by PT lice, but

  14. 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. PMID:25705194

  15. BK channels: multiple sensors, one activation gate

    PubMed Central

    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 Ca2+ 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. PMID:25705194

  16. Estrogen-related receptor γ (ERRγ) regulates oxygen-dependent expression of voltage-gated potassium (K+) channels and tissue kallikrein during human trophoblast differentiation.

    PubMed

    Luo, Yanmin; Kumar, Premlata; Mendelson, Carole R

    2013-06-01

    Estrogen-related receptor γ (ERRγ) serves a critical O2-dependent regulatory role in the differentiation of human cytotrophoblasts to syncytiotrophoblast. In this study, we investigated expression of genes encoding tissue kallikrein (KLK1) and voltage-gated K(+) channels (KV7) during differentiation of human trophoblasts in culture and the roles of ERRγ and O2 tension in their regulation. Expression of KLK1 and the KV7 channel subunits, KCNQ1, KCNE1, KCNE3, and KCNE5, increased during differentiation of cultured human trophoblast cells in a 20% O2 environment. Notably, together with ERRγ, expression of KLK1, KCNQ1, KCNE1, KCNE3, and KCNE5 was markedly reduced when cells were cultured in a hypoxic environment (2% O2). Moreover, upon transduction of trophoblast cells with short hairpin RNAs for endogenous ERRγ, KLK1, KCNQ1, KCNE1, and KCNE3 expression was significantly decreased. Promoter and site-directed mutagenesis studies in transfected cells identified putative ERRγ response elements within the KLK1 and KCNE1 5'-flanking regions required for ERRγ-stimulated transcriptional activity. Binding of endogenous ERRγ to these ERRγ response elements increased during trophoblast differentiation in culture and was inhibited by hypoxia. The KV7 blocker linopirdine reduced human chorionic gonadotropin secretion and aggregation of cultured human trophoblasts, suggesting a possible role of KV7 channels in cell fusion and differentiation. Illumina gene expression arrays of cultured human trophoblast cells revealed several genes upregulated during syncytiotrophoblast differentiation and downregulated upon ERRγ knockdown involved in cell differentiation, adhesion, and synthesis of steroid and peptide hormones required for placental development and function. Collectively, these findings suggest that ERRγ mediates O2-dependent expression of genes involved in human trophoblast differentiation, function, and vascular homeostasis. PMID:23584901

  17. Estrogen-Related Receptor γ (ERRγ) Regulates Oxygen-Dependent Expression of Voltage-gated Potassium (K+) Channels and Tissue Kallikrein during Human Trophoblast Differentiation

    PubMed Central

    Luo, Yanmin; Kumar, Premlata

    2013-01-01

    Estrogen-related receptor γ (ERRγ) serves a critical O2-dependent regulatory role in the differentiation of human cytotrophoblasts to syncytiotrophoblast. In this study, we investigated expression of genes encoding tissue kallikrein (KLK1) and voltage-gated K+ channels (KV7) during differentiation of human trophoblasts in culture and the roles of ERRγ and O2 tension in their regulation. Expression of KLK1 and the KV7 channel subunits, KCNQ1, KCNE1, KCNE3, and KCNE5, increased during differentiation of cultured human trophoblast cells in a 20% O2 environment. Notably, together with ERRγ, expression of KLK1, KCNQ1, KCNE1, KCNE3, and KCNE5 was markedly reduced when cells were cultured in a hypoxic environment (2% O2). Moreover, upon transduction of trophoblast cells with short hairpin RNAs for endogenous ERRγ, KLK1, KCNQ1, KCNE1, and KCNE3 expression was significantly decreased. Promoter and site-directed mutagenesis studies in transfected cells identified putative ERRγ response elements within the KLK1 and KCNE1 5′-flanking regions required for ERRγ-stimulated transcriptional activity. Binding of endogenous ERRγ to these ERRγ response elements increased during trophoblast differentiation in culture and was inhibited by hypoxia. The KV7 blocker linopirdine reduced human chorionic gonadotropin secretion and aggregation of cultured human trophoblasts, suggesting a possible role of KV7 channels in cell fusion and differentiation. Illumina gene expression arrays of cultured human trophoblast cells revealed several genes upregulated during syncytiotrophoblast differentiation and downregulated upon ERRγ knockdown involved in cell differentiation, adhesion, and synthesis of steroid and peptide hormones required for placental development and function. Collectively, these findings suggest that ERRγ mediates O2-dependent expression of genes involved in human trophoblast differentiation, function, and vascular homeostasis. PMID:23584901

  18. 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

  19. The desensitization gate of inhibitory Cys-loop receptors

    NASA Astrophysics Data System (ADS)

    Gielen, Marc; Thomas, Philip; Smart, Trevor G.

    2015-04-01

    Cys-loop neurotransmitter-gated ion channels are vital for communication throughout the nervous system. Following activation, these receptors enter into a desensitized state in which the ion channel shuts even though the neurotransmitter molecules remain bound. To date, the molecular determinants underlying this most fundamental property of Cys-loop receptors have remained elusive. Here we present a generic mechanism for the desensitization of Cys-loop GABAA (GABAARs) and glycine receptors (GlyRs), which both mediate fast inhibitory synaptic transmission. Desensitization is regulated by interactions between the second and third transmembrane segments, which affect the ion channel lumen near its intracellular end. The GABAAR and GlyR pore blocker picrotoxin prevented desensitization, consistent with its deep channel-binding site overlapping a physical desensitization gate.

  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. A gating mechanism of pentameric ligand-gated ion channels

    PubMed Central

    Calimet, Nicolas; Simoes, Manuel; Changeux, Jean-Pierre; Karplus, Martin; Taly, Antoine; Cecchini, Marco

    2013-01-01

    Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communication in the nervous system and are involved in fundamental processes such as attention, learning, and memory. They are oligomeric protein assemblies that convert a chemical signal into an ion flux through the postsynaptic membrane, but the molecular mechanism of gating ions has remained elusive. Here, we present atomistic molecular dynamics simulations of the prokaryotic channels from Gloeobacter violaceus (GLIC) and Erwinia chrysanthemi (ELIC), whose crystal structures are thought to represent the active and the resting states of pLGICs, respectively, and of the eukaryotic glutamate-gated chloride channel from Caenorhabditis elegans (GluCl), whose open-channel structure was determined complexed with the positive allosteric modulator ivermectin. Structural observables extracted from the trajectories of GLIC and ELIC are used as progress variables to analyze the time evolution of GluCl, which was simulated in the absence of ivermectin starting from the structure with bound ivermectin. The trajectory of GluCl with ivermectin removed shows a sequence of structural events that couple agonist unbinding from the extracellular domain to ion-pore closing in the transmembrane domain. Based on these results, we propose a structural mechanism for the allosteric communication leading to deactivation/activation of the GluCl channel. This model of gating emphasizes the coupling between the quaternary twisting and the opening/closing of the ion pore and is likely to apply to other members of the pLGIC family. PMID:24043807

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

    PubMed Central

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

    2014-01-01

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

  3. Inhibition of voltage-gated sodium channels by sumatriptan bioisosteres

    PubMed Central

    Carbonara, Roberta; Carocci, Alessia; Roussel, Julien; Crescenzo, Giuseppe; Buonavoglia, Canio; Franchini, Carlo; Lentini, Giovanni; Camerino, Diana Conte; Desaphy, Jean-François

    2015-01-01

    Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7–1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogs, namely 20b, (R)-31b, and (S)-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S)-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S)-22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that use-dependent sodium channel blockade contributes to the analgesic activity of (R)-31b and (S)-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors. PMID:26257653

  4. Modal gating of muscle nicotinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Vij, Ridhima

    Many ion channels exhibit multiple patterns of kinetic activity in single-channel currents. This behavior is rare in WT mouse muscle nicotinic acetylcholine receptors (AChRs), where A2C↔A2O gating events are well-described by single exponentials. Also, single-channel open probability (PO) is essentially homogeneous at a given agonist concentration in the WT receptors. Here I report that perturbations of almost all the residues in loop C (alpha188-alpha199, at the agonist binding site) generate heterogeneity in PO ('modes'). Such unsettled activity was apparent with an alanine substitution at all positions in loop C (except alphaY190 and alphaY198) and with different side chain substitutions at alphaP197 for both adult- and fetal-type AChRs. I used single channel electrophysiology along with site-directed mutagenesis to study modal gating in AChRs consequent to mutations/deletions in loop C. The multiple patterns of kinetic activity arose from the difference in agonist affinity rather than in intrinsic AChR gating. Out of the four different agonists used to study the modal behavior, acetylcholine (ACh) showed a higher degree of kinetic heterogeneity compared to others. The time constant for switching between modes was long (~mins), suggesting that they arise from alternative, stable protein conformations. By studying AChRs having only 1 functional binding site, I attempted to find the source of the affinity difference, which was traced mainly to the alphadelta agonist site. Affinity at the neurotransmitter binding site is mainly determined by a core of five aromatic residues (alphaY93, alphaW149, alphaY190, alphaY198 and deltaW57). Phenylalanine substitutions at all aromatic residues except alphaY93 resulted in elimination of modes. Modes were also eliminated by alanine mutation at deltaW57 on the complementary side but not at other aromatics. Also, by substituting four gamma subunit residues into the delta subunit on the complementary beta sheet, I found that

  5. Voltage-Gated Sodium Channels: Evolutionary History and Distinctive Sequence Features.

    PubMed

    Kasimova, M A; Granata, D; Carnevale, V

    2016-01-01

    Voltage-gated sodium channels (Nav) are responsible for the rising phase of the action potential. Their role in electrical signal transmission is so relevant that their emergence is believed to be one of the crucial factors enabling development of nervous system. The presence of voltage-gated sodium-selective channels in bacteria (BacNav) has raised questions concerning the evolutionary history of the ones in animals. Here we review some of the milestones in the field of Nav phylogenetic analysis and discuss some of the most important sequence features that distinguish these channels from voltage-gated potassium channels and transient receptor potential channels. PMID:27586287

  6. 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

  7. Hysteresis of gating underlines sensitization of TRPV3 channels

    PubMed Central

    Liu, Beiying; Yao, Jing; Zhu, Michael X.

    2011-01-01

    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 Ca2+. 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 Ca2+-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. PMID:22006988

  8. 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

  9. Design and synthesis of skeletal analogues of gambierol: attenuation of amyloid-β and tau pathology with voltage-gated potassium channel and N-methyl-D-aspartate receptor implications.

    PubMed

    Alonso, Eva; Fuwa, Haruhiko; Vale, Carmen; Suga, Yuto; Goto, Tomomi; Konno, Yu; Sasaki, Makoto; LaFerla, Frank M; Vieytes, Mercedes R; Giménez-Llort, Lydia; Botana, Luis M

    2012-05-01

    Gambierol is a potent neurotoxin that belongs to the family of marine polycyclic ether natural products and primarily targets voltage-gated potassium channels (K(v) channels) in excitable membranes. Previous work in the chemistry of marine polycyclic ethers has suggested the critical importance of the full length of polycyclic ether skeleton for potent biological activity. Although we have previously investigated structure-activity relationships (SARs) of the peripheral functionalities of gambierol, it remained unclear whether the whole polycyclic ether skeleton is needed for its cellular activity. In this work, we designed and synthesized two truncated skeletal analogues of gambierol comprising the EFGH- and BCDEFGH-rings of the parent compound, both of which surprisingly showed similar potency to gambierol on voltage-gated potassium channels (K(v)) inhibition. Moreover, we examined the effect of these compounds in an in vitro model of Alzheimer's disease (AD) obtained from triple transgenic (3xTg-AD) mice, which expresses amyloid beta (Aβ) accumulation and tau hyperphosphorylation. In vitro preincubation of the cells with the compounds resulted in significant inhibition of K(+) currents, a reduction in the extra- and intracellular levels of Aβ, and a decrease in the levels of hyperphosphorylated tau. In addition, pretreatment with these compounds reduced the steady-state level of the N-methyl-D-aspartate (NMDA) receptor subunit 2A without affecting the 2B subunit. The involvement of glutamate receptors was further suggested by the blockage of the effect of gambierol on tau hyperphosphorylation by glutamate receptor antagonists. The present study constitutes the first discovery of skeletally simplified, designed polycyclic ethers with potent cellular activity and demonstrates the utility of gambierol and its synthetic analogues as chemical probes for understanding the function of K(v) channels as well as the molecular mechanism of Aβ metabolism modulated by

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-08-21

    Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca(2+) channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP3R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3(-/-)/Nrl(-/-) mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP3R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca(2+) channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency. PMID:26124274

  12. Inactivation Gating of Kv4 Potassium Channels

    PubMed Central

    Jerng, Henry H.; Shahidullah, Mohammad; Covarrubias, Manuel

    1999-01-01

    Kv4 channels represent the main class of brain A-type K+ channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996. J. Neurophysiol. 75:2174– 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH2- and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997. Biophys. J. 72:163–174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993. J. Gen. Physiol. 101:603–626; Shieh, C.-C., and G.E. Kirsch. 1994. Biophys. J. 67:2316–2325). A double mutation V[404,406]I in the distal section of the S6 region of the protein drastically slowed channel inactivation and deactivation, and significantly reduced the blockade by 4-AP. In addition, recovery from inactivation was slightly faster, but the pore properties were not significantly affected. Consistent with a more stable open state and disrupted closed state inactivation, V[404,406]I also caused hyperpolarizing and depolarizing shifts of the peak conductance–voltage curve (∼5 mV) and the prepulse inactivation curve (>10 mV), respectively. By contrast, the analogous mutations (V[556,558]I) in a K+ channel that undergoes N- and C-type inactivation (Kv1.4) did not affect macroscopic inactivation but dramatically slowed deactivation and recovery from inactivation, and eliminated open-channel blockade by 4-AP. Mutation of a Kv4-specifc residue in the S4–S5 loop (C322S) of Kv4.1 also altered gating and 4-AP sensitivity in a manner that closely resembles the effects of V[404,406]I. However, this mutant did not exhibit

  13. 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.

  14. Elucidating ligand binding and channel gating mechanisms in pentameric ligand-gated ion channels by atomistic simulations.

    PubMed

    Comitani, Federico; Melis, Claudio; Molteni, Carla

    2015-04-01

    Pentameric ligand-gated ion channels (pLGICs) are important biomolecules that mediate fast synaptic transmission. Their malfunctions are linked to serious neuronal disorders and they are major pharmaceutical targets; in invertebrates, they are involved in insecticide resistance. The complexity of pLGICs and the limited crystallographic information available prevent a detailed understanding of how they function. State-of-the-art computational techniques are therefore crucial to build an accurate picture at the atomic level of the mechanisms which drive the activation of pLGICs, complementing the available experimental data. We have used a series of simulation methods, including homology modelling, ligand-protein docking, density functional theory, molecular dynamics and metadynamics, a powerful scheme for accelerating rare events, with the guidance of mutagenesis electrophysiology experiments, to explore ligand-binding mechanisms, the effects of mutations and the potential role of a proline molecular switch for the gating of the ion channels. Results for the insect RDL receptor, the GABAC receptor, the 5-HT3 receptor and the nicotinic acetylcholine receptor will be reviewed. PMID:25849909

  15. A Direct Interaction between the Sigma-1 Receptor and the hERG Voltage-gated K+ Channel Revealed by Atomic Force Microscopy and Homogeneous Time-resolved Fluorescence (HTRF®)*

    PubMed Central

    Balasuriya, Dilshan; D'Sa, Lauren; Talker, Ronel; Dupuis, Elodie; Maurin, Fabrice; Martin, Patrick; Borgese, Franck; Soriani, Olivier; Edwardson, J. Michael

    2014-01-01

    The sigma-1 receptor is an endoplasmic reticulum chaperone protein, widely expressed in central and peripheral tissues, which can translocate to the plasma membrane and modulate the function of various ion channels. The human ether-à-go-go-related gene encodes hERG, a cardiac voltage-gated K+ channel that is abnormally expressed in many human cancers and is known to interact functionally with the sigma-1 receptor. Our aim was to investigate the nature of the interaction between the sigma-1 receptor and hERG. We show that the two proteins can be co-isolated from a detergent extract of stably transfected HEK-293 cells, consistent with a direct interaction between them. Atomic force microscopy imaging of the isolated protein confirmed the direct binding of the sigma-1 receptor to hERG monomers, dimers, and tetramers. hERG dimers and tetramers became both singly and doubly decorated by sigma-1 receptors; however, hERG monomers were only singly decorated. The distribution of angles between pairs of sigma-1 receptors bound to hERG tetramers had two peaks, at ∼90 and ∼180° in a ratio of ∼2:1, indicating that the sigma-1 receptor interacts with hERG with 4-fold symmetry. Homogeneous time-resolved fluorescence (HTRF®) allowed the detection of the interaction between the sigma-1 receptor and hERG within the plane of the plasma membrane. This interaction was resistant to sigma ligands, but was decreased in response to cholesterol depletion of the membrane. We suggest that the sigma-1 receptor may bind to hERG in the endoplasmic reticulum, aiding its assembly and trafficking to the plasma membrane. PMID:25266722

  16. 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.

  17. 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

  18. 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

  19. Acid-sensitive ion channels and receptors.

    PubMed

    Holzer, Peter

    2009-01-01

    Acidosis is a noxious condition associated with inflammation, ischaemia or defective acid containment. As a consequence, acid sensing has evolved as an important property of afferent neurons with unmyelinated and thinly myelinated nerve fibres. Protons evoke multiple currents in primary afferent neurons, which are carried by several acid-sensitive ion channels. Among these, acid-sensing ion channels (ASICs) and transient receptor potential (TRP) vanilloid-1 (TRPV1) ion channels have been most thoroughly studied. ASICs survey moderate decreases in extracellular pH, whereas TRPV1 is activated only by severe acidosis resulting in pH values below 6. Two-pore-domain K(+) (K(2P)) channels are differentially regulated by small deviations of extra- or intracellular pH from physiological levels. Other acid-sensitive channels include TRPV4, TRPC4, TRPC5, TRPP2 (PKD2L1), ionotropic purinoceptors (P2X), inward rectifier K(+) channels, voltage-activated K(+) channels, L-type Ca(2+) channels, hyperpolarization-activated cyclic nucleotide gated channels, gap junction channels, and Cl(-) channels. In addition, acid-sensitive G protein coupled receptors have also been identified. Most of these molecular acid sensors are expressed by primary sensory neurons, although to different degrees and in various combinations. Emerging evidence indicates that many of the acid-sensitive ion channels and receptors play a role in acid sensing, acid-induced pain and acid-evoked feedback regulation of homeostatic reactions. The existence and apparent redundancy of multiple pH surveillance systems attests to the concept that acid-base regulation is a vital issue for cell and tissue homeostasis. Since upregulation and overactivity of acid sensors appear to contribute to various forms of chronic pain, acid-sensitive ion channels and receptors are considered as targets for novel analgesic drugs. This approach will only be successful if the pathological implications of acid sensors can be differentiated

  20. Acid-sensitive ion channels and receptors

    PubMed Central

    Holzer, Peter

    2015-01-01

    Acidosis is a noxious condition associated with inflammation, ischaemia or defective acid containment. As a consequence, acid sensing has evolved as an important property of afferent neurons with unmyelinated and thinly myelinated nerve fibres. Protons evoke multiple currents in primary afferent neurons, which are carried by several acid-sensitive ion channels. Among these, acid-sensing ion channels (ASICs) and transient receptor potential (TRP) vanilloid-1 (TRPV1) ion channels have been most thoroughly studied. ASICs survey moderate decreases in extracellular pH whereas TRPV1 is activated only by severe acidosis resulting in pH values below 6. Two-pore domain K+ (K2P) channels are differentially regulated by small deviations of extra- or intracellular pH from physiological levels. Other acid-sensitive channels comprise TRPV4, TRPC4, TRPC5, TRPP2 (PKD2L1), ionotropic purinoceptors (P2X), inward rectifier K+ channels, voltage-activated K+ channels, L-type Ca2+ channels, hyperpolarization-activated cyclic nucleotide-gated channels, gap junction channels, and Cl− channels. In addition, acid-sensitive G protein-coupled receptors have also been identified. Most of these molecular acid sensors are expressed by primary sensory neurons, although to different degrees and in various combinations. Emerging evidence indicates that many of the acid-sensitive ion channels and receptors play a role in acid sensing, acid-induced pain and acid-evoked feedback regulation of homeostatic reactions. The existence and apparent redundancy of multiple pH surveillance systems attests to the concept that acid-base regulation is a vital issue for cell and tissue homeostasis. Since upregulation and overactivity of acid sensors appear to contribute to various forms of chronic pain, acid-sensitive ion channels and receptors are considered as targets for novel analgesic drugs. This approach will only be successful if the pathological implications of acid sensors can be differentiated

  1. GABRB3 mutation, G32R, associated with childhood absence epilepsy alters α1β3γ2L γ-aminobutyric acid type A (GABAA) receptor expression and channel gating.

    PubMed

    Gurba, Katharine N; Hernandez, Ciria C; Hu, Ningning; Macdonald, Robert L

    2012-04-01

    A GABA(A) receptor β3 subunit mutation, G32R, has been associated with childhood absence epilepsy. We evaluated the possibility that this mutation, which is located adjacent to the most N-terminal of three β3 subunit N-glycosylation sites, might reduce GABAergic inhibition by increasing glycosylation of β3 subunits. The mutation had three major effects on GABA(A) receptors. First, coexpression of β3(G32R) subunits with α1 or α3 and γ2L subunits in HEK293T cells reduced surface expression of γ2L subunits and increased surface expression of β3 subunits, suggesting a partial shift from ternary αβ3γ2L receptors to binary αβ3 and homomeric β3 receptors. Second, β3(G32R) subunits were more likely than β3 subunits to be N-glycosylated at Asn-33, but increases in glycosylation were not responsible for changes in subunit surface expression. Rather, both phenomena could be attributed to the presence of a basic residue at position 32. Finally, α1β3(G32R)γ2L receptors had significantly reduced macroscopic current density. This reduction could not be explained fully by changes in subunit expression levels (because γ2L levels decreased only slightly) or glycosylation (because reduction persisted in the absence of glycosylation at Asn-33). Single channel recording revealed that α1β3(G32R)γ2L receptors had impaired gating with shorter mean open time. Homology modeling indicated that the mutation altered salt bridges at subunit interfaces, including regions important for subunit oligomerization. Our results suggest both a mechanism for mutation-induced hyperexcitability and a novel role for the β3 subunit N-terminal α-helix in receptor assembly and gating. PMID:22303015

  2. Predicted structure of the extracellular region of ligand-gated ion-channel receptors shows SH2-like and SH3-like domains forming the ligand-binding site.

    PubMed Central

    Gready, J. E.; Ranganathan, S.; Schofield, P. R.; Matsuo, Y.; Nishikawa, K.

    1997-01-01

    Fast synaptic neurotransmission is mediated by ligand-gated ion-channel (LGIC) receptors, which include receptors for acetylcholine, serotonin, GABA, glycine, and glutamate. LGICs are pentamers with extracellular ligand-binding domains and form integral membrane ion channels that are selective for cations (acetylcholine and serotonin 5HT3 receptors) or anions (GABAA and glycine receptors and the invertebrate glutamate-binding chloride channel). They form a protein superfamily with no sequence similarity to any protein of known structure. Using a 1D-3D structure mapping approach, we have modeled the extracellular ligand-binding domain based on a significant match with the SH2 and SH3 domains of the biotin repressor structure. Refinement of the model based on knowledge of the large family of SH2 and SH3 structures, sequence alignments, and use of structure templates for loop building, allows the prediction of both monomer and pentamer models. These are consistent with medium-resolution electron microscopy structures and with experimental structure/function data from ligand-binding, antibody-binding, mutagenesis, protein-labeling and subunit-linking studies, and glycosylation sites. Also, the predicted polarity of the channel pore calculated from electrostatic potential maps of pentamer models of superfamily members is consistent with known ion selectivities. Using the glycine receptor alpha 1 subunit, which forms homopentamers, the monomeric and pentameric models define the agonist and antagonist (strychnine) binding sites to a deep crevice formed by an extended loop, which includes the invariant disulfide bridge, between the SH2 and SH3 domains. A detailed binding site for strychnine is reported that is in strong agreement with known structure/function data. A site for interaction of the extracellular ligand-binding domain with the activation of the M2 transmembrane helix is also suggested. PMID:9144769

  3. Comparative sequence analysis suggests a conserved gating mechanism for TRP channels

    PubMed Central

    Palovcak, Eugene; Delemotte, Lucie; Klein, Michael L.

    2015-01-01

    The transient receptor potential (TRP) channel superfamily plays a central role in transducing diverse sensory stimuli in eukaryotes. Although dissimilar in sequence and domain organization, all known TRP channels act as polymodal cellular sensors and form tetrameric assemblies similar to those of their distant relatives, the voltage-gated potassium (Kv) channels. Here, we investigated the related questions of whether the allosteric mechanism underlying polymodal gating is common to all TRP channels, and how this mechanism differs from that underpinning Kv channel voltage sensitivity. To provide insight into these questions, we performed comparative sequence analysis on large, comprehensive ensembles of TRP and Kv channel sequences, contextualizing the patterns of conservation and correlation observed in the TRP channel sequences in light of the well-studied Kv channels. We report sequence features that are specific to TRP channels and, based on insight from recent TRPV1 structures, we suggest a model of TRP channel gating that differs substantially from the one mediating voltage sensitivity in Kv channels. The common mechanism underlying polymodal gating involves the displacement of a defect in the H-bond network of S6 that changes the orientation of the pore-lining residues at the hydrophobic gate. PMID:26078053

  4. Thermosensitive TRPV Channel Subunits Coassemble into Heteromeric Channels with Intermediate Conductance and Gating Properties

    PubMed Central

    Cheng, Wei; Yang, Fan; Takanishi, Christina L.; Zheng, Jie

    2007-01-01

    Heat-sensitive transient receptor potential (TRP) channels (TRPV1–4) form the major cellular sensors for detecting temperature increases. Homomeric channels formed by thermosensitive TRPV subunits exhibit distinct temperature thresholds. While these subunits do share significant sequence similarity, whether they can coassemble into heteromeric channels has been controversial. In the present study we investigated the coassembly of TRPV subunits using both spectroscopy-based fluorescence resonance energy transfer (FRET) and single-channel recordings. Fluorescent protein–tagged TRPV subunits were coexpressed in HEK 293 cells; FRET between different subunits was measured as an indication of the formation of heteromeric channels. We observed strong FRET when fluorescence signals were collected selectively from the plasma membrane using a “spectra FRET” approach but much weaker or no FRET from intracellular fluorescence. In addition, no FRET was detected when TRPV subunits were coexpressed with members of the TRPM subfamily or CLC-0 chloride channel subunits. These results indicate that a substantial fraction of TRP channels in the plasma membrane of cotransfected cells were heteromeric. Single-channel recordings confirmed the existence of multiple heteromeric channel forms. Interestingly, heteromeric TRPV channels exhibit intermediate conductance levels and gating kinetic properties. As these subunits coexpress both in sensory neurons and in other tissues, including heart and brain, coassembly between TRPV subunits may contribute to greater functional diversity. PMID:17325193

  5. ThermoTRP channels as modular proteins with allosteric gating.

    PubMed

    Latorre, Ramon; Brauchi, Sebastian; Orta, Gerardo; Zaelzer, Cristián; Vargas, Guillermo

    2007-01-01

    Ion channels activate by sensing stimuli such as membrane voltage, ligand binding or temperature and transduce this information into conformational changes that open the channel pore. Thus, a key question in understanding ion channel function is how do the protein domains involved in sensing stimuli (sensors) and opening the pore (gates) communicate. In this regard, transient receptor potential (TRP) channels that confer thermosensation [A. Dhaka, V. Viswanath, A. Patapoutian, TRP ion channels and temperature sensation, Annu. Rev. Neurosci. 29 (2006) 135-161; I.S. Ramsey, M. Delling, D.E. Clapham, An introduction to TRP channels, Annu. Rev. Physiol. 68 (2006) 619-647] (thermoTRP; Q(10)>10) are unique to the extent that they integrate a variety of physical and chemical stimuli. In some cases such as, for example, the vanilloid receptor TRPV1 [M.J. Caterina, M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, D. Julius, The capsaicin receptor: a heat-activated ion channel in the pain pathway, Nature 389 (1997) 816-824] and TRPA1 [G.M. Story, A.M. Peier, A.J. Reeve, S.R. Eid, J. Mosbacher, T.R. Hricik, T.J. Earley, A.C. Hergarden, D.A. Andersson, S.W. Hwang, P. McIntyre, T. Jegla, S. Bevan, A. Patapoutian, ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures, Cell 112 (2003) 819-829; S. Jordt, D. Julius, Molecular basis for species-specific sensitivity to "hot" chilli peppers, Cell 108 (2002) 421-430] the integration of these stimuli elicit pain [M. Tominaga, M.J. Caterina, A.B. Malmberg, T.A. Rosen, H. Gilbert, K. Skinner, B.E. Raumann, A.I. Basbaum, D. Julius, The cloned capsaicin receptor integrates multiple pain-producing stimuli, Neuron 21 (1998) 531-543; M. Bandell, A. Dubin, M. Petrus, A. Orth, J. Mathur, S. Hwang, A. Patapoutian, High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol, Nat. Neurosci. 9 (2006) 466-468; S. Zurborg, B. Yurgionas, JA. Jira, O

  6. Voltage-Gated R-Type Calcium Channel Inhibition via Human μ-, δ-, and κ-opioid Receptors Is Voltage-Independently Mediated by Gβγ Protein Subunits.

    PubMed

    Berecki, Géza; Motin, Leonid; Adams, David J

    2016-01-01

    Elucidating the mechanisms that modulate calcium channels via opioid receptor activation is fundamental to our understanding of both pain perception and how opioids modulate pain. Neuronal voltage-gated N-type calcium channels (Cav2.2) are inhibited by activation of G protein-coupled opioid receptors (ORs). However, inhibition of R-type (Cav2.3) channels by μ- or κ-ORs is poorly defined and has not been reported for δ-ORs. To investigate such interactions, we coexpressed human μ-, δ-, or κ-ORs with human Cav2.3 or Cav2.2 in human embryonic kidney 293 cells and measured depolarization-activated Ba(2+) currents (IBa). Selective agonists of μ-, δ-, and κ-ORs inhibited IBa through Cav2.3 channels by 35%. Cav2.2 channels were inhibited to a similar extent by κ-ORs, but more potently (60%) via μ- and δ-ORs. Antagonists of δ- and κ-ORs potentiated IBa amplitude mediated by Cav2.3 and Cav2.2 channels. Consistent with G protein βγ (Gβγ) interaction, modulation of Cav2.2 was primarily voltage-dependent and transiently relieved by depolarizing prepulses. In contrast, Cav2.3 modulation was voltage-independent and unaffected by depolarizing prepulses. However, Cav2.3 inhibition was sensitive to pertussis toxin and to intracellular application of guanosine 5'-[β-thio]diphosphate trilithium salt and guanosine 5'-[γ-thio]triphosphate tetralithium salt. Coexpression of Gβγ-specific scavengers-namely, the carboxyl terminus of the G protein-coupled receptor kinase 2 or membrane-targeted myristoylated-phosducin-attenuated or abolished Cav2.3 modulation. Our study reveals the diversity of OR-mediated signaling at Cav2 channels and identifies neuronal Cav2.3 channels as potential targets for opioid analgesics. Their novel modulation is dependent on pre-existing OR activity and mediated by membrane-delimited Gβγ subunits in a voltage-independent manner. PMID:26490245

  7. 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.

  8. 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

  9. The influence of lipids on voltage-gated ion channels

    PubMed Central

    Jiang, Qiu-Xing; Gonen, Tamir

    2012-01-01

    Voltage-gated ion channels are responsible for transmitting electrochemical signals in both excitable and non-excitable cells. Structural studies of voltage-gated potassium and sodium channels by X-ray crystallography have revealed atomic details on their voltage-sensor domains and pore domains, and were put in context of disparate mechanistic views on the voltage-driven conformational changes in these proteins. Functional investigation of voltage-gated channels in membranes, however, showcased a mechanism of lipid-dependent gating for voltage-gated channels, suggesting that the lipids play an indispensible and critical role in the proper gating of many of these channels. Structure determination of membrane-embedded voltage-gated ion channels appears to be the next frontier in fully addressing the mechanism by which the voltage sensor domains control channel opening. Currently electron crystallography is the only structural biology method in which a membrane protein of interest is crystallized within a complete lipid-bilayer mimicking the native environment of a biological membrane. At a sufficiently high resolution, an electron crystallographic structure could reveal lipids, the channel and their mutual interactions at the atomic level. Electron crystallography is therefore a promising avenue toward understanding how lipids modulate channel activation through close association with the voltage sensor domains. PMID:22483432

  10. Alkanols inhibit voltage-gated K+ channels via a distinct gating modifying mechanism that prevents gate opening

    PubMed Central

    Martínez-Morales, Evelyn; Kopljar, Ivan; Snyders, Dirk J.; Labro, Alain J.

    2015-01-01

    Alkanols are small aliphatic compounds that inhibit voltage-gated K+ (Kv) channels through a yet unresolved gating mechanism. Kv channels detect changes in the membrane potential with their voltage-sensing domains (VSDs) that reorient and generate a transient gating current. Both 1-Butanol (1-BuOH) and 1-Hexanol (1-HeOH) inhibited the ionic currents of the Shaker Kv channel in a concentration dependent manner with an IC50 value of approximately 50 mM and 3 mM, respectively. Using the non-conducting Shaker-W434F mutant, we found that both alkanols immobilized approximately 10% of the gating charge and accelerated the deactivating gating currents simultaneously with ionic current inhibition. Thus, alkanols prevent the final VSD movement(s) that is associated with channel gate opening. Applying 1-BuOH and 1-HeOH to the Shaker-P475A mutant, in which the final gating transition is isolated from earlier VSD movements, strengthened that neither alkanol affected the early VSD movements. Drug competition experiments showed that alkanols do not share the binding site of 4-aminopyridine, a drug that exerts a similar effect at the gating current level. Thus, alkanols inhibit Shaker-type Kv channels via a unique gating modifying mechanism that stabilizes the channel in its non-conducting activated state. PMID:26616025

  11. Structure, dynamics and implied gating mechanism of a human cyclic nucleotide-gated channel.

    PubMed

    Gofman, Yana; Schärfe, Charlotta; Marks, Debora S; Haliloglu, Turkan; Ben-Tal, Nir

    2014-12-01

    Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels, essential for visual and olfactory sensory transduction. Although the channels include voltage-sensor domains (VSDs), their conductance is thought to be independent of the membrane potential, and their gating regulated by cytosolic cyclic nucleotide-binding domains. Mutations in these channels result in severe, degenerative retinal diseases, which remain untreatable. The lack of structural information on CNG channels has prevented mechanistic understanding of disease-causing mutations, precluded structure-based drug design, and hampered in silico investigation of the gating mechanism. To address this, we built a 3D model of the cone tetrameric CNG channel, based on homology to two distinct templates with known structures: the transmembrane (TM) domain of a bacterial channel, and the cyclic nucleotide-binding domain of the mouse HCN2 channel. Since the TM-domain template had low sequence-similarity to the TM domains of the CNG channels, and to reconcile conflicts between the two templates, we developed a novel, hybrid approach, combining homology modeling with evolutionary coupling constraints. Next, we used elastic network analysis of the model structure to investigate global motions of the channel and to elucidate its gating mechanism. We found the following: (i) In the main mode of motion, the TM and cytosolic domains counter-rotated around the membrane normal. We related this motion to gating, a proposition that is supported by previous experimental data, and by comparison to the known gating mechanism of the bacterial KirBac channel. (ii) The VSDs could facilitate gating (supplementing the pore gate), explaining their presence in such 'voltage-insensitive' channels. (iii) Our elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently, but are coupled to each

  12. Polyunsaturated Fatty Acids Modify the Gating of Kv Channels

    PubMed Central

    Moreno, Cristina; Macias, Alvaro; Prieto, Angela; De La Cruz, Alicia; Valenzuela, Carmen

    2012-01-01

    Polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, which are attributed to their capability to modulate ion channels. This PUFAs ability has been reported to be due to their effects on the gating properties of ion channels. In the present review, we will focus on the role of PUFAs on the gating of two Kv channels, Kv1.5 and Kv11.1. Kv1.5 channels are blocked by n−3 PUFAs of marine [docosahexaenoic acid (DHA) and eicosapentaenoic acid] and plant origin (alpha-linolenic acid, ALA) at physiological concentrations. The blockade of Kv1.5 channels by PUFAs steeply increased in the range of membrane potentials coinciding with those of Kv1.5 channel activation, suggesting that PUFAs-channel binding may derive a significant fraction of its voltage sensitivity through the coupling to channel gating. A similar shift in the activation voltage was noted for the effects of n–6 arachidonic acid (AA) and DHA on Kv1.1, Kv1.2, and Kv11.1 channels. PUFAs-Kv1.5 channel interaction is time-dependent, producing a fast decay of the current upon depolarization. Thus, Kv1.5 channel opening is a prerequisite for the PUFA-channel interaction. Similar to the Kv1.5 channels, the blockade of Kv11.1 channels by AA and DHA steeply increased in the range of membrane potentials that coincided with the range of Kv11.1 channel activation, suggesting that the PUFAs-Kv channel interactions are also coupled to channel gating. Furthermore, AA regulates the inactivation process in other Kv channels, introducing a fast voltage-dependent inactivation in non-inactivating Kv channels. These results have been explained within the framework that AA closes voltage-dependent potassium channels by inducing conformational changes in the selectivity filter, suggesting that Kv channel gating is lipid dependent. PMID:22973228

  13. Kinetic contributions to gating by interactions unique to N-methyl-D-aspartate (NMDA) receptors.

    PubMed

    Borschel, William F; Cummings, Kirstie A; Tindell, LeeAnn K; Popescu, Gabriela K

    2015-10-30

    Among glutamate-gated channels, NMDA receptors produce currents that subside with unusually slow kinetics, and this feature is essential to the physiology of central excitatory synapses. Relative to the homologous AMPA and kainate receptors, NMDA receptors have additional intersubunit contacts in the ligand binding domain that occur at both conserved and non-conserved sites. We examined GluN1/GluN2A single-channel currents with kinetic analyses and modeling to probe these class-specific intersubunit interactions for their role in glutamate binding and receptor gating. We found that substitutions that eliminate such interactions at non-conserved sites reduced stationary gating, accelerated deactivation, and imparted sensitivity to aniracetam, an AMPA receptor-selective positive modulator. Abolishing unique contacts at conserved sites also reduced stationary gating and accelerated deactivation. These results show that contacts specific to NMDA receptors, which brace the heterodimer interface within the ligand binding domain, stabilize actively gating receptor conformations and result in longer bursts and slower deactivations. They support the view that the strength of the heterodimer interface modulates gating in both NMDA and non-NMDA receptors and that unique interactions at this interface are responsible in part for basic differences between the kinetics of NMDA and non-NMDA currents at glutamatergic synapses. PMID:26370091

  14. Strategies for Investigating G-Protein Modulation of Voltage-Gated Ca2+ Channels.

    PubMed

    Lu, Van B; Ikeda, Stephen R

    2016-01-01

    G-protein-coupled receptor modulation of voltage-gated ion channels is a common means of fine-tuning the response of channels to changes in membrane potential. Such modulation impacts physiological processes such as synaptic transmission, and hence therapeutic strategies often directly or indirectly target these pathways. As an exemplar of channel modulation, we examine strategies for investigating G-protein modulation of CaV2.2 or N-type voltage-gated Ca(2+) channels. We focus on biochemical and genetic tools for defining the molecular mechanisms underlying the various forms of CaV2.2 channel modulation initiated following ligand binding to G-protein-coupled receptors. PMID:27140924

  15. Agonist-dependent single channel current and gating in alpha4beta2delta and alpha1beta2gamma2S GABAA receptors.

    PubMed

    Keramidas, Angelo; Harrison, Neil L

    2008-02-01

    The family of gamma-aminobutyric acid type A receptors (GABA(A)Rs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic GABA(A)Rs that are mainly comprised of alpha(1), beta(2), and gamma(2) subunits, whereas tonic inhibition is mediated by extrasynaptic GABA(A)Rs comprised of alpha(4/6), beta(2), and delta subunits. We investigated the activation properties of recombinant alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs in response to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at alpha(4)beta(2)delta GABA(A)Rs (beta approximately 1600 s(-1)) than at alpha(1)beta(2)gamma(2S) GABA(A)Rs (beta approximately 460 s(-1)), whereas GABA activated alpha(1)beta(2)gamma(2S) GABA(A)Rs more rapidly (beta approximately 1800 s(-1)) than alpha(4)beta(2)delta GABA(A)Rs (beta < 440 s(-1)). Single channel recordings of alpha(1)beta(2)gamma(2S) and alpha(4)beta(2)delta GABA(A)Rs showed that both channels open to a main conductance state of approximately 25 pS at -70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited approximately 36 pS openings at both channels. Saturating concentrations of GABA elicited brief (<10 ms) openings with low intraburst open probability (P(O) approximately 0.3) at alpha(4)beta(2)delta GABA(A)Rs and at least two "modes" of single channel bursting activity, lasting approximately 100 ms at alpha(1)beta(2)gamma(2S) GABA(A)Rs. The most prevalent bursting mode had a P(O) of approximately 0.7 and was described by a reaction scheme with three open and three shut states, whereas the "high" P(O) mode ( approximately 0.9) was characterized by two shut and three open states. Single channel activity elicited by THIP in alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA

  16. Three Homologous Subunits Form a High Affinity Peptide-gated Ion Channel in Hydra*

    PubMed Central

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D.; Williamson, Michael; Kalbacher, Hubert; Grimmelikhuijzen, Cornelis J. P.; Holstein, Thomas W.; Gründer, Stefan

    2010-01-01

    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. PMID:20159980

  17. Oxidative Modulation of Voltage-Gated Potassium Channels

    PubMed Central

    Sahoo, Nirakar; Hoshi, Toshinori

    2014-01-01

    Abstract Significance: Voltage-gated K+ channels are a large family of K+-selective ion channel protein complexes that open on membrane depolarization. These K+ channels are expressed in diverse tissues and their function is vital for numerous physiological processes, in particular of neurons and muscle cells. Potentially reversible oxidative regulation of voltage-gated K+ channels by reactive species such as reactive oxygen species (ROS) represents a contributing mechanism of normal cellular plasticity and may play important roles in diverse pathologies including neurodegenerative diseases. Recent Advances: Studies using various protocols of oxidative modification, site-directed mutagenesis, and structural and kinetic modeling provide a broader phenomenology and emerging mechanistic insights. Critical Issues: Physicochemical mechanisms of the functional consequences of oxidative modifications of voltage-gated K+ channels are only beginning to be revealed. In vivo documentation of oxidative modifications of specific amino-acid residues of various voltage-gated K+ channel proteins, including the target specificity issue, is largely absent. Future Directions: High-resolution chemical and proteomic analysis of ion channel proteins with respect to oxidative modification combined with ongoing studies on channel structure and function will provide a better understanding of how the function of voltage-gated K+ channels is tuned by ROS and the corresponding reducing enzymes to meet cellular needs. Antioxid. Redox Signal. 21, 933–952. PMID:24040918

  18. 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

  19. Supramolecular Assemblies and Localized Regulation of Voltage-Gated Ion Channels

    PubMed Central

    Dai, Shuiping; Hall, Duane D.; Hell, Johannes W.

    2009-01-01

    This review addresses the localized regulation of voltage-gated ion channels by phosphorylation. Comprehensive data on channel regulation by associated protein kinases, phosphatases, and related regulatory proteins are mainly available for voltage-gated Ca2+ channels, which form the main focus of this review. Other voltage-gated ion channels and especially Kv7.1-3 (KCNQ1-3), the large- and small-conductance Ca2+-activated K+ channels BK and SK2, and the inward-rectifying K+ channels Kir3 have also been studied to quite some extent and will be included. Regulation of the L-type Ca2+ channel Cav1.2 by PKA has been studied most thoroughly as it underlies the cardiac fight-or-flight response. A prototypical Cav1.2 signaling complex containing the β2 adrenergic receptor, the heterotrimeric G protein Gs, adenylyl cyclase, and PKA has been identified that supports highly localized via cAMP. The type 2 ryanodine receptor as well as AMPA- and NMDA-type glutamate receptors are in close proximity to Cav1.2 in cardiomyocytes and neurons, respectively, yet independently anchor PKA, CaMKII, and the serine/threonine phosphatases PP1, PP2A, and PP2B, as is discussed in detail. Descriptions of the structural and functional aspects of the interactions of PKA, PKC, CaMKII, Src, and various phosphatases with Cav1.2 will include comparisons with analogous interactions with other channels such as the ryanodine receptor or ionotropic glutamate receptors. Regulation of Na+ and K+ channel phosphorylation complexes will be discussed in separate papers. This review is thus intended for readers interested in ion channel regulation or in localization of kinases, phosphatases, and their upstream regulators. PMID:19342611

  20. 2. INTAKE CHANNEL LOOKING NORTHWEST; GATES 1, 2, AND 3 ...

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

    2. INTAKE CHANNEL LOOKING NORTHWEST; GATES 1, 2, AND 3 SHOWING. - Hondius Water Line, 1.6 miles Northwest of Park headquarters building & 1 mile Northwest of Beaver Meadows entrance station, Estes Park, Larimer County, CO

  1. 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

  2. 4. GATES 1 AND 2, INTAKE CHANNEL LOOKING EAST. ...

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

    4. GATES 1 AND 2, INTAKE CHANNEL LOOKING EAST. - Hondius Water Line, 1.6 miles Northwest of Park headquarters building & 1 mile Northwest of Beaver Meadows entrance station, Estes Park, Larimer County, CO

  3. The CFTR ion channel: gating, regulation, and anion permeation.

    PubMed

    Hwang, Tzyh-Chang; Kirk, Kevin L

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated anion channel with two remarkable distinctions. First, it is the only ATP-binding cassette (ABC) transporter that is known to be an ion channel--almost all others function as transport ATPases. Second, CFTR is the only ligand-gated channel that consumes its ligand (ATP) during the gating cycle--a consequence of its enzymatic activity as an ABC transporter. We discuss these special properties of CFTR in the context of its evolutionary history as an ABC transporter. Other topics include the mechanisms by which CFTR gating is regulated by phosphorylation of its unique regulatory domain and our current view of the CFTR permeation pathway (or pore). Understanding these basic operating principles of the CFTR channel is central to defining the mechanisms of action of prospective cystic fibrosis drugs and to the development of new, rational treatment strategies. PMID:23284076

  4. Chloride dependence of hyperpolarization-activated chloride channel gates.

    PubMed

    Pusch, M; Jordt, S E; Stein, V; Jentsch, T J

    1999-03-01

    1. 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. 2. 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. 3. With all these channels, reducing [Cl-]i shifted activation to more negative voltages and reduced the maximal activation at most negative voltages. 4. 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. 5. 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. 6. 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-. 7. 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 for the

  5. 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

  6. Voltage-gated sodium channels in neurological disorders.

    PubMed

    Chahine, Mohamed; Chatelier, Aurélien; Babich, Olga; Krupp, Johannes J

    2008-04-01

    Voltage-gated sodium channels play an essential biophysical role in many excitable cells such as neurons. They transmit electrical signals through action potential (AP) generation and propagation in the peripheral (PNS) and central nervous systems (CNS). Each sodium channel is formed by one alpha-subunit and one or more beta-subunits. There is growing evidence indicating that mutations, changes in expression, or inappropriate modulation of these channels can lead to electrical instability of the cell membrane and inappropriate spontaneous activity observed during pathological states. This review describes the biochemical, biophysical and pharmacological properties of neuronal voltage-gated sodium channels (VGSC) and their implication in several neurological disorders. PMID:18537643

  7. 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

  8. Clustered voltage-gated Na+ channels in Aplysia axons.

    PubMed

    Johnston, W L; Dyer, J R; Castellucci, V F; Dunn, R J

    1996-03-01

    Clustering of voltage-gated Na+ channels is critical for the fast saltatory conduction of action potentials in vertebrate myelinated axons. However, the mechanisms responsible for the generation and maintenance of Na+ channel clustering are not well understood. In this study we have raised an antibody against the cloned SCAP-1 voltage-gated Na+ channel of the marine invertebrate Aplysia californica and used it to examine Na+ channel localization in Aplysia ganglia and in cultured Aplysia sensory neurons. Our results show that there is a large cytoplasmic pool of Na+ channels in the soma of Aplysia neurons. Furthermore, we show that Na+ channels in Aplysia axons are not homogeneously distributed but, rather, are present in distinct clusters. Theoretical considerations indicate that Na+ channel clustering may enhance action potential conduction. We propose that clustered Na+ channels may be a fundamental property of many axons, and perhaps of many membranes that conduct Na(+)-dependent action potentials. PMID:8774441

  9. Voltage-Gated Potassium Channels: A Structural Examination of Selectivity and Gating.

    PubMed

    Kim, Dorothy M; Nimigean, Crina M

    2016-01-01

    Voltage-gated potassium channels play a fundamental role in the generation and propagation of the action potential. The discovery of these channels began with predictions made by early pioneers, and has culminated in their extensive functional and structural characterization by electrophysiological, spectroscopic, and crystallographic studies. With the aid of a variety of crystal structures of these channels, a highly detailed picture emerges of how the voltage-sensing domain reports changes in the membrane electric field and couples this to conformational changes in the activation gate. In addition, high-resolution structural and functional studies of K(+) channel pores, such as KcsA and MthK, offer a comprehensive picture on how selectivity is achieved in K(+) channels. Here, we illustrate the remarkable features of voltage-gated potassium channels and explain the mechanisms used by these machines with experimental data. PMID:27141052

  10. 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

  11. Electrophysiological Signature of Homomeric and Heteromeric Glycine Receptor Channels.

    PubMed

    Raltschev, Constanze; Hetsch, Florian; Winkelmann, Aline; Meier, Jochen C; Semtner, Marcus

    2016-08-19

    Glycine receptors are chloride-permeable, ligand-gated ion channels and contribute to the inhibition of neuronal firing in the central nervous system or to facilitation of neurotransmitter release if expressed at presynaptic sites. Recent structure-function studies have provided detailed insights into the mechanisms of channel gating, desensitization, and ion permeation. However, most of the work has focused only on comparing a few isoforms, and among studies, different cellular expression systems were used. Here, we performed a series of experiments using recombinantly expressed homomeric and heteromeric glycine receptor channels, including their splice variants, in the same cellular expression system to investigate and compare their electrophysiological properties. Our data show that the current-voltage relationships of homomeric channels formed by the α2 or α3 subunits change upon receptor desensitization from a linear to an inwardly rectifying shape, in contrast to their heteromeric counterparts. The results demonstrate that inward rectification depends on a single amino acid (Ala(254)) at the inner pore mouth of the channels and is closely linked to chloride permeation. We also show that the current-voltage relationships of glycine-evoked currents in primary hippocampal neurons are inwardly rectifying upon desensitization. Thus, the alanine residue Ala(254) determines voltage-dependent rectification upon receptor desensitization and reveals a physio-molecular signature of homomeric glycine receptor channels, which provides unprecedented opportunities for the identification of these channels at the single cell level. PMID:27382060

  12. Bell-shaped calcium-response curves of lns(l,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum

    NASA Astrophysics Data System (ADS)

    Bezprozvanny, Llya; Watras, James; Ehrlich, Barbara E.

    1991-06-01

    RELEASE of calcium from intracellular stores occurs by two pathways, an inositol 1,4,5-trisphosphate (InsP3)-gated channel1-3 and a calcium-gated channel (ryanodine receptor)4-6. Using specific antibodies, both receptors were found in Purkinje cells of cerebellum7,8. We have now compared the functional properties of the channels corresponding to the two receptors by incorporating endoplasmic reticulum vesicles from canine cerebellum into planar bilayers. InsP3-gated channels were observed most frequently. Another channel type was activated by adenine nucleotides or caffeine, inhibited by ruthenium red, and modified by ryanodine, characteristics of the ryanodine receptor/channel6. The open probability of both channel types displayed a bell-shaped curve for dependence on calcium. For the InsP3-gated channel, the maximum probability of opening occurred at 0.2 µM free calcium, with sharp decreases on either side of the maximum. Maximum activity for the ryanodine receptor/channel was maintained between 1 and 100 µM calcium. Thus, within the physiological range of cytoplasmic calcium, the InsP3-gated channel itself allows positive feed-back and then negative feedback for calcium release, whereas the ryanodine receptor/channel behaves solely as a calcium-activated channel. The existence in the same cell of two channels with different responses to calcium and different ligand sensitivities provides a basis for complex patterns of intracellular calcium regulation.

  13. The CFTR Ion Channel: Gating, Regulation, and Anion Permeation

    PubMed Central

    Hwang, Tzyh-Chang; Kirk, Kevin L.

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated anion channel with two remarkable distinctions. First, it is the only ATP-binding cassette (ABC) transporter that is known to be an ion channel—almost all others function as transport ATPases. Second, CFTR is the only ligand-gated channel that consumes its ligand (ATP) during the gating cycle—a consequence of its enzymatic activity as an ABC transporter. We discuss these special properties of CFTR in the context of its evolutionary history as an ABC transporter. Other topics include the mechanisms by which CFTR gating is regulated by phosphorylation of its unique regulatory domain and our current view of the CFTR permeation pathway (or pore). Understanding these basic operating principles of the CFTR channel is central to defining the mechanisms of action of prospective cystic fibrosis drugs and to the development of new, rational treatment strategies. PMID:23284076

  14. Cys-loop ligand-gated chloride channels in dorsal unpaired median neurons of Locusta migratoria.

    PubMed

    Janssen, Daniel; Derst, Christian; Rigo, Jean-Michel; Van Kerkhove, Emmy

    2010-05-01

    In insects, inhibitory neurotransmission is generally associated with members of the cys-loop ligand-gated anion channels, such as the glutamate-gated chloride channel (GluCl), the GABA-gated chloride channels (GABACl), and the histamine-gated chloride channels (HisCl). These ionotropic receptors are considered established target sites for the development of insecticides, and therefore it is necessary to obtain a better insight in their distribution, structure, and functional properties. Here, by combining electrophysiology and molecular biology techniques, we identified and characterized GluCl, GABACl, and HisCl in dorsal unpaired median (DUM) neurons of Locust migratoria. In whole cell patch-clamp recordings, application of glutamate, GABA, or histamine induced rapidly activating ionic currents. GluCls were sensitive to ibotenic acid and blocked by picrotoxin and fipronil. The pharmacological profile of the L. migratoria GABACl fitted neither the vertebrate GABA(A) nor GABA(C) receptor and was similar to the properties of the cloned Drosophila melanogaster GABA receptor subunit (Rdl). The expression of Rdl-like subunit-containing GABA receptors was shown at the molecular level using RT-PCR. Sequencing analysis indicated that the orthologous GABACl of D. melanogaster CG10357-A is expressed in DUM neurons of L. migratoria. Histamine-induced currents exhibited a fast onset and desensitized completely on continuous application of histamine. In conclusion, within the DUM neurons of L. migratoria, we identified three different cys-loop ligand-gated anion channels that use GABA, glutamate, or histamine as their neurotransmitter. PMID:20200125

  15. The gain-of-function enhancement of IP3-receptor channel gating by familial Alzheimer's disease-linked presenilin mutants increases the open probability of mitochondrial permeability transition pore.

    PubMed

    Toglia, Patrick; Ullah, Ghanim

    2016-07-01

    Mutants in presenilins (PS1 or PS2) are the major cause of familial Alzheimer's disease (FAD). They affect intracellular Ca(2+) homeostasis by increasing the open probability (Po) of inositol 1,4,5-trisposphate (IP3) receptor (IP3R) Ca(2+) release channel located on the endoplasmic reticulum (ER) leading to exaggerated Ca(2+) release into a cytoplasmic microdomain formed by neighboring cluster of a few IP3R channels and mitochondrial Ca(2+) uniporter (MCU). Ca(2+) concentration in the microdomain ( [Formula: see text] ) depends on the distance between the cluster and MCU (r); the number of IP3R in the cluster releasing Ca(2+) to the cytoplasm ( [Formula: see text] ), and Po of IP3R. Using experimental whole-cell IP3R-mediated cytosolic Ca(2+) data, in conjunction with a computational model of cell bioenergetics, a data-driven Markov chain model for IP3R gating, and a model for the dynamics of the mitochondrial permeability transition pore (PTP), we explore differences in mitochondrial Ca(2+) uptake in cells expressing wild type (PS1-WT) and FAD-causing mutant (PS1-M146L) PS. We find that increased mitochondrial [Formula: see text] due to the gain-of-function enhancement of IP3R channels in the cells expressing PS1-M146L leads to the opening of PTP in high conductance state (PTPh), where the latency of opening is inversely correlated with r and proportional to [Formula: see text] . Furthermore, we observe diminished inner mitochondrial membrane potential (ΔΨm), [NADH], [Formula: see text] , and [ATP] when PTP opens. Additionally, we explore how parameters such as the pH gradient, inorganic phosphate concentration, and the rate of the Na(+)/Ca(2+)-exchanger affect the latency of PTP to open in PTPh. PMID:27184076

  16. Voltage Sensor in Voltage-gated ion channels

    NASA Astrophysics Data System (ADS)

    Bezanilla, Francisco

    2006-03-01

    Voltage-gated ion channels are intrinsic membrane proteins that play a fundamental role in the generation and propagation of the nerve impulse. Their salient characteristic is that the probability of the ion channel of being open depends steeply on the voltage across the membrane where those channels are inserted. Thus, in a membrane containing many channels, the ionic conductance is controlled by the membrane potential. The voltage exerts its control on the channel by reorienting intrinsic charges in the protein, generally arginine or lysine residues located in the 4th transmembrane segment of the channel protein, a region that has been called the voltage sensor. Upon changing the membrane potential, the charged groups reorient in the field generating a transient current (gating current). The properties of the gating current may be studied with a small number of channels to infer the operation of the sensor at the single molecule level by noise analysis or with a large number of channels to infer the details of the energy landscape the sensor traverses in opening the pore. This information is global in nature and cannot pinpoint the exact origin of the charge movement that generates the gating current. The movement of physical charges in the protein has been inferred with site-directed mutagenesis of the charged residues to histidine that allows the study of proton accessibility. The actual movement has been studied with fluorescence spectroscopy, fluorescence resonance energy transfer. The combined information of site-directed mutagenesis, gating currents, fluorescence studies and emerging crystal structures have started to delineate a physical representation of the conformational changes responsible for voltage sensing that lead to the opening of the conduction pore in voltage-gated ion channels.

  17. 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

  18. High temperature sensitivity is intrinsic to voltage-gated potassium channels.

    PubMed

    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. PMID:25030910

  19. 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

  20. Inositol Trisphosphate Receptor Ca2+ Release Channels

    PubMed Central

    FOSKETT, J. KEVIN; WHITE, CARL; CHEUNG, KING-HO; MAK, DON-ON DANIEL

    2010-01-01

    The inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are a family of Ca2+ release channels localized predominately in the endoplasmic reticulum of all cell types. They function to release Ca2+ into the cytoplasm in response to InsP3 produced by diverse stimuli, generating complex local and global Ca2+ signals that regulate numerous cell physiological processes ranging from gene transcription to secretion to learning and memory. The InsP3R is a calcium-selective cation channel whose gating is regulated not only by InsP3, but by other ligands as well, in particular cytoplasmic Ca2+. Over the last decade, detailed quantitative studies of InsP3R channel function and its regulation by ligands and interacting proteins have provided new insights into a remarkable richness of channel regulation and of the structural aspects that underlie signal transduction and permeation. Here, we focus on these developments and review and synthesize the literature regarding the structure and single-channel properties of the InsP3R. PMID:17429043

  1. 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

  2. The Concise Guide to PHARMACOLOGY 2015/16: Ligand-gated ion channels.

    PubMed

    Alexander, Stephen Ph; Peters, John A; Kelly, Eamonn; Marrion, Neil; Benson, Helen E; Faccenda, Elena; Pawson, Adam J; Sharman, Joanna L; Southan, Christopher; Davies, Jamie A

    2015-12-01

    The Concise Guide to PHARMACOLOGY 2015/16 provides concise overviews of the key properties of over 1750 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13349/full. Ligand-gated ion channels are one of the eight major pharmacological targets into which the Guide is divided, with the others being: ligand-gated ion channels, voltage-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The Concise Guide is published in landscape format in order to facilitate comparison of related targets. It is a condensed version of material contemporary to late 2015, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in the previous Guides to Receptors & Channels and the Concise Guide to PHARMACOLOGY 2013/14. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates. PMID:26650440

  3. 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.

  4. 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.

  5. 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

  6. Emerging Roles of L-Type Voltage-Gated and Other Calcium Channels in T Lymphocytes

    PubMed Central

    Badou, Abdallah; Jha, Mithilesh K.; Matza, Didi; Flavell, Richard A.

    2013-01-01

    In T lymphocytes, calcium ion controls a variety of biological processes including development, survival, proliferation, and effector functions. These distinct and specific roles are regulated by different calcium signals, which are generated by various plasma membrane calcium channels. The repertoire of calcium-conducting proteins in T lymphocytes includes store-operated CRAC channels, transient receptor potential channels, P2X channels, and L-type voltage-gated calcium (Cav1) channels. In this paper, we will focus mainly on the role of the Cav1 channels found expressed by T lymphocytes, where these channels appear to operate in a T cell receptor stimulation-dependent and voltage sensor independent manner. We will review their expression profile at various differentiation stages of CD4 and CD8 T lymphocytes. Then, we will present crucial genetic evidence in favor of a role of these Cav1 channels and related regulatory proteins in both CD4 and CD8 T cell functions such as proliferation, survival, cytokine production, and cytolysis. Finally, we will provide evidence and speculate on how these voltage-gated channels might function in the T lymphocyte, a non-excitable cell. PMID:24009608

  7. Structural elements in the Girk1 subunit that potentiate G protein-gated potassium channel activity.

    PubMed

    Wydeven, Nicole; Young, Daniele; Mirkovic, Kelsey; Wickman, Kevin

    2012-12-26

    G protein-gated inwardly rectifying K(+) (Girk/K(IR)3) channels mediate the inhibitory effect of many neurotransmitters on excitable cells. Girk channels are tetramers consisting of various combinations of four mammalian Girk subunits (Girk1 to -4). Although Girk1 is unable to form functional homomeric channels, its presence in cardiac and neuronal channel complexes correlates with robust channel activity. This study sought to better understand the potentiating influence of Girk1, using the GABA(B) receptor and Girk1/Girk2 heteromer as a model system. Girk1 did not increase the protein levels or alter the trafficking of Girk2-containing channels to the cell surface in transfected cells or hippocampal neurons, indicating that its potentiating influence involves enhancement of channel activity. Structural elements in both the distal carboxyl-terminal domain and channel core were identified as key determinants of robust channel activity. In the distal carboxyl-terminal domain, residue Q404 was identified as a key determinant of receptor-induced channel activity. In the Girk1 core, three unique residues in the pore (P) loop (F137, A142, Y150) were identified as a collective potentiating influence on both receptor-dependent and receptor-independent channel activity, exerting their influence, at least in part, by enhancing mean open time and single-channel conductance. Interestingly, the potentiating influence of the Girk1 P-loop is tempered by residue F162 in the second membrane-spanning domain. Thus, discontinuous and sometime opposing elements in Girk1 underlie the Girk1-dependent potentiation of receptor-dependent and receptor-independent heteromeric channel activity. PMID:23236146

  8. 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.

  9. Inherent Dynamics of the Acid-Sensing Ion Channel 1 Correlates with the Gating Mechanism

    PubMed Central

    Li, Wei-Guang; Yu, Fang; Cao, Hui; Xu, Tian-Le; Jiang, Hualiang

    2009-01-01

    The acid-sensing ion channel 1 (ASIC1) is a key receptor for extracellular protons. Although numerous structural and functional studies have been performed on this channel, the structural dynamics underlying the gating mechanism remains unknown. We used normal mode analysis, mutagenesis, and electrophysiological methods to explore the relationship between the inherent dynamics of ASIC1 and its gating mechanism. Here we show that a series of collective motions among the domains and subdomains of ASIC1 correlate with its acid-sensing function. The normal mode analysis result reveals that the intrinsic rotation of the extracellular domain and the collective motions between the thumb and finger induced by proton binding drive the receptor to experience a deformation from the extracellular domain to the transmembrane domain, triggering the channel pore to undergo “twist-to-open” motions. The movements in the transmembrane domain indicate that the likely position of the channel gate is around Leu440. These motion modes are compatible with a wide body of our complementary mutations and electrophysiological data. This study provides the dynamic fundamentals of ASIC1 gating. PMID:19597538

  10. Glutamate Receptor Ion Channels: Structure, Regulation, and Function

    PubMed Central

    Wollmuth, Lonnie P.; McBain, Chris J.; Menniti, Frank S.; Vance, Katie M.; Ogden, Kevin K.; Hansen, Kasper B.; Yuan, Hongjie; Myers, Scott J.; Dingledine, Ray

    2010-01-01

    The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors. PMID:20716669

  11. Voltage-dependent gating and gating charge measurements in the Kv1.2 potassium channel

    PubMed Central

    Ishida, Itzel G.; Rangel-Yescas, Gisela E.; Carrasco-Zanini, Julia

    2015-01-01

    Much has been learned about the voltage sensors of ion channels since the x-ray structure of the mammalian voltage-gated potassium channel Kv1.2 was published in 2005. High resolution structural data of a Kv channel enabled the structural interpretation of numerous electrophysiological findings collected in various ion channels, most notably Shaker, and permitted the development of meticulous computational simulations of the activation mechanism. The fundamental premise for the structural interpretation of functional measurements from Shaker is that this channel and Kv1.2 have the same characteristics, such that correlation of data from both channels would be a trivial task. We tested these assumptions by measuring Kv1.2 voltage-dependent gating and charge per channel. We found that the Kv1.2 gating charge is near 10 elementary charges (eo), ∼25% less than the well-established 13–14 eo in Shaker. Next, we neutralized positive residues in the Kv1.2 S4 transmembrane segment to investigate the cause of the reduction of the gating charge and found that, whereas replacing R1 with glutamine decreased voltage sensitivity to ∼50% of the wild-type channel value, mutation of the subsequent arginines had a much smaller effect. These data are in marked contrast to the effects of charge neutralization in Shaker, where removal of the first four basic residues reduces the gating charge by roughly the same amount. In light of these differences, we propose that the voltage-sensing domains (VSDs) of Kv1.2 and Shaker might undergo the same physical movement, but the septum that separates the aqueous crevices in the VSD of Kv1.2 might be thicker than Shaker’s, accounting for the smaller Kv1.2 gating charge. PMID:25779871

  12. Voltage-dependent gating and gating charge measurements in the Kv1.2 potassium channel.

    PubMed

    Ishida, Itzel G; Rangel-Yescas, Gisela E; Carrasco-Zanini, Julia; Islas, León D

    2015-04-01

    Much has been learned about the voltage sensors of ion channels since the x-ray structure of the mammalian voltage-gated potassium channel Kv1.2 was published in 2005. High resolution structural data of a Kv channel enabled the structural interpretation of numerous electrophysiological findings collected in various ion channels, most notably Shaker, and permitted the development of meticulous computational simulations of the activation mechanism. The fundamental premise for the structural interpretation of functional measurements from Shaker is that this channel and Kv1.2 have the same characteristics, such that correlation of data from both channels would be a trivial task. We tested these assumptions by measuring Kv1.2 voltage-dependent gating and charge per channel. We found that the Kv1.2 gating charge is near 10 elementary charges (eo), ∼25% less than the well-established 13-14 eo in Shaker. Next, we neutralized positive residues in the Kv1.2 S4 transmembrane segment to investigate the cause of the reduction of the gating charge and found that, whereas replacing R1 with glutamine decreased voltage sensitivity to ∼50% of the wild-type channel value, mutation of the subsequent arginines had a much smaller effect. These data are in marked contrast to the effects of charge neutralization in Shaker, where removal of the first four basic residues reduces the gating charge by roughly the same amount. In light of these differences, we propose that the voltage-sensing domains (VSDs) of Kv1.2 and Shaker might undergo the same physical movement, but the septum that separates the aqueous crevices in the VSD of Kv1.2 might be thicker than Shaker's, accounting for the smaller Kv1.2 gating charge. PMID:25779871

  13. 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

  14. Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel.

    PubMed

    Heusser, Stephanie A; Yoluk, Özge; Klement, Göran; Riederer, Erika A; Lindahl, Erik; Howard, Rebecca J

    2016-07-01

    The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region's role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. In this study, we elucidate the validity of a modified glutamate-gated

  15. Voltage gated sodium channels as drug discovery targets

    PubMed Central

    Bagal, Sharan K; Marron, Brian E; Owen, Robert M; Storer, R Ian; Swain, Nigel A

    2015-01-01

    Voltage-gated sodium (NaV) channels are a family of transmembrane ion channel proteins. They function by forming a gated, water-filled pore to help establish and control cell membrane potential via control of the flow of ions between the intracellular and the extracellular environments. Blockade of NaVs has been successfully accomplished in the clinic to enable control of pathological firing patterns that occur in a diverse range of conditions such as chronic pain, epilepsy, and cardiac arrhythmias. First generation sodium channel modulator drugs, despite low inherent subtype selectivity, preferentially act on over-excited cells which reduces undesirable side effects in the clinic. However, the limited therapeutic indices observed with the first generation demanded a new generation of sodium channel inhibitors. The structure, function and the state of the art in sodium channel modulator drug discovery are discussed in this chapter. PMID:26646477

  16. Emerging Molecular Mechanisms of Signal Transduction in Pentameric Ligand-Gated Ion Channels.

    PubMed

    Nemecz, Ákos; Prevost, Marie S; Menny, Anaïs; Corringer, Pierre-Jean

    2016-05-01

    Nicotinic acetylcholine, serotonin type 3, γ-amminobutyric acid type A, and glycine receptors are major players of human neuronal communication. They belong to the family of pentameric ligand-gated ion channels, sharing a highly conserved modular 3D structure. Recently, high-resolution structures of both open- and closed-pore conformations have been solved for a bacterial, an invertebrate, and a vertebrate receptor in this family. These data suggest that a common gating mechanism occurs, coupling neurotransmitter binding to pore opening, but they also pinpoint significant differences among subtypes. In this Review, we summarize the structural and functional data in light of these gating models and speculate about their mechanistic consequences on ion permeation, pathological mutations, as well as functional regulation by orthosteric and allosteric effectors. PMID:27151638

  17. Magnesium gating of cardiac gap junction channels.

    PubMed

    Matsuda, Hiroyuki; Kurata, Yasutaka; Oka, Chiaki; Matsuoka, Satoshi; Noma, Akinori

    2010-09-01

    We aimed to study kinetics of modulation by intracellular Mg(2+) of cardiac gap junction (Mg(2+) gate). Paired myocytes of guinea-pig ventricle were superfused with solutions containing various concentrations of Mg(2+). In order to rapidly apply Mg(2+) to one aspect of the gap junction, the non-junctional membrane of one of the pair was perforated at nearly the connecting site by pulses of nitrogen laser beam. The gap junction conductance (G(j)) was measured by clamping the membrane potential of the other cell using two-electrode voltage clamp method. The laser perforation immediately increased G(j), followed by slow G(j) change with time constant of 3.5 s at 10 mM Mg(2+). Mg(2+) more than 1.0 mM attenuated dose-dependently the gap junction conductance and lower Mg(2+) (0.6 mM) increased G(j) with a Hill coefficient of 3.4 and a half-maximum effective concentration of 0.6 mM. The time course of G(j) changes was fitted by single exponential function, and the relationship between the reciprocal of time constant and Mg(2+) concentration was almost linear. Based on the experimental data, a mathematical model of Mg(2+) gate with one open state and three closed states well reproduced experimental results. One-dimensional cable model of thirty ventricular myocytes connected to the Mg(2+) gate model suggested a pivotal role of the Mg(2+) gate of gap junction under pathological conditions. PMID:20553744

  18. 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

  19. 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

  20. Mechanisms of lipid regulation and lipid gating in TRPC channels.

    PubMed

    Svobodova, Barbora; Groschner, Klaus

    2016-06-01

    TRPC proteins form cation channels that integrate and relay cellular signals by mechanisms involving lipid recognition and lipid-dependent gating. The lipohilic/amphiphilic molecules that function as cellular activators or modulators of TRPC proteins span a wide range of chemical structures. In this context, cellular redox balance is likely linked to the lipid recognition/gating features of TRPC channels. Both classical ligand-protein interactions as well as indirect and promiscuous sensory mechanisms have been proposed. Some of the recognition processes are suggested to involve ancillary lipid-binding scaffolds or regulators as well as dynamic protein-protein interactions determined by bilayer architecture. A complex interplay of protein-protein and protein-lipid interactions is likely to govern the gating and/or plasma membrane recruitment of TRPC channels, thereby providing a distinguished platform for signal integration and coincident signal detection. Both the primary molecular event(s) of lipid recognition by TRPC channels as well as the transformation of these events into distinct gating movements is poorly understood at the molecular level, and it remains elusive whether lipid sensing in TRPCs is conferred to a distinct sensor domain. Recent structural information on the molecular action of lipophilic activators in distantly related members of the TRP superfamily encourages speculations on TRPC gating mechanisms involved in lipid recognition/gating. This review aims to provide an update on the current understanding of the lipid-dependent control of TRPC channels with focus on the TRPC lipid sensing, signal-integration hub and a short discussion of potential links to redox signaling. PMID:27125985

  1. 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. PMID:27129924

  2. Electrostatics and the gating pore of Shaker potassium channels.

    PubMed

    Islas, L D; Sigworth, F J

    2001-01-01

    Various experiments have suggested that the S4 segment in voltage-dependent Na(+) and K(+) channels is in contact with a solvent-accessible cavity. We explore the consequences of the existence of such a cavity through the electrostatic effects on the gating currents of Shaker K(+) channels under conditions of reduced ionic strength S. We observe that approximately 10-fold reductions of intracellular S produce reductions of the measured gating charge of approximately 10%. These effects continue at even lower values of S. The reduction of gating charge when S is reduced by 10-fold at the extracellular surface is much smaller (approximately 2%). Shifts of the Q(V) curve because of a reduced S are small (<10 mV in size), which is consistent with very little fixed surface charge. Continuum electrostatic calculations show that the S effects on gating charge can be explained by the alteration of the local potential in an intracellular conical cavity of 20-24-A depth and 12-A aperture, and a smaller extracellular cavity of 3-A depth and the same aperture. In this case, the attenuation of the membrane potential at low S leads to reduction of the apparent gating charge. We suggest that this cavity is made by a bundle of transmembrane helices, and that the gating charge movement occurs by translocation of charged residues across a thin septum of approximately 3-7 A thickness. PMID:11134232

  3. Crystal structure of the ATP-gated P2X[subscript 4] ion channel in the closed state

    SciTech Connect

    Kawate, Toshimitsu; Michel, Jennifer Carlisle; Birdsong, William T.; Gouaux, Eric

    2009-08-13

    P2X receptors are cation-selective ion channels gated by extracellular ATP, and are implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Because P2X receptors are not related to other ion channel proteins of known structure, there is at present no molecular foundation for mechanisms of ligand-gating, allosteric modulation and ion permeation. Here we present crystal structures of the zebrafish P2X{sub 4} receptor in its closed, resting state. The chalice-shaped, trimeric receptor is knit together by subunit-subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in {beta}-strands, have large acidic patches that may attract cations, through fenestrations, to vestibules near the ion channel. In the transmembrane pore, the 'gate' is defined by an {approx}8 {angstrom} slab of protein. We define the location of three non-canonical, intersubunit ATP-binding sites, and suggest that ATP binding promotes subunit rearrangement and ion channel opening.

  4. Electrophysiological evidence for 4-isobutyl-3-isopropylbicyclophosphorothionate as a selective blocker of insect GABA-gated chloride channels.

    PubMed

    Akiyoshi, Yuki; Ju, Xiu-Lian; Furutani, Shogo; Matsuda, Kazuhiko; Ozoe, Yoshihisa

    2013-06-01

    Invertebrate γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and glutamate-gated chloride channels (GluCls), which function as inhibitory neurotransmitter receptors, are important targets of insecticides and antiparasitic agents. The antagonism of GABACls and GluCls by 4-isobutyl-3-isopropylbicyclophosphorothionate (PS-14) was examined in cultured cockroach and rat neurons using a whole-cell patch-clamp method. The results indicated that PS-14 selectively blocks cockroach GABACls relative to cockroach GluCls and rat GABACls. PS-14 represents a useful probe for the study of insect GABA receptors. PMID:23591113

  5. Calcium Channels and Associated Receptors in Malignant Brain Tumor Therapy.

    PubMed

    Morrone, Fernanda B; Gehring, Marina P; Nicoletti, Natália F

    2016-09-01

    Malignant brain tumors are highly lethal and aggressive. Despite recent advances in the current therapies, which include the combination of surgery and radio/chemotherapy, the average survival rate remains poor. Altered regulation of ion channels is part of the neoplastic transformation, which suggests that ion channels are involved in cancer. Distinct classes of calcium-permeable channels are abnormally expressed in cancer and are likely involved in the alterations underlying malignant growth. Specifically, cytosolic Ca(2+) activity plays an important role in the regulation of cell proliferation, and Ca(2+) signaling is altered in proliferating tumor cells. A series of previous studies emphasized the importance of the T-type low-voltage-gated calcium channels (VGCC) in different cancer types, including gliomas, and remarkably, pharmacologic inhibition of T-type VGCC caused antiproliferative effects and triggered apoptosis of human glioma cells. Other calcium permeable channels, such as transient receptor potential (TRP) channels, contribute to changes in Ca(2+) by modulating the driving force for Ca(2+) entry, and some TRP channels are required for proliferation and migration in gliomas. Furthermore, recent evidence shows that TRP channels contribute to the progression and survival of the glioblastoma patients. Likewise, the purinergic P2X7 receptor acts as a direct conduit for Ca(2+)-influx and an indirect activator of voltage-gated Ca(2+)-channel. Evidence also shows that P2X7 receptor activation is linked to elevated expression of inflammation promoting factors, tumor cell migration, an increase in intracellular mobilization of Ca(2+), and membrane depolarization in gliomas. Therefore, this review summarizes the recent findings on calcium channels and associated receptors as potential targets to treat malignant gliomas. PMID:27418672

  6. A family of acetylcholine-gated chloride channel subunits in Caenorhabditis elegans.

    PubMed

    Putrenko, Igor; Zakikhani, Mahvash; Dent, Joseph A

    2005-02-25

    The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by alpha-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors. PMID:15579462

  7. The Concise Guide to PHARMACOLOGY 2015/16: Voltage-gated ion channels.

    PubMed

    Alexander, Stephen Ph; Catterall, William A; Kelly, Eamonn; Marrion, Neil; Peters, John A; Benson, Helen E; Faccenda, Elena; Pawson, Adam J; Sharman, Joanna L; Southan, Christopher; Davies, Jamie A

    2015-12-01

    The Concise Guide to PHARMACOLOGY 2015/16 provides concise overviews of the key properties of over 1750 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13350/full. Voltage-gated ion channels are one of the eight major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ligand-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The Concise Guide is published in landscape format in order to facilitate comparison of related targets. It is a condensed version of material contemporary to late 2015, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in the previous Guides to Receptors & Channels and the Concise Guide to PHARMACOLOGY 2013/14. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates. PMID:26650441

  8. EPR Studies of Gating Mechanisms in Ion Channels

    PubMed Central

    Chakrapani, Sudha

    2015-01-01

    Ion channels open and close in response to diverse stimuli, and the molecular events underlying these processes are extensively modulated by ligands of both endogenous and exogenous origin. In the past decade, high-resolution structures of several channel types have been solved, providing unprecedented details of the molecular architecture of these membrane proteins. Intrinsic conformational flexibility of ion channels critically governs their functions. However, the dynamics underlying gating mechanisms and modulations are obscured in the information from crystal structures. While nuclear magnetic resonance spectroscopic methods allow direct measurements of protein dynamics, they are limited by the large size of these membrane protein assemblies in detergent micelles or lipid membranes. Electron paramagnetic resonance (EPR) spectroscopy has emerged as a key biophysical tool to characterize structural dynamics of ion channels and to determine stimulus-driven conformational transition between functional states in a physiological environment. This review will provide an overview of the recent advances in the field of voltage- and ligand-gated channels and highlight some of the challenges and controversies surrounding the structural information available. It will discuss general methods used in site-directed spin labeling and EPR spectroscopy and illustrate how findings from these studies have narrowed the gap between high-resolution structures and gating mechanisms in membranes, and have thereby helped reconcile seemingly disparate models of ion channel function. PMID:25950970

  9. 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

  10. 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

  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. 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

  13. 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

  14. Voltage-Dependent Gating: Novel Insights from KCNQ1 Channels.

    PubMed

    Cui, Jianmin

    2016-01-01

    Gating of voltage-dependent cation channels involves three general molecular processes: voltage sensor activation, sensor-pore coupling, and pore opening. KCNQ1 is a voltage-gated potassium (Kv) channel whose distinctive properties have provided novel insights on fundamental principles of voltage-dependent gating. 1) Similar to other Kv channels, KCNQ1 voltage sensor activation undergoes two resolvable steps; but, unique to KCNQ1, the pore opens at both the intermediate and activated state of voltage sensor activation. The voltage sensor-pore coupling differs in the intermediate-open and the activated-open states, resulting in changes of open pore properties during voltage sensor activation. 2) The voltage sensor-pore coupling and pore opening require the membrane lipid PIP2 and intracellular ATP, respectively, as cofactors, thus voltage-dependent gating is dependent on multiple stimuli, including the binding of intracellular signaling molecules. These mechanisms underlie the extraordinary KCNE1 subunit modification of the KCNQ1 channel and have significant physiological implications. PMID:26745405

  15. 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

  16. Ca channel gating during cardiac action potentials.

    PubMed

    Mazzanti, M; DeFelice, L J

    1990-10-01

    How do Ca channels conduct Ca ions during the cardiac action potential? We attempt to answer this question by applying a two-microelectrode technique, previously used for Na and K currents, in which we record the patch current and the action potential at the same time (Mazzanti, M., and L. J. DeFelice. 1987. Biophys. J. 12:95-100, and 1988. Biophys. J. 54:1139-1148; Wellis, D., L. J. DeFelice, and M. Mazzanti. 1990. Biophys. J. 57:41-48). In this paper, we also compare the action currents obtained by the technique with the step-protocol currents obtained during standard voltage-clamp experiments. Individual Ca channels were measured in 10 mM Ca/1 Ba and 10 mM Ba. To describe part of our results, we use the nomenclature introduced by Hess, P., J. B. Lansman, and R. W. Tsien (1984. Nature (Lond.). 311:538-544). With Ba as the charge carrier, Ca channel kinetics convert rapidly from long to short open times as the patch voltage changes from 20 to -20 mV. This voltage-dependent conversion occurs during action potentials and in step-protocol experiments. With Ca as the charge carrier, the currents are brief at all voltages, and it is difficult to define either the number of channels in the patch or the conductance of the individual channels. Occasionally, however, Ca-conducting channels spontaneously convert to long-open-time kinetics (in Hess et al., 1984, notation, mode 2). When this happens, which is about once in every 100beats, there usually appears to be only one channel in the patch. In this rare configuration, the channel is open long enough to measure its conductance in 10 Ca/ 1 Ba. The value is 8-10 pS, which is about half the conductance in Ba. Because the long openings occur so infrequently with Ca as the charge carrier, they contribute negligibly to the average Ca current at any particular time during an action potential. However, the total number of Ca ions entering during these long openings may be significant when compared to the number entering by the

  17. Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors*

    PubMed Central

    Liang, Xin; Samways, Damien S. K.; Wolf, Kyle; Bowles, Elizabeth A.; Richards, Jennifer P.; Bruno, Jonathan; Dutertre, Sébastien; DiPaolo, Richard J.; Egan, Terrance M.

    2015-01-01

    ATP-gated P2X7 receptors are prominently expressed in inflammatory cells and play a key role in the immune response. A major consequence of receptor activation is the regulated influx of Ca2+ through the self-contained cation non-selective channel. Although the physiological importance of the resulting rise in intracellular Ca2+ is universally acknowledged, the biophysics of the Ca2+ flux responsible for the effects are poorly understood, largely because traditional methods of measuring Ca2+ permeability are difficult to apply to P2X7 receptors. Here we use an alternative approach, called dye-overload patch-clamp photometry, to quantify the agonist-gated Ca2+ flux of recombinant P2X7 receptors of dog, guinea pig, human, monkey, mouse, rat, and zebrafish. We find that the magnitude of the Ca2+ component of the ATP-gated current depends on the species of origin, the splice variant, and the concentration of the purinergic agonist. We also measured a significant contribution of Ca2+ to the agonist-gated current of the native P2X7Rs of mouse and human immune cells. Our results provide cross-species quantitative measures of the Ca2+ current of the P2X7 receptor for the first time, and suggest that the cytoplasmic N terminus plays a meaningful role in regulating the flow of Ca2+ through the channel. PMID:25645917

  18. Chemical Synthesis of Tetracyclic Terpenes and Evaluation of Antagonistic Activity on Endothelin-A Receptors and Voltage-gated Calcium Channels

    PubMed Central

    Lu, Jianyu; Aguilar, Angelo; Zou, Bende; Bao, Weier; Koldas, Serkan; Aibin, Shi; Desper, John; Wangemann, Philine; Xie, Xinmin Simon; Hua, Duy H.

    2015-01-01

    A class of tetracyclic terpenes was synthesized and evaluated for antagonistic activity of endothelin-1 (ET-1) induced vasoconstriction and inhibitory activity of voltage-activated Ca2+ channels. Three repeated Robinson annulation reactions were utilized to construct the tetracyclic molecules. A stereoselective reductive Robinson annulation was discovered for the formation of optically pure tricyclic terpenes. Stereoselective addition of cyanide to the hindered α-face of tetracyclic enone (-)-18 was found and subsequent transformation into the aldehyde function was affected by the formation of bicyclic hemiiminal (-)-4. Six selected synthetic tetracyclic terpenes show inhibitory activities in ET-1 induced vasoconstriction in the gerbil spiral modiolar artery with putative affinity constants ranging between 93 and 319 nM. Moreover, one compound, (-)-3, was evaluated further and found to inhibit voltage-activated Ca2+ currents but not to affect Na+ or K+ currents in dorsal root ganglion cells under similar concentrations. These observations imply a dual mechanism of action. In conclusion, tetracyclic terpenes represent a new class of hit molecules for the discovery of new drugs for the treatment of pulmonary hypertension and vascular related diseases. PMID:26190460

  19. Chemical synthesis of tetracyclic terpenes and evaluation of antagonistic activity on endothelin-A receptors and voltage-gated calcium channels.

    PubMed

    Lu, Jianyu; Aguilar, Angelo; Zou, Bende; Bao, Weier; Koldas, Serkan; Shi, Aibin; Desper, John; Wangemann, Philine; Xie, Xinmin Simon; Hua, Duy H

    2015-09-01

    A class of tetracyclic terpenes was synthesized and evaluated for antagonistic activity of endothelin-1 (ET-1) induced vasoconstriction and inhibitory activity of voltage-activated Ca(2+) channels. Three repeated Robinson annulation reactions were utilized to construct the tetracyclic molecules. A stereoselective reductive Robinson annulation was discovered for the formation of optically pure tricyclic terpenes. Stereoselective addition of cyanide to the hindered α-face of tetracyclic enone (-)-18 was found and subsequent transformation into the aldehyde function was affected by the formation of bicyclic hemiiminal (-)-4. Six selected synthetic tetracyclic terpenes show inhibitory activities in ET-1 induced vasoconstriction in the gerbil spiral modiolar artery with putative affinity constants ranging between 93 and 319 nM. Moreover, one compound, (-)-3, was evaluated further and found to inhibit voltage-activated Ca(2+) currents but not to affect Na(+) or K(+) currents in dorsal root ganglion cells under similar concentrations. These observations imply a dual mechanism of action. In conclusion, tetracyclic terpenes represent a new class of hit molecules for the discovery of new drugs for the treatment of pulmonary hypertension and vascular related diseases. PMID:26190460

  20. Stochastic 16-state model of voltage gating of gap-junction channels enclosing fast and slow gates.

    PubMed

    Paulauskas, Nerijus; Pranevicius, Henrikas; Mockus, Jonas; Bukauskas, Feliksas F

    2012-06-01

    Gap-junction (GJ) channels formed of connexin (Cx) proteins provide a direct pathway for electrical and metabolic cell-cell interaction. Each hemichannel in the GJ channel contains fast and slow gates that are sensitive to transjunctional voltage (Vj). We developed a stochastic 16-state model (S16SM) that details the operation of two fast and two slow gates in series to describe the gating properties of homotypic and heterotypic GJ channels. The operation of each gate depends on the fraction of Vj that falls across the gate (VG), which varies depending on the states of three other gates in series, as well as on parameters of the fast and slow gates characterizing 1), the steepness of each gate's open probability on VG; 2), the voltage at which the open probability of each gate equals 0.5; 3), the gating polarity; and 4), the unitary conductances of the gates and their rectification depending on VG. S16SM allows for the simulation of junctional current dynamics and the dependence of steady-state junctional conductance (gj,ss) on Vj. We combined global coordinate optimization algorithms with S16SM to evaluate the gating parameters of fast and slow gates from experimentally measured gj,ss-Vj dependencies in cells expressing different Cx isoforms and forming homotypic and/or heterotypic GJ channels. PMID:22713562

  1. Stochastic 16-State Model of Voltage Gating of Gap-Junction Channels Enclosing Fast and Slow Gates

    PubMed Central

    Paulauskas, Nerijus; Pranevicius, Henrikas; Mockus, Jonas; Bukauskas, Feliksas F.

    2012-01-01

    Gap-junction (GJ) channels formed of connexin (Cx) proteins provide a direct pathway for electrical and metabolic cell-cell interaction. Each hemichannel in the GJ channel contains fast and slow gates that are sensitive to transjunctional voltage (Vj). We developed a stochastic 16-state model (S16SM) that details the operation of two fast and two slow gates in series to describe the gating properties of homotypic and heterotypic GJ channels. The operation of each gate depends on the fraction of Vj that falls across the gate (VG), which varies depending on the states of three other gates in series, as well as on parameters of the fast and slow gates characterizing 1), the steepness of each gate's open probability on VG; 2), the voltage at which the open probability of each gate equals 0.5; 3), the gating polarity; and 4), the unitary conductances of the gates and their rectification depending on VG. S16SM allows for the simulation of junctional current dynamics and the dependence of steady-state junctional conductance (gj,ss) on Vj. We combined global coordinate optimization algorithms with S16SM to evaluate the gating parameters of fast and slow gates from experimentally measured gj,ss-Vj dependencies in cells expressing different Cx isoforms and forming homotypic and/or heterotypic GJ channels. PMID:22713562

  2. 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.

  3. 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

  4. Novel Activation of Voltage-gated K+ Channels by Sevoflurane*

    PubMed Central

    Barber, Annika F.; Liang, Qiansheng; Covarrubias, Manuel

    2012-01-01

    Voltage-gated ion channels are modulated by halogenated inhaled general anesthetics, but the underlying molecular mechanisms are not understood. Alkanols and halogenated inhaled anesthetics such as halothane and isoflurane inhibit the archetypical voltage-gated Kv3 channel homolog K-Shaw2 by stabilizing the resting/closed states. By contrast, sevoflurane, a more heavily fluorinated ether commonly used in general anesthesia, specifically activates K-Shaw2 currents at relevant concentrations (0.05–1 mm) in a rapid and reversible manner. The concentration dependence of this modulation is consistent with the presence of high and low affinity interactions (KD = 0.06 and 4 mm, respectively). Sevoflurane (<1 mm) induces a negative shift in the conductance-voltage relation and increases the maximum conductance. Furthermore, suggesting possible roles in general anesthesia, mammalian Kv1.2 and Kv1.5 channels display similar changes. Quantitative description of the observations by an economical allosteric model indicates that sevoflurane binding favors activation gating and eliminates an unstable inactivated state outside the activation pathway. This study casts light on the mechanism of the novel sevoflurane-dependent activation of Kv channels, which helps explain how closely related inhaled anesthetics achieve specific actions and suggests strategies to develop novel Kv channel activators. PMID:23038249

  5. Imaging ligand-gated ion channels with quantum dots

    NASA Astrophysics Data System (ADS)

    Tomlinson, I. D.; Orndorff, Rebecca L.; Gussin, Hélène; Mason, John N.; Blakely, Randy D.; Pepperberg, David R.; Rosenthal, Sandra J.

    2007-02-01

    In this paper we report two different methodologies for labeling ligand-gated receptors. The first of these builds upon our earlier work with serotonin conjugated quantum dots and our studies with pegilated quantum dots to reduce non specific binding. In this approach a pegilated derivative of muscimol was synthesized and attached via an amide linkage to quantum dots coated in an amphiphillic polymer derivative of poly acrylamide. These conjugates were used to image the GABA C receptor in oocytes. An alternative approach was used to image tissue sections to study nicotinic acetylcholine receptors in the neuro muscular junction with biotinylated Bungerotoxin and streptavidin coated quantum dots.

  6. 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.

  7. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins.

    PubMed

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel's ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  8. State-Dependent Modification of Voltage-Gated Sodium Channels by Pyrethroids

    PubMed Central

    Soderlund, David M.

    2009-01-01

    Pyrethroids disrupt nerve function by altering the rapid kinetic transitions between conducting and nonconducting states of voltage-gated sodium channels that underlie the generation of nerve action potentials. Recent studies of pyrethroid action on cloned insect and mammalian sodium channel isoforms expressed in Xenopus laevis oocytes show that in some cases pyrethroid modification is either absolutely dependent on or significantly enhanced by repeated channel activation. These use-dependent effects have been interpreted as evidence of preferential binding of at least some pyrethroids to the open, rather than resting, state of the sodium channel. This paper reviews the evidence for state-dependent modification of insect and mammalian sodium channels expressed in oocytes by pyrethroids and considers the implications of state-dependent effects for understanding the molecular mechanism of pyrethroid action and the development and testing of models of the pyrethroid receptor. PMID:20652092

  9. Effect of Charge Substitutions at Residue His-142 on Voltage Gating of Connexin43 Channels

    PubMed Central

    Shibayama, Junko; Gutiérrez, Cristina; González, Daniel; Kieken, Fabien; Seki, Akiko; Requena Carrión, Jesus; Sorgen, Paul L.; Taffet, Steven M.; Barrio, Luis C.; Delmar, Mario

    2006-01-01

    Previous studies indicate that the carboxyl terminal of connexin43 (Cx43CT) is involved in fast transjunctional voltage gating. Separate studies support the notion of an intramolecular association between Cx43CT and a region of the cytoplasmic loop (amino acids 119–144; referred to as “L2”). Structural analysis of L2 shows two α-helical domains, each with a histidine residue in its sequence (H126 and H142). Here, we determined the effect of H142 replacement by lysine, alanine, and glutamate on the voltage gating of Cx43 channels. Mutation H142E led to a significant reduction in the frequency of occurrence of the residual state and a prolongation of dwell open time. Macroscopically, there was a large reduction in the fast component of voltage gating. These results resembled those observed for a mutant lacking the carboxyl terminal (CT) domain. NMR experiments showed that mutation H142E significantly decreased the Cx43CT-L2 interaction and disrupted the secondary structure of L2. Overall, our data support the hypothesis that fast voltage gating involves an intramolecular particle-receptor interaction between CT and L2. Some of the structural constrains of fast voltage gating may be shared with those involved in the chemical gating of Cx43. PMID:16963503

  10. Gating machinery of InsP3R channels revealed by electron cryomicroscopy

    PubMed Central

    Fan, Guizhen; Baker, Matthew L.; Wang, Zhao; Baker, Mariah R.; Sinyagovskiy, Pavel A.; Chiu, Wah; Ludtke, Steven J.; Serysheva, Irina I.

    2016-01-01

    Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are ubiquitous ion channels responsible for cytosolic Ca2+ signalling and essential for a broad array of cellular processes ranging from contraction to secretion, and from proliferation to cell death. Despite decades of research on InsP3Rs, a mechanistic understanding of their structure–function relationship is lacking. Here we present the first, to our knowledge, near-atomic (4.7 Å) resolution electron cryomicroscopy structure of the tetrameric mammalian type 1 InsP3R channel in its apo-state. At this resolution, we are able to trace unambiguously ~85% of the protein backbone, allowing us to identify the structural elements involved in gating and modulation of this 1.3-megadalton channel. Although the central Ca2+-conduction pathway is similar to other ion channels, including the closely related ryanodine receptor, the cytosolic carboxy termini are uniquely arranged in a left-handed α-helical bundle, directly interacting with the amino-terminal domains of adjacent subunits. This configuration suggests a molecular mechanism for allosteric regulation of channel gating by intracellular signals. PMID:26458101

  11. Gating machinery of InsP3R channels revealed by electron cryomicroscopy.

    PubMed

    Fan, Guizhen; Baker, Matthew L; Wang, Zhao; Baker, Mariah R; Sinyagovskiy, Pavel A; Chiu, Wah; Ludtke, Steven J; Serysheva, Irina I

    2015-11-19

    Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are ubiquitous ion channels responsible for cytosolic Ca(2+) signalling and essential for a broad array of cellular processes ranging from contraction to secretion, and from proliferation to cell death. Despite decades of research on InsP3Rs, a mechanistic understanding of their structure-function relationship is lacking. Here we present the first, to our knowledge, near-atomic (4.7 Å) resolution electron cryomicroscopy structure of the tetrameric mammalian type 1 InsP3R channel in its apo-state. At this resolution, we are able to trace unambiguously ∼85% of the protein backbone, allowing us to identify the structural elements involved in gating and modulation of this 1.3-megadalton channel. Although the central Ca(2+)-conduction pathway is similar to other ion channels, including the closely related ryanodine receptor, the cytosolic carboxy termini are uniquely arranged in a left-handed α-helical bundle, directly interacting with the amino-terminal domains of adjacent subunits. This configuration suggests a molecular mechanism for allosteric regulation of channel gating by intracellular signals. PMID:26458101

  12. Allosteric regulation of pentameric ligand-gated ion channels

    PubMed Central

    Taly, Antoine; Hénin, Jérôme; Changeux, Jean-Pierre; Cecchini, Marco

    2014-01-01

    Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger—a neurotransmitter—into an ion flux through the postsynaptic membrane. They are oligomeric assemblies that provide prototypical examples of allosterically regulated integral membrane proteins. Here, we present an overview of the most recent advances on the signal transduction mechanism based on the X-ray structures of both prokaryotic and invertebrate eukaryotic pLGICs and on atomistic Molecular Dynamics simulations. The present results suggest that ion gating involves a large structural reorganization of the molecule mediated by two distinct quaternary transitions, a global twisting and the blooming of the extracellular domain, which can be modulated by ligand binding at the topographically distinct orthosteric and allosteric sites. The emerging model of gating is consistent with a wealth of functional studies and will boost the development of novel pharmacological strategies. PMID:25478624

  13. Gating of Acoustic Transducer Channels Is Shaped by Biomechanical Filter Processes.

    PubMed

    Hummel, Jennifer; Schöneich, Stefan; Kössl, Manfred; Scherberich, Jan; Hedwig, Berthold; Prinz, Simone; Nowotny, Manuela

    2016-02-24

    Mechanoelectrical transduction of acoustic signals is the fundamental process for hearing in all ears across the animal kingdom. Here, we performed in vivo laser-vibrometric and electrophysiological measurements at the transduction site in an insect ear (Mecopoda elongata) to relate the biomechanical tonotopy along the hearing organ to the frequency tuning of the corresponding sensory cells. Our mechanical and electrophysiological map revealed a biomechanical filter process that considerably sharpens the neuronal response. We demonstrate that the channel gating, which acts on chordotonal stretch receptor neurons, is based on a mechanical directionality of the sound-induced motion. Further, anatomical studies of the transduction site support our finding of a stimulus-relevant tilt. In conclusion, we were able to show, in an insect ear, that directionality of channel gating considerably sharpens the neuronal frequency selectivity at the peripheral level and have identified a mechanism that enhances frequency discrimination in tonotopically organized ears. PMID:26911686

  14. Single-channel, macroscopic, and gating currents from sodium channels in the squid giant axon.

    PubMed Central

    Vandenberg, C A; Bezanilla, F

    1991-01-01

    Single-channel, macroscopic ionic, and macroscopic gating currents were recorded from the voltage-dependent sodium channel using patch-clamp techniques on the cut-open squid giant axon. To obtain a complete set of physiological measurements of sodium channel gating under identical conditions, and to facilitate comparison with previous work, comparison was made between currents recorded in the absence of extracellular divalent cations and in the presence of physiological concentrations of extracellular Ca2+ (10 mM) and Mg2+ (50 mM). The single-channel currents were well resolved when divalent cations were not included in the extracellular solution, but were decreased in amplitude in the presence of Ca2+ and Mg2+ ions. The instantaneous current-voltage relationship obtained from macroscopic tail current measurements similarly was depressed by divalents, and showed a negative slope-conductance region for inward current at negative potentials. Voltage dependent parameters of channel gating were shifted 9-13 mV towards depolarized potentials by external divalent cations, including the peak fraction of channels open versus voltage, the time constant of tail current decline, the prepulse inactivation versus voltage relationship, and the charge-voltage relationship for gating currents. The effects of divalent cations are consistent with open channel block by Ca2+ and Mg2+ together with divalent screening of membrane charges. PMID:1663795

  15. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

    PubMed Central

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  16. 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

  17. 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

  18. Adaptive evolution of voltage-gated sodium channels: The first 800 million years

    PubMed Central

    Zakon, Harold H.

    2012-01-01

    Voltage-gated Na+-permeable (Nav) channels form the basis for electrical excitability in animals. Nav channels evolved from Ca2+ channels and were present in the common ancestor of choanoflagellates and animals, although this channel was likely permeable to both Na+ and Ca2+. Thus, like many other neuronal channels and receptors, Nav channels predated neurons. Invertebrates possess two Nav channels (Nav1 and Nav2), whereas vertebrate Nav channels are of the Nav1 family. Approximately 500 Mya in early chordates Nav channels evolved a motif that allowed them to cluster at axon initial segments, 50 million years later with the evolution of myelin, Nav channels “capitalized” on this property and clustered at nodes of Ranvier. The enhancement of conduction velocity along with the evolution of jaws likely made early gnathostomes fierce predators and the dominant vertebrates in the ocean. Later in vertebrate evolution, the Nav channel gene family expanded in parallel in tetrapods and teleosts (∼9 to 10 genes in amniotes, 8 in teleosts). This expansion occurred during or after the late Devonian extinction, when teleosts and tetrapods each diversified in their respective habitats, and coincided with an increase in the number of telencephalic nuclei in both groups. The expansion of Nav channels may have allowed for more sophisticated neural computation and tailoring of Nav channel kinetics with potassium channel kinetics to enhance energy savings. Nav channels show adaptive sequence evolution for increasing diversity in communication signals (electric fish), in protection against lethal Nav channel toxins (snakes, newts, pufferfish, insects), and in specialized habitats (naked mole rats). PMID:22723361

  19. 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.

  20. Voltage-Gated Proton Channel in Human Glioblastoma Multiforme Cells.

    PubMed

    Ribeiro-Silva, Luisa; Queiroz, Fernanda Oliveira; da Silva, Annielle Mendes Brito; Hirata, Aparecida Emiko; Arcisio-Miranda, Manoel

    2016-07-20

    Solid tumors tend to have a more glycolytic metabolism leading to an accumulation of acidic metabolites in their cytosol, and consequently, their intracellular pH (pHi) turns critically lower if the cells do not handle the acid excess. Recently, it was proposed that the voltage gated proton channels (HV1) can regulate the pHi in several cancers. Here we report the functional expression of voltage gated proton channels in a human glioblastoma multiforme (GBM) cell line, the most common and lethal brain tumor. T98G cells presented an outward, slow activating voltage-dependent proton current, which was also ΔpH-dependent and inhibited by ZnCl2, characterizing it as being conducted by HV1 channels. Furthermore, blocking HV1 channels with ZnCl2 significantly reduced the pHi, cell survival, and migration, indicating an important role for HV1 for tumor proliferation and progression in GBM. Overall, our results suggest that HV1 channels can be a new therapeutic target for GBM. PMID:27225904

  1. A single amino acid gates the KcsA channel.

    PubMed

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

    2014-08-01

    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. PMID:25019991

  2. 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.

  3. [Ion channels and action potentials in olfactory receptor cells].

    PubMed

    Kawai, Fusao; Miyachi, Ei-ichi

    2007-11-01

    The first step in olfactory sensation involves the binding of odorant molecules to specific receptor proteins on the ciliary surface of olfactory receptor cells (ORCs). Odorant receptors coupled to G-proteins activate adenylyl cyclase leading to the generation of cAMP, which directly gates a cyclic nucleotide-gated cationic channel in the ciliary membrane. This initial excitation causes a slow and graded depolarizing voltage change, which is encoded into a train of action potentials. Action potentials of ORCs are generated by voltage-gated Na- currents and T-type Ca2- currents in the somatic membrane. Isolated ORCs that have lost their cilia during the dissociation procedure are known to exhibit spike frequency accommodation by injecting the steady current. This raises the possibility that somatic ionic channels in ORCs may serve for odor adaptation at the level of spike encoding, although odor adaptation is mainly accomplished by the ciliary transduction machinery. This review discusses current knowledge concerning the mechanisms of spike generation in ORCs. It also reviews how neurotransmitters and hormones modulate ionic currents and action potentials in ORCs. PMID:18154041

  4. Regulation of CFTR ion channel gating by MgATP.

    PubMed

    Aleksandrov, A A; Riordan, J R

    1998-07-10

    Single channel currents of wild-type CFTR reconstituted in lipid bilayers were recorded to study the temperature dependence of channel gating between +20 degrees C and +40 degrees C. The opening of the channel was highly temperature dependent and required an activation energy of about 100 kJ/mol. Closing of the channel was only weakly temperature dependent with an activation energy close to that of diffusion in water. We found no significant difference in the free energy between the open and closed states. Most of the excess energy needed to activate channel opening is used to diminish the entropy of the open state. This structural reorganization is initiated by ATP binding followed by interconversion to the open channel structure as the CFTR-ATP-Mg complex passes to the transition state for hydrolysis. The energy of the CFTR-ATP-Mg interaction in the transition state is responsible for the CFTR ion channel opening rather than the energy of ATP hydrolysis. Channel closing is a diffusion limited process and does not require additional ATP binding. PMID:9684873

  5. Structural insights into Ca(2+)-activated long-range allosteric channel gating of RyR1.

    PubMed

    Wei, Risheng; Wang, Xue; Zhang, Yan; Mukherjee, Saptarshi; Zhang, Lei; Chen, Qiang; Huang, Xinrui; Jing, Shan; Liu, Congcong; Li, Shuang; Wang, Guangyu; Xu, Yaofang; Zhu, Sujie; Williams, Alan J; Sun, Fei; Yin, Chang-Cheng

    2016-09-01

    Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca(2+)-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 Å and a resolution of 4.2 Å for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca(2+) activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family. PMID:27573175

  6. Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels*

    PubMed Central

    Carswell, Casey L.; Sun, Jiayin; Baenziger, John E.

    2015-01-01

    Although the Torpedo nicotinic acetylcholine receptor (nAChR) reconstituted into phosphatidylcholine (PC) membranes lacking cholesterol and anionic lipids adopts a conformation where agonist binding is uncoupled from channel gating, the underlying mechanism remains to be defined. Here, we examine the mechanism behind lipid-dependent uncoupling by comparing the propensities of two prokaryotic homologs, Gloebacter and Erwinia ligand-gated ion channel (GLIC and ELIC, respectively), to adopt a similar uncoupled conformation. Membrane-reconstituted GLIC and ELIC both exhibit folded structures in the minimal PC membranes that stabilize an uncoupled nAChR. GLIC, with a large number of aromatic interactions at the interface between the outermost transmembrane α-helix, M4, and the adjacent transmembrane α-helices, M1 and M3, retains the ability to flux cations in this uncoupling PC membrane environment. In contrast, ELIC, with a level of aromatic interactions intermediate between that of the nAChR and GLIC, does not undergo agonist-induced channel gating, although it does not exhibit the expected biophysical characteristics of the uncoupled state. Engineering new aromatic interactions at the M4-M1/M3 interface to promote effective M4 interactions with M1/M3, however, increases the stability of the transmembrane domain to restore channel function. Our data provide direct evidence that M4 interactions with M1/M3 are modulated during lipid sensing. Aromatic residues strengthen M4 interactions with M1/M3 to reduce the sensitivities of pentameric ligand-gated ion channels to their surrounding membrane environment. PMID:25519904

  7. Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps*

    PubMed Central

    Tol, Menno B.; Deluz, Cédric; Hassaine, Gherici; Graff, Alexandra; Stahlberg, Henning; Vogel, Horst

    2013-01-01

    Pentameric ligand-gated ion channels (LGICs) play an important role in fast synaptic signal transduction. Binding of agonists to the β-sheet-structured extracellular domain opens an ion channel in the transmembrane α-helical region of the LGIC. How the structurally distinct and distant domains are functionally coupled for such central transmembrane signaling processes remains an open question. To obtain detailed information about the stability of and the coupling between these different functional domains, we analyzed the thermal unfolding of a homopentameric LGIC, the 5-hydroxytryptamine receptor (ligand binding, secondary structure, accessibility of Trp and Cys residues, and aggregation), in plasma membranes as well as during detergent extraction, purification, and reconstitution into artificial lipid bilayers. We found a large loss in thermostability correlating with the loss of the lipid bilayer during membrane solubilization and purification. Thermal unfolding of the 5-hydroxytryptamine receptor occurred in consecutive steps at distinct protein locations. A loss of ligand binding was detected first, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally lead to the formation receptor aggregates. Structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Our results are important because they quantify the stability of LGICs during detergent extraction and purification and can be used to create stabilized receptor proteins for structural and functional studies. PMID:23275379

  8. Mechanism of voltage-gated channel formation in lipid membranes.

    PubMed

    Guidelli, Rolando; Becucci, Lucia

    2016-04-01

    Although several molecular models for voltage-gated ion channels in lipid membranes have been proposed, a detailed mechanism accounting for the salient features of experimental data is lacking. A general treatment accounting for peptide dipole orientation in the electric field and their nucleation and growth kinetics with ion channel formation is provided. This is the first treatment that explains all the main features of the experimental current-voltage curves of peptides forming voltage-gated channels available in the literature. It predicts a regime of weakly voltage-dependent conductance, followed by one of strong voltage-dependent conductance at higher voltages. It also predicts values of the parameters expressing the exponential dependence of conductance upon voltage and peptide bulk concentration for both regimes, in good agreement with those reported in the literature. Most importantly, the only two adjustable parameters involved in the kinetics of nucleation and growth of ion channels can be varied over broad ranges without affecting the above predictions to a significant extent. Thus, the fitting of experimental current-voltage curves stems naturally from the treatment and depends only slightly upon the choice of the kinetic parameters. PMID:26768224

  9. 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. PMID:26836092

  10. Cardiac voltage-gated calcium channel macromolecular complexes.

    PubMed

    Rougier, Jean-Sébastien; Abriel, Hugues

    2016-07-01

    Over the past 20years, a new field of research, called channelopathies, investigating diseases caused by ion channel dysfunction has emerged. Cardiac ion channels play an essential role in the generation of the cardiac action potential. Investigators have largely determined the physiological roles of different cardiac ion channels, but little is known about the molecular determinants of their regulation. The voltage-gated calcium channel Cav1.2 shapes the plateau phase of the cardiac action potential and allows the influx of calcium leading to cardiomyocyte contraction. Studies suggest that the regulation of Cav1.2 channels is not uniform in working cardiomyocytes. The notion of micro-domains containing Cav1.2 channels and different calcium channel interacting proteins, called macro-molecular complex, has been proposed to explain these observations. The objective of this review is to summarize the currently known information on the Cav1.2 macromolecular complexes in the cardiac cell and discuss their implication in cardiac function and disorder. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. PMID:26707467

  11. Peptide-gated ion channels and the simple nervous system of Hydra.

    PubMed

    Gründer, Stefan; Assmann, Marc

    2015-02-15

    Neurons either use electrical or chemical synapses to communicate with each other. Transmitters at chemical synapses are either small molecules or neuropeptides. After binding to their receptors, transmitters elicit postsynaptic potentials, which can either be fast and transient or slow and longer lasting, depending on the type of receptor. Fast transient potentials are mediated by ionotropic receptors and slow long-lasting potentials by metabotropic receptors. Transmitters and receptors are well studied for animals with a complex nervous system such as vertebrates and insects, but much less is known for animals with a simple nervous system like Cnidaria. As cnidarians arose early in animal evolution, nervous systems might have first evolved within this group and the study of neurotransmission in cnidarians might reveal an ancient mechanism of neuronal communication. The simple nervous system of the cnidarian Hydra extensively uses neuropeptides and, recently, we cloned and functionally characterized an ion channel that is directly activated by neuropeptides of the Hydra nervous system. These results demonstrate the existence of peptide-gated ion channels in Hydra, suggesting they mediate fast transmission in its nervous system. As related channels are also present in the genomes of the cnidarian Nematostella, of placozoans and of ctenophores, it should be considered that the early nervous systems of cnidarians and ctenophores have co-opted neuropeptides for fast transmission at chemical synapses. PMID:25696818

  12. Ligand-specific opening of a gated-porin channel in the outer membrane of living bacteria.

    PubMed

    Jiang, X; Payne, M A; Cao, Z; Foster, S B; Feix, J B; Newton, S M; Klebba, P E

    1997-05-23

    Ligand-gated membrane channels selectively facilitate the entry of iron into prokaryotic cells. The essential role of iron in metabolism makes its acquisition a determinant of bacterial pathogenesis and a target for therapeutic strategies. In Gram-negative bacteria, TonB-dependent outer membrane proteins form energized, gated pores that bind iron chelates (siderophores) and internalize them. The time-resolved operation of the Escherichia coli ferric enterobactin receptor FepA was observed in vivo with electron spin resonance spectroscopy by monitoring the mobility of covalently bound nitroxide spin labels. A ligand-binding surface loop of FepA, which normally closes its transmembrane channel, exhibited energy-dependent structural changes during iron and toxin (colicin) transport. These changes were not merely associated with ligand binding, but occurred during ligand uptake through the outer membrane bilayer. The results demonstrate by a physical method that gated-porin channels open and close during membrane transport in vivo. PMID:9157886

  13. 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

  14. 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

  15. Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels in Drosophila melanogaster.

    PubMed

    Remnant, Emily J; Williams, Adam; Lumb, Chris; Yang, Ying Ting; Chan, Janice; Duchêne, Sebastian; Daborn, Phillip J; Batterham, Philip; Perry, Trent

    2016-01-01

    Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927, and CG11340, are highly expressed in nonneuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here, we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization to examine cell type and tissue-specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including the salivary gland, ejaculatory duct, malpighian tubules, and early midgut. CG11340 is found in malpighian tubules and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific nonneuronal role for this subunit in D. melanogaster Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets. PMID:27172217

  16. Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels in Drosophila melanogaster

    PubMed Central

    Remnant, Emily J.; Williams, Adam; Lumb, Chris; Yang, Ying Ting; Chan, Janice; Duchêne, Sebastian; Daborn, Phillip J.; Batterham, Philip; Perry, Trent

    2016-01-01

    Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927, and CG11340, are highly expressed in nonneuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here, we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization to examine cell type and tissue-specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including the salivary gland, ejaculatory duct, malpighian tubules, and early midgut. CG11340 is found in malpighian tubules and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific nonneuronal role for this subunit in D. melanogaster. Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets. PMID:27172217

  17. An amiloride-sensitive H+-gated Na+ channel in Caenorhabditis elegans body wall muscle cells

    PubMed Central

    Jospin, Maëlle; Allard, Bruno

    2004-01-01

    About 30 genes are predicted to encode degenerin/epithelial sodium channels (DEG/ENaCs) in Caenorhabditis elegans but the gating mode of these channels has not been determined. Using the whole-cell configuration of the patch-clamp technique in acutely dissected C. elegans, we investigated the effects of H+ as a potential activating factor of DEG/ENaCs on electrical properties of body wall muscle cells. Under current-clamp conditions, decreasing external pH from 7.2 to 6.1 led to a reversible depolarization of muscle cells associated with a decrease in input resistance which was partially inhibited by amiloride. Under voltage-clamp conditions, extracellular acidification activated an inward desensitizing current at −60 mV. In the absence of external Ca2+, H+-gated channels were found to be slightly more permeable to Na+ than to K+ and were blocked by amiloride with a K0.5 of 31 μm at −60 mV. An inward current could be also activated by protons in a GABA receptor null mutant in the presence of d-tubocurare and in an unc-105 null mutant. These results demonstrate that ion channels sharing common properties with mammalian acid-sensing ion channels (ASICs) are functional in C. elegans muscle which should prove useful for understanding proton sensing in animals. PMID:15254157

  18. LRRK2 Regulates Voltage-Gated Calcium Channel Function

    PubMed Central

    Bedford, Cade; Sears, Catherine; Perez-Carrion, Maria; Piccoli, Giovanni; Condliffe, Steven B.

    2016-01-01

    Voltage-gated Ca2+ (CaV) channels enable Ca2+ influx in response to membrane depolarization. CaV2.1 channels are localized to the presynaptic membrane of many types of neurons where they are involved in triggering neurotransmitter release. Several signaling proteins have been identified as important CaV2.1 regulators including protein kinases, G-proteins and Ca2+ binding proteins. Recently, we discovered that leucine rich repeat kinase 2 (LRRK2), a protein associated with inherited Parkinson’s disease, interacts with specific synaptic proteins and influences synaptic transmission. Since synaptic proteins functionally interact with CaV2.1 channels and synaptic transmission is triggered by Ca2+ entry via CaV2.1, we investigated whether LRRK2 could impact CaV2.1 channel function. CaV2.1 channel properties were measured using whole cell patch clamp electrophysiology in HEK293 cells transfected with CaV2.1 subunits and various LRRK2 constructs. Our results demonstrate that both wild type (wt) LRRK2 and the G2019S LRRK2 mutant caused a significant increase in whole cell Ca2+ current density compared to cells expressing only the CaV2.1 channel complex. In addition, LRRK2 expression caused a significant hyperpolarizing shift in voltage-dependent activation while having no significant effect on inactivation properties. These functional changes in CaV2.1 activity are likely due to a direct action of LRRK2 as we detected a physical interaction between LRRK2 and the β3 CaV channel subunit via coimmunoprecipitation. Furthermore, effects on CaV2.1 channel function are dependent on LRRK2 kinase activity as these could be reversed via treatment with a LRRK2 inhibitor. Interestingly, LRRK2 also augmented endogenous voltage-gated Ca2+ channel function in PC12 cells suggesting other CaV channels could also be regulated by LRRK2. Overall, our findings support a novel physiological role for LRRK2 in regulating CaV2.1 function that could have implications for how mutations in LRRK2

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

    PubMed

    Kruger, Larisa C; Isom, Lori L

    2016-01-01

    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. PMID:27252364

  20. Structure and gating of CLC channels and exchangers.

    PubMed

    Accardi, Alessio

    2015-09-15

    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

  1. Structure-Driven Pharmacology of Transient Receptor Potential Channel Vanilloid 1.

    PubMed

    Díaz-Franulic, Ignacio; Caceres-Molina, Javier; Sepulveda, Romina V; Gonzalez-Nilo, Fernando; Latorre, Ramon

    2016-09-01

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal receptor that mediates the flux of cations across the membrane in response to several stimuli, including heat, voltage, and ligands. The best known agonist of TRPV1 channels is capsaicin, the pungent component of "hot" chili peppers. In addition, peptides found in the venom of poisonous animals, along with the lipids phosphatidylinositol 4,5-biphosphate, lysophosphatidic acid, and cholesterol, bind to TRPV1 with high affinity to modulate channel gating. Here, we discuss the functional evidence regarding ligand-dependent activation of TRPV1 channels in light of structural data recently obtained by cryoelectron microscopy. This review focuses on the mechanistic insights into ligand binding and allosteric gating of TRPV1 channels and the relevance of accurate polymodal receptor biophysical characterization for drug design in novel pain therapies. PMID:27335334

  2. Structural Basis for Xenon Inhibition in a Cationic Pentameric Ligand-Gated Ion Channel

    PubMed Central

    Sauguet, Ludovic; Fourati, Zeineb; Prangé, Thierry; Delarue, Marc; Colloc'h, Nathalie

    2016-01-01

    GLIC receptor is a bacterial pentameric ligand-gated ion channel whose action is inhibited by xenon. Xenon has been used in clinical practice as a potent gaseous anaesthetic for decades, but the molecular mechanism of interactions with its integral membrane receptor targets remains poorly understood. Here we characterize by X-ray crystallography the xenon-binding sites within both the open and “locally-closed” (inactive) conformations of GLIC. Major binding sites of xenon, which differ between the two conformations, were identified in three distinct regions that all belong to the trans-membrane domain of GLIC: 1) in an intra-subunit cavity, 2) at the interface between adjacent subunits, and 3) in the pore. The pore site is unique to the locally-closed form where the binding of xenon effectively seals the channel. A putative mechanism of the inhibition of GLIC by xenon is proposed, which might be extended to other pentameric cationic ligand-gated ion channels. PMID:26910105

  3. Molecular basis for convergent evolution of glutamate recognition by pentameric ligand-gated ion channels

    PubMed Central

    Lynagh, Timothy; Beech, Robin N.; Lalande, Maryline J.; Keller, Kevin; Cromer, Brett A.; Wolstenholme, Adrian J.; Laube, Bodo

    2015-01-01

    Glutamate is an indispensable neurotransmitter, triggering postsynaptic signals upon recognition by postsynaptic receptors. We questioned the phylogenetic position and the molecular details of when and where glutamate recognition arose in the glutamate-gated chloride channels. Experiments revealed that glutamate recognition requires an arginine residue in the base of the binding site, which originated at least three distinct times according to phylogenetic analysis. Most remarkably, the arginine emerged on the principal face of the binding site in the Lophotrochozoan lineage, but 65 amino acids upstream, on the complementary face, in the Ecdysozoan lineage. This combined experimental and computational approach throws new light on the evolution of synaptic signalling. PMID:25708000

  4. 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

  5. Lipid bilayer modification alters the gating properties and pharmacological sensitivity of voltage-gated sodium channel.

    PubMed

    Zhu, Yan; Wu, Bin; Feng, Yi-Jun; Tao, Jie; Ji, Yong-Hua

    2015-06-25

    Voltage-gated sodium channels (VGSCs) are widely distributed in most cells and tissues, performing many physiological functions. As one kind of membrane proteins in the lipid bilayer, whether lipid composition plays a role in the gating and pharmacological sensitivity of VGSCs still remains unknown. Through the application of sphingomyelinase D (SMaseD), the gating and pharmacological sensitivity of the endogenous VGSCs in neuroblastoma ND7-23 cell line to BmK I and BmK AS, two sodium channel-specific modulators from the venom of Buthus martensi Karsch (BmK), were assessed before and after lipid modification. The results showed that, in ND7-23 cells, SMaseD did not change the gating properties of VGSCs. However, SMaseD application altered the slope factor of activation with the treatment of 30 nmol/L BmK I, but caused no significant effects at 100 and 500 nmol/L BmK I. With low concentration of BmK I (30 and 100 nmol/L) treatment, the application of SMaseD exerted hyperpolarizing effects on both slow-inactivation and steady-state inactivation, and increased the recovery time constant, whereas total inactivation and recovery remained unaltered at 500 nmol/L BmK I. Meanwhile, SMaseD modulation hyperpolarized the voltage dependence of slow-inactivation at 0.1 nmol/L BmK AS and altered the slope factor of slow-inactivation at 10 nmol/L BmK AS, whereas other parameters remained unchanged. These results indicated a possibility that the lipid bilayer would disturb the pharmacological sensitivity of VGSCs for the first time, which might open a new way of developing new drugs for treating sodium channelopathies. PMID:26109300

  6. Cellular Functions of Transient Receptor Potential channels

    PubMed Central

    Dadon, Daniela; Minke, Baruch

    2010-01-01

    Transient Receptor Potential channels are polymodal cellular sensors involved in a wide variety of cellular processes, mainly by increasing cellular Ca2+. In this review we focus on the roles of these channels in: i) cell death ii) proliferation and differentiation and iii) synaptic vesicle release. Cell death Ca2+ influx participates in apoptotic and necrotic cell death. The Ca2+ permeability and high sensitivity of part of these channels to oxidative/metabolic stress make them important participants in cell death. Several examples are given. Transient Receptor Potential Melastatin 2 is activated by H2O2, inducing cell death through an increase in cellular Ca2+ and activation of Poly ADP-Ribose Polymerase. Exposure of cultured cortical neurons to oxygen-glucose deprivation, in vitro, causes cell death via cation influx, mediated by Transient Receptor Potential Melastatin 7. Metabolic stress constitutively activates the Ca2+ permeable Transient Receptor Potential channels of Drosophila photoreceptor in the dark, potentially leading to retinal degeneration. Similar sensitivity to metabolic stress characterizes several mammalian Transient Receptor Potential Canonical channels. Proliferation and differentiation The rise in cytosolic Ca2+ induces cell growth, differentiation and proliferation via activation of several transcription factors. Activation a variety of store operated and Transient Receptor Potential channels cause a rise in cytosolic Ca2+, making these channels components involved in proliferation and differentiation. Synaptic vesicle release Transient Receptor Potential Melastatin 7 channels reside in synaptic vesicles and regulate neurotransmitter release by a mechanism that is not entirely clear. All the above features of Transient Receptor Potential channels make them crucial components in important, sometimes conflicting, cellular processes that still need to be explored. PMID:20399884

  7. [Computational analysis of a cys-loop ligand gated ion channel from the green alga Chlamydomonas reinhardtii].

    PubMed

    Mukherjee, Ashutosh

    2015-01-01

    Plants possess several neurotransmitters with well-known physiological roles. Currently only receptors for glutamate were reported to be found in plants, while receptors for acetylcholine, serotonin and GABA have not yet been reported. In animals, these neurotransmitters act via one class of ligand binding ion channels called Cys-loop receptors which play a major role in fast synaptic transmission. They show the presence of two domains namely Neurotransmitter-gated ion-channel ligand-binding domain (Pfam: PF02931) and Neurotransmitter-gated transmembrane domain (Pfam: PF02932). Cys-loop receptors are also known in prokaryotes. No cys-loop receptor has been characterized from plants yet. In this study, the Ensembl plants database was searched for proteins with these two domains in the sequenced plant genomes, what resulted in only one protein (LIC1) from the alga Chlamydomonas reinhardtii. BLAST and profile HMM searches against the pdb structure database showed that this protein is related to animal and prokaryotic cys-loop receptors, although the cysteine residues characteristic of the cys-loop are absent. Physico-chemical and sequence analysis indicate that LIC1 is an anionic receptor. A model of this protein was generated using homology modeling based on a nicotinic acetylcholine receptor of Torpedo marmorata. The characteristic extracellular domain (ECD) and transmembrane domain (TMD) are well structured but the intercellular region is poorly formed. This is the first report on a detailed characterization of a cys-loop receptor from the plant kingdom. PMID:26510602

  8. 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. PMID:27268784

  9. CFTR (ABCC7) is a hydrolyzable-ligand-gated channel.

    PubMed

    Aleksandrov, Andrei A; Aleksandrov, Luba A; Riordan, John R

    2007-02-01

    As the product of the gene mutated in cystic fibrosis, the most common genetic disease of Caucasians, CFTR is an atypical ABC protein. From an evolutionary perspective, it is apparently a relatively young member of the ABC family, present only in metazoans where it plays a critical role in epithelial salt and fluid homeostasis. Functionally, the membrane translocation process it mediates, the passive bidirectional diffusion of small inorganic anions, is simpler than the vectorial transport of larger more complex substrates ("allocrites") by most ABC transporters. However, the control of the permeation pathway which cannot go unchecked is necessarily more stringent than in the case of the transporters. There is tight regulation by the phosphorylation/dephosphorylation of the unique CFTR R domain superimposed on the basic ABC regulation mode of ATP binding and hydrolysis at the dual nucleotide binding sites. As with other ABCC subfamily members, only the second of these sites is hydrolytic in CFTR. The phosphorylation and ATP binding/hydrolysis events do not strongly influence each other; rather, R domain phosphorylation appears to enable transduction of the nucleotide binding allosteric signal to the responding channel gate. ATP hydrolysis is not required for either the opening or closing gating transitions but efficiently clears the ligand-binding site enabling a new gating cycle to be initiated. PMID:17021796

  10. 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

  11. Conformational Changes During the Gating of a Potassium Channel Revealed by Structural Mass Spectrometry

    SciTech Connect

    Gupta, S.; Bavro, V; D' Mello, R; Tucker, S; Venien-Bryan, C; Chance, M

    2010-01-01

    Potassium channels are dynamic proteins that undergo large conformational changes to regulate the flow of K{sup +} ions across the cell membrane. Understanding the gating mechanism of these channels therefore requires methods for probing channel structure in both their open and closed conformations. Radiolytic footprinting is used to study the gating mechanism of the inwardly-rectifying potassium channel KirBac3.1. The purified protein stabilized in either open or closed conformations was exposed to focused synchrotron X-ray beams on millisecond timescales to modify solvent accessible amino acid side chains. These modifications were identified and quantified using high-resolution mass spectrometry. The differences observed between the closed and open states were then used to reveal local conformational changes that occur during channel gating. The results provide support for a proposed gating mechanism of the Kir channel and demonstrate a method of probing the dynamic gating mechanism of other integral membrane proteins and ion channels.

  12. 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

  13. Mechanism of voltage-dependent gating in skeletal muscle chloride channels.

    PubMed Central

    Fahlke, C; Rosenbohm, A; Mitrovic, N; George, A L; Rüdel, R

    1996-01-01

    Voltage-dependent gating was investigated in a recombinant human skeletal muscle Cl- channel, hCIC-1, heterologously expressed in human embryonic kidney (HEK-293) cells. Gating was found to be mediated by two qualitatively distinct processes. One gating step operates on a microsecond time scale and involves the rapid rearrangement of two identical intramembranous voltage sensors, each consisting of a single titratable residue. The second process occurs on a millisecond time scale and is due to a blocking-unblocking reaction mediated by a cytoplasmic gate that interacts with the ion pore of the channel. These results illustrate a rather simple structural basis for voltage sensing that has evolved in skeletal muscle Cl- channels and provides evidence for the existence of a cytoplasmic gating mechanism in an anion channel analogous to the "ball and chain" mechanism of voltage-gated cation channels. Images FIGURE 3 PMID:8842208

  14. Dynamic phospholipid interaction of β2e subunit regulates the gating of voltage-gated Ca2+ channels

    PubMed Central

    Kim, Dong-Il; Park, Yongsoo; Jang, Deok-Jin

    2015-01-01

    High voltage-activated Ca2+ (CaV) channels are protein complexes containing pore-forming α1 and auxiliary β and α2δ subunits. The subcellular localization and membrane interactions of the β subunits play a crucial role in regulating CaV channel inactivation and its lipid sensitivity. Here, we investigated the effects of membrane phosphoinositide (PI) turnover on CaV2.2 channel function. The β2 isoform β2e associates with the membrane through electrostatic and hydrophobic interactions. Using chimeric β subunits and liposome-binding assays, we determined that interaction between the N-terminal 23 amino acids of β2e and anionic phospholipids was sufficient for β2e membrane targeting. Binding of the β2e subunit N terminus to liposomes was significantly increased by inclusion of 1% phosphatidylinositol 4,5-bisphosphate (PIP2) in the liposomes, suggesting that, in addition to phosphatidylserine, PIs are responsible for β2e targeting to the plasma membrane. Membrane binding of the β2e subunit slowed CaV2.2 current inactivation. When membrane phosphatidylinositol 4-phosphate and PIP2 were depleted by rapamycin-induced translocation of pseudojanin to the membrane, however, channel opening was decreased and fast inactivation of CaV2.2(β2e) currents was enhanced. Activation of the M1 muscarinic receptor elicited transient and reversible translocation of β2e subunits from membrane to cytosol, but not that of β2a or β3, resulting in fast inactivation of CaV2.2 channels with β2e. These results suggest that membrane targeting of the β2e subunit, which is mediated by nonspecific electrostatic insertion, is dynamically regulated by receptor stimulation, and that the reversible association of β2e with membrane PIs results in functional changes in CaV channel gating. The phospholipid–protein interaction observed here provides structural insight into mechanisms of membrane–protein association and the role of phospholipids in ion channel regulation. PMID

  15. Inhibition of Voltage-Gated Calcium Channels by RGK Proteins.

    PubMed

    Buraei, Zafir; Yang, Jian

    2015-01-01

    Due to their essential biological roles, voltage-gated calcium channels (VGCCs) are regulated by a myriad of molecules and mechanisms. Fifteen years ago, RGK proteins were discovered to bind the VGCC β subunit (Cavβ) and potently inhibit high-voltage activated Ca(2+) channels. RGKs (Rad, Rem, Rem2 and Gem/Kir) are a family of monomeric small GTPases belonging to the superfamily of Ras GTPases. They exert dual inhibitory effects on VGCCs, decreasing surface expression and suppressing surface channels through immobilization of the voltage sensor or reduction of channel open probability. While Cavβ is required for all forms of RGK inhibition, not all inhibition is mediated by the RGK-Cavβ interaction. Some RGK proteins also interact directly with the pore-forming α1 subunit of some types of VGCCs (Cavα1). Importantly, RGK proteins tonically inhibit VGCCs in native cells, regulating cardiac and neural functions. This minireview summarizes the mechanisms, molecular determinants, and physiological impact of RGK inhibition of VGCCs. PMID:25966691

  16. Successive openings of the same acetylcholine receptor channel are correlated in open time.

    PubMed Central

    Jackson, M B; Wong, B S; Morris, C E; Lecar, H; Christian, C N

    1983-01-01

    Previous analysis of single-channel current records has shown that both the opening and closing transitions of chemically activated ion channels are operated by fast and slow kinetic processes. The fast component in the kinetics of channel opening has been interpreted as the reopening of a channel that has just closed. The fast component in the kinetics of channel closure has many possible explanations and is therefore more difficult to interpret. We can gain insight into the closing process by asking whether the lifetimes of successive openings of an acetylcholine receptor channel are correlated in open-state lifetime. Five kinetic models of channel closure are considered. Two of these models predict uncorrelated open-state lifetimes, one predicts correlated open-state lifetimes, and for two others a range of behavior is possible. Acetylcholine receptor channel data from cultured rat muscle are analyzed to show that open-state lifetimes are correlated, eliminating two models of channel gating. PMID:6301575

  17. Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms.

    PubMed

    Wanke, Enzo; Zaharenko, André Junqueira; Redaelli, Elisa; Schiavon, Emanuele

    2009-12-15

    As voltage-gated Na(+) channels are responsible for the conduction of electrical impulses in most excitable tissues in the majority of animals (except nematodes), they have become important targets for the toxins of venomous animals, from sea anemones to molluscs, scorpions, spiders and even fishes. During their evolution, different animals have developed a set of cysteine-rich peptides capable of binding different extracellular sites of this channel protein. A fundamental question concerning the mechanism of action of these toxins is whether they act at a common receptor site in Na(+) channels when exerting their different pharmacological effects, or at distinct receptor sites in different Na(v) channels subtypes whose particular properties lead to these pharmacological differences. The alpha-subunits of voltage-gated Na(+) channels (Na(v)1.x) have been divided into at least nine subtypes on the basis of amino acid sequences. Sea anemones have been extensively studied from the toxinological point of view for more than 40 years. There are about 40 sea anemone type 1 peptides known to be active on Na(v)1.x channels and all are 46-49 amino acid residues long, with three disulfide bonds and their molecular weights range between 3000 and 5000 Da. About 12 years ago a general model of Na(v)1.2-toxin interaction, developed for the alpha-scorpion toxins, was shown to fit also to action of sea anemone toxin such as ATX-II. According to this model these peptides are specifically acting on the type 3 site known to be between segments 3 and 4 in domain IV of the Na(+) channel protein. This region is indeed responsible for the normal Na(+) currents fast inactivation that is potently slowed by these toxins. This fundamental "gain-of-function" mechanism is responsible for the strong increase in the action potential duration. They constitute a class of tools by means of which physiologists and pharmacologists can study the structure/function relationships of channel proteins. As

  18. Receptor for protons: First observations on Acid Sensing Ion Channels.

    PubMed

    Krishtal, Oleg

    2015-07-01

    The history of ASICs began in 1980 with unexpected observation. The concept of highly selective Na(+) current gated by specific receptors for protons was not easily accepted. It took 16 years to get these receptor/channels cloned and start a new stage in their investigation. "The receptor for protons" became ASIC comprising under this name a family of receptor/channels ubiquitous for mammalian nervous system, both peripheral and central. The role of ASICs as putative nociceptors was suggested almost immediately after their discovery. This role subsequently was proven in many forms of pain-related phenomena. Many other functions of ASICs have been also found or primed for speculations both in physiology and in disease. Despite the width of field and strength of efforts, numerous basic questions are to be answered before we understand how the local changes in pH in the nervous tissue transform into electric and messenger signaling via ASICs as transducers. This article is part of the Special Issue entitled 'Acid-Sensing Ion Channels in the Nervous System'. PMID:25582296

  19. Cys-loop ligand-gated ion channel gene discovery in the Locusta migratoria manilensis through the neuron transcriptome.

    PubMed

    Wang, Xin; Meng, Xiangkun; Liu, Chuanjun; Gao, Hongli; Zhang, Yixi; Liu, Zewen

    2015-05-01

    As an ideal model, Locusta migratoria manilensis (Meyen) has been widely used in the study of endocrinological and neurobiological processes. Here we created a large transcriptome of the locust neurons, which enriched ion channels whose potential for functional genetic experiments is currently limited. With high-throughput Illumina sequencing technology, we obtained more than 50 million raw reads, which were assembled into 61,056 unique sequences with average size of 737bp. Among the unigenes, a total 24,884 sequences had significant similarities with proteins in the five public databases (NR, SwissProt, GO, COG and KEGG) with a cut-off E-value of 10(-5) using BLASTx. Moreover, the number of potential genes of the cys-loop ligand-gated ion channels (LGICs) was manually curated, including 39 putative nicotinic acetylcholine receptors (nAChRs), 6 putative γ-aminobutyric acid (GABA) gated anion channels, 21 putative glutamate-gated chloride channels (GluCls) and 1 histamine-gated chloride channels (HisCls). In addition, the full-length of 11 nAChRs subunits (9 alpha and 2 beta) were obtained by RACE technique that would be helpful to further studies on nAChR neurochemistry and pharmacological aspects. To our knowledge, this is the first study to characterize the locust neuron transcriptome, which will provide a useful resource especially for future studies on the neuro-function and behavior of the locust. PMID:25701599

  20. Back-gated split-gate transistor - A one-dimensional ballistic channel with variable Fermi energy

    NASA Astrophysics Data System (ADS)

    Hamilton, A. R.; Frost, J. E. F.; Smith, C. G.; Kelly, M. J.; Linfield, E. H.; Ford, C. J. B.; Ritchie, D. A.; Jones, G. A. C.; Pepper, M.; Hasko, D. G.

    1992-06-01

    The quantized conductance of a one-dimensional ballistic channel in the two-dimensional electron gas of a back-gated GaAs/AlGaAs heterostructure is studied. A standard Schottky split-gate fabricated with electron-beam lithography techniques is used to define the one-dimensional channel, but an epitaxially grown in situ back-gate, situated about 1 micron below the electron gas is incorporated to provide additional control of the carrier density. Quantized conductance steps can be induced by changing the bias on either gate, highlighting the self-consistent nature of the electrostatics involved. In principle, independent control of the one-dimensional carrier density and channel width can be achieved.

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

    PubMed

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

    2016-02-01

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

  2. 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.

  3. 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.

  4. Local postsynaptic voltage-gated sodium channel activation in dendritic spines of olfactory bulb granule cells.

    PubMed

    Bywalez, Wolfgang G; Patirniche, Dinu; Rupprecht, Vanessa; Stemmler, Martin; Herz, Andreas V M; Pálfi, Dénes; Rózsa, Balázs; Egger, Veronica

    2015-02-01

    Neuronal dendritic spines have been speculated to function as independent computational units, yet evidence for active electrical computation in spines is scarce. Here we show that strictly local voltage-gated sodium channel (Nav) activation can occur during excitatory postsynaptic potentials in the spines of olfactory bulb granule cells, which we mimic and detect via combined two-photon uncaging of glutamate and calcium imaging in conjunction with whole-cell recordings. We find that local Nav activation boosts calcium entry into spines through high-voltage-activated calcium channels and accelerates postsynaptic somatic depolarization, without affecting NMDA receptor-mediated signaling. Hence, Nav-mediated boosting promotes rapid output from the reciprocal granule cell spine onto the lateral mitral cell dendrite and thus can speed up recurrent inhibition. This striking example of electrical compartmentalization both adds to the understanding of olfactory network processing and broadens the general view of spine function. PMID:25619656

  5. An epilepsy mutation in the beta1 subunit of the voltage-gated sodium channel results in reduced channel sensitivity to phenytoin.

    PubMed

    Lucas, Paul T; Meadows, Laurence S; Nicholls, Jane; Ragsdale, David S

    2005-05-01

    The antiepileptic drug phenytoin inhibits voltage-gated sodium channels. Phenytoin block is enhanced at depolarized membrane potentials and during high frequency channel activation. These properties, which are important for the clinical efficacy of the drug, depend on voltage-dependent channel gating. In this study, we examined the action of phenytoin on sodium channels, comprising a mutant auxiliary beta1 subunit (mutation C121Wbeta1), which causes the inherited epilepsy syndrome, generalized epilepsy with febrile seizures plus (GEFS+). Whole cell sodium currents in Chinese hamster ovary (CHO) cells coexpressing human Na(v)1.3 sodium channels and C121Wbeta1 exhibited altered gating properties, compared to currents in cells coexpressing Na(v)1.3 and wild type beta1. In addition mutant channels were less sensitive to inhibition by phenytoin, showing reduced tonic block at -70mV (EC(50)=26microM for C121Wbeta1 versus 11microM for wild type beta1) and less frequency-dependent inhibition in response to a 20Hz pulse train ( approximately 40% inhibition for C121Wbeta1 versus approximately 70% inhibition for wild type beta1, with 50microM phenytoin). Mutant and wild type channels did not differ in inactivated state affinity for phenytoin, suggesting that their pharmacological differences were secondary to their differences in voltage-dependent gating, rather than being caused by direct effects of the mutation on the drug receptor. Together, these data show that a sodium channel mutation responsible for epilepsy can also alter channel response to antiepileptic drugs. PMID:15922564

  6. Gambierol inhibition of voltage-gated potassium channels augments spontaneous Ca2+ oscillations in cerebrocortical neurons.

    PubMed

    Cao, Zhengyu; Cui, Yanjun; Busse, Eric; Mehrotra, Suneet; Rainier, Jon D; Murray, Thomas F

    2014-09-01

    Gambierol is a marine polycyclic ether toxin produced by the marine dinoflagellate Gambierdiscus toxicus and is a member of the ciguatoxin toxin family. Gambierol has been demonstrated to be either a low-efficacy partial agonist/antagonist of voltage-gated sodium channels or a potent blocker of voltage-gated potassium channels (Kvs). Here we examined the influence of gambierol on intact cerebrocortical neurons. We found that gambierol produced both a concentration-dependent augmentation of spontaneous Ca(2+) oscillations, and an inhibition of Kv channel function with similar potencies. In addition, an array of selective as well as universal Kv channel inhibitors mimicked gambierol in augmenting spontaneous Ca(2+) oscillations in cerebrocortical neurons. These data are consistent with a gambierol blockade of Kv channels underlying the observed increase in spontaneous Ca(2+) oscillation frequency. We also found that gambierol produced a robust stimulation of phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2). Gambierol-stimulated ERK1/2 activation was dependent on both inotropic [N-methyl-d-aspartate (NMDA)] and type I metabotropic glutamate receptors (mGluRs) inasmuch as MK-801 [NMDA receptor inhibitor; (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate], S-(4)-CGP [S-(4)-carboxyphenylglycine], and MTEP [type I mGluR inhibitors; 3-((2-methyl-4-thiazolyl)ethynyl) pyridine] attenuated the response. In addition, 2-aminoethoxydiphenylborane, an inositol 1,4,5-trisphosphate receptor inhibitor, and U73122 (1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), a phospholipase C inhibitor, both suppressed gambierol-induced ERK1/2 activation, further confirming the role of type I mGluR-mediated signaling in the observed ERK1/2 activation. Finally, we found that gambierol produced a concentration-dependent stimulation of neurite outgrowth that was mimicked by 4-aminopyridine, a universal

  7. Gambierol Inhibition of Voltage-Gated Potassium Channels Augments Spontaneous Ca2+ Oscillations in Cerebrocortical Neurons

    PubMed Central

    Cao, Zhengyu; Cui, Yanjun; Busse, Eric; Mehrotra, Suneet; Rainier, Jon D.

    2014-01-01

    Gambierol is a marine polycyclic ether toxin produced by the marine dinoflagellate Gambierdiscus toxicus and is a member of the ciguatoxin toxin family. Gambierol has been demonstrated to be either a low-efficacy partial agonist/antagonist of voltage-gated sodium channels or a potent blocker of voltage-gated potassium channels (Kvs). Here we examined the influence of gambierol on intact cerebrocortical neurons. We found that gambierol produced both a concentration-dependent augmentation of spontaneous Ca2+ oscillations, and an inhibition of Kv channel function with similar potencies. In addition, an array of selective as well as universal Kv channel inhibitors mimicked gambierol in augmenting spontaneous Ca2+ oscillations in cerebrocortical neurons. These data are consistent with a gambierol blockade of Kv channels underlying the observed increase in spontaneous Ca2+ oscillation frequency. We also found that gambierol produced a robust stimulation of phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2). Gambierol-stimulated ERK1/2 activation was dependent on both inotropic [N-methyl-d-aspartate (NMDA)] and type I metabotropic glutamate receptors (mGluRs) inasmuch as MK-801 [NMDA receptor inhibitor; (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate], S-(4)-CGP [S-(4)-carboxyphenylglycine], and MTEP [type I mGluR inhibitors; 3-((2-methyl-4-thiazolyl)ethynyl) pyridine] attenuated the response. In addition, 2-aminoethoxydiphenylborane, an inositol 1,4,5-trisphosphate receptor inhibitor, and U73122 (1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), a phospholipase C inhibitor, both suppressed gambierol-induced ERK1/2 activation, further confirming the role of type I mGluR-mediated signaling in the observed ERK1/2 activation. Finally, we found that gambierol produced a concentration-dependent stimulation of neurite outgrowth that was mimicked by 4-aminopyridine, a universal potassium

  8. Voltage-Gated Sodium Channels: Biophysics, Pharmacology, and Related Channelopathies

    PubMed Central

    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 (INa) 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. PMID:22798951

  9. Nonvolatile Quantum Dot Gate Memory (NVQDM): Tunneling Rate from Quantum Well Channel to Quantum Dot Gate

    NASA Astrophysics Data System (ADS)

    Hasaneen, El-Sayed; Heller, Evan; Bansal, Rajeev; Jain, Faquir

    2003-10-01

    In this paper, we compute the tunneling of electrons in a nonvolatile quantum dot memory (NVQDM) cell during the WRITE operation. The transition rate of electrons from a quantum well channel to the quantum dots forming the floating gate is calculated using a recently reported method by Chuang et al.[1]. Tunneling current is computed based on transport of electrons from the channel to the floating quantum dots. The maximum number of electrons on a dot is calculated using surface electric field and break down voltage of the tunneling dielectric material. Comparison of tunneling for silicon oxide and high-k dielectric gate insulators is also described. Capacitance-Voltage characteristics of a NVQDM device are calculated by solving the Schrodinger and Poisson equations self-consistently. In addition, the READ operation of the memory has been investigated analytically. Results for 70 nm channel length Si NVQDMs are presented. Threshold voltage is calculated including the effect of the charge on nanocrystal quantum dots. Current-voltage characteristics are obtained using BSIM3v3 model [2-3]. This work is supported by Office of Navel Research (N00014210883, Dr. D. Purdy, Program Monitor), Connecticut Innovations Inc./TranSwitch (CII # 00Y17), and National Science Foundation (CCR-0210428) grants. [1] S. L. Chuang and N. Holonyak, Appl. Phys. Lett., 80, pp. 1270, 2002. [2] Y. Chen et. al., BSIM3v3 Manual, Elect. Eng. and Comp. Dept., U. California, Berkeley, CA, 1996. [3] W. Liu, MOSFET Models for SPICE Simulation, John Wiley & Sons, Inc., 2001.

  10. Transient Receptor Potential Channels in the Vasculature

    PubMed Central

    Earley, Scott; Brayden, Joseph E.

    2015-01-01

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

  11. 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.

  12. 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

  13. 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. PMID:27586280

  14. Molecular identity of dendritic voltage-gated sodium channels.

    PubMed

    Lorincz, Andrea; Nusser, Zoltan

    2010-05-14

    Active invasion of the dendritic tree by action potentials (APs) generated in the axon is essential for associative synaptic plasticity and neuronal ensemble formation. In cortical pyramidal cells (PCs), this AP back-propagation is supported by dendritic voltage-gated Na+ (Nav) channels, whose molecular identity is unknown. Using a highly sensitive electron microscopic immunogold technique, we revealed the presence of the Nav1.6 subunit in hippocampal CA1 PC proximal and distal dendrites. Here, the subunit density is lower by a factor of 35 to 80 than that found in axon initial segments. A gradual decrease in Nav1.6 density along the proximodistal axis of the dendritic tree was also detected without any labeling in dendritic spines. Our results reveal the characteristic subcellular distribution of the Nav1.6 subunit, identifying this molecule as a key substrate enabling dendritic excitability. PMID:20466935

  15. Modulation of voltage-gated K+ channels by the sodium channel β1 subunit

    PubMed Central

    Nguyen, Hai M.; Miyazaki, Haruko; Hoshi, Naoto; Smith, Brian J.; Nukina, Nobuyuki; Goldin, Alan L.; Chandy, K. George

    2012-01-01

    Voltage-gated sodium (NaV) and potassium (KV) channels are critical components of neuronal action potential generation and propagation. Here, we report that NaVβ1 encoded by SCN1b, an integral subunit of NaV channels, coassembles with and modulates the biophysical properties of KV1 and KV7 channels, but not KV3 channels, in an isoform-specific manner. Distinct domains of NaVβ1 are involved in modulation of the different KV channels. Studies with channel chimeras demonstrate that NaVβ1-mediated changes in activation kinetics and voltage dependence of activation require interaction of NaVβ1 with the channel’s voltage-sensing domain, whereas changes in inactivation and deactivation require interaction with the channel’s pore domain. A molecular model based on docking studies shows NaVβ1 lying in the crevice between the voltage-sensing and pore domains of KV channels, making significant contacts with the S1 and S5 segments. Cross-modulation of NaV and KV channels by NaVβ1 may promote diversity and flexibility in the overall control of cellular excitability and signaling. PMID:23090990

  16. Insights into the structural nature of the transition state in the Kir channel gating pathway.

    PubMed

    Fowler, Philip W; Bollepalli, Murali K; Rapedius, Markus; Nematian-Ardestani, Ehsan; Shang, Lijun; Sansom, Mark Sp; Tucker, Stephen J; Baukrowitz, Thomas

    2014-01-01

    In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a 'catalytic' effect upon the channel gating kinetics. PMID:25483285

  17. Insights into the structural nature of the transition state in the Kir channel gating pathway

    PubMed Central

    Fowler, Philip W; Bollepalli, Murali K; Rapedius, Markus; Nematian-Ardestani, Ehsan; Shang, Lijun; Sansom, Mark SP; Tucker, Stephen J; Baukrowitz, Thomas

    2014-01-01

    In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a ‘catalytic’ effect upon the channel gating kinetics. PMID:25483285

  18. Spontaneous openings of the acetylcholine receptor channel.

    PubMed Central

    Jackson, M B

    1984-01-01

    Patch clamp recordings from embryonic mouse muscle cells in culture revealed spontaneous openings of the acetylcholine receptor channel in the absence of exogenously applied cholinergic agent. The conductance of the spontaneous channel currents was, within experimental error, identical with the conductance of suberyldicholine-activated channel currents. The comparison of channel conductance was made with sodium and with cesium, each at two concentrations, with the same result. Treatment of the cells with alpha-bungarotoxin blocked the spontaneous channel currents. To determine whether the spontaneous openings were caused by an endogenous agent with cholinergic activity a reactive disulfide bond near the receptor binding site was reduced with dithiothreitol and alkylated with N-ethylmaleimide. This chemical modification reduced the effectiveness with which suberyldicholine and curare activated channel currents but did not reduce the frequency of spontaneous openings. These experiments indicate that the acetylcholine receptor briefly and infrequently fluctuates into an active state in the absence of agonist. Agonist activation of the receptor presumably accelerates this spontaneously occurring process. PMID:6328531

  19. Voltage-gated sodium channels in the mammalian heart

    PubMed Central

    Zimmer, Thomas; Haufe, Volker; Blechschmidt, Steve

    2014-01-01

    Mammalian species express nine functional voltage-gated Na+ channels. Three of them, the cardiac-specific isoform Nav1.5 and the neuronal isoforms Nav1.8 and Nav1.9, are relatively resistant to the neurotoxin tetrodotoxin (TTX; IC50 ≥ 1 μM). The other six isoforms are highly sensitive to TTX with IC50 values in the nanomolar range. These isoforms are expressed in the central nervous system (Nav1.1, Nav1.2, Nav1.3, Nav1.6), in the skeletal muscle (Nav1.4), and in the peripheral nervous system (Nav1.6, Nav1.7). The isoform Nav1.5, encoded by the SCN5A gene, is responsible for the upstroke of the action potential in the heart. Mutations in SCN5A are associated with a variety of life-threatening arrhythmias, like long QT syndrome type 3 (LQT3), Brugada syndrome (BrS) or cardiac conduction disease (CCD). Previous immunohistochemical and electrophysiological assays demonstrated the cardiac expression of neuronal and skeletal muscle Na+ channels in the heart of various mammals, which led to far-reaching speculations on their function. However, when comparing the Na+ channel mRNA patterns in the heart of various mammalian species, only minute quantities of transcripts for TTX-sensitive Na+ channels were detectable in whole pig and human hearts, suggesting that these channels are not involved in cardiac excitation phenomena in higher mammals. This conclusion is strongly supported by the fact that mutations in TTX-sensitive Na+ channels were associated with epilepsy or skeletal muscle diseases, rather than with a pathological cardiac phenotype. Moreover, previous data from TTX-intoxicated animals and from cases of human tetrodotoxication showed that low TTX dosages caused at most little alterations of both the cardiac output and the electrocardiogram. Recently, genome-wide association studies identified SCN10A, the gene encoding Nav1.8, as a determinant of cardiac conduction parameters, and mutations in SCN10A have been associated with BrS. These novel findings opened a

  20. 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.

  1. 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

    PubMed Central

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

    2011-01-01

    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. PMID:20561903

  2. 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.

    PubMed

    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. PMID:20561903

  3. Propranolol blocks cardiac and neuronal voltage-gated sodium channels.

    PubMed

    Wang, Dao W; Mistry, Akshitkumar M; Kahlig, Kristopher M; Kearney, Jennifer A; Xiang, Jizhou; George, Alfred L

    2010-01-01

    Propranolol is a widely used, non-selective β-adrenergic receptor antagonist with proven efficacy in treating cardiovascular disorders and in the prevention of migraine headaches. At plasma concentrations exceeding those required for β-adrenergic receptor inhibition, propranolol also exhibits anti-arrhythmic ("membrane stabilizing") effects that are not fully explained by β-blockade. Previous in vitro studies suggested that propranolol may have local anesthetic effects. We directly tested the effects of propranolol on heterologously expressed recombinant human cardiac (NaV1.5) and brain (NaV1.1, NaV1.2, NaV1.3) sodium channels using whole-cell patch-clamp recording. We found that block was not stereospecific as we observed approximately equal IC50 values for tonic and use-dependent block by R-(+) and S-(-) propranolol (tonic block: R: 21.4 μM vs S: 23.6 μM; use-dependent block: R: 2.7 μM vs S: 2.6 μM). Metoprolol and nadolol did not block NaV1.5 indicating that sodium channel block is not a class effect of β-blockers. The biophysical effects of R-(+)-propranolol on NaV1.5 and NaV1.1 resembled that of the prototypical local anesthetic lidocaine including the requirement for a critical phenylalanine residue (F1760 in NaV1.5) in the domain 4 S6 segment. Finally, we observed that brain sodium channels exhibited less sensitivity to R-(+)-propranolol than NaV1.5 channels. Our findings establish sodium channels as targets for propranolol and may help explain some beneficial effects of the drug in treating cardiac arrhythmias, and may explain certain adverse central nervous system effects. PMID:21833183

  4. Temperature-sensitive gating of TRPV1 channel as probed by atomistic simulations of its trans- and juxtamembrane domains

    PubMed Central

    Chugunov, Anton O.; Volynsky, Pavel E.; Krylov, Nikolay A.; Nolde, Dmitry E.; Efremov, Roman G.

    2016-01-01

    Heat-activated transient receptor potential channel TRPV1 is one of the most studied eukaryotic proteins involved in temperature sensation. Upon heating, it exhibits rapid reversible pore gating, which depolarizes neurons and generates action potentials. Underlying molecular details of such effects in the pore region of TRPV1 is of a crucial importance to control temperature responses of the organism. Despite the spatial structure of the channel in both open (O) and closed (C) states is known, microscopic nature of channel gating and mechanism of thermal sensitivity are still poorly understood. In this work, we used unrestrained atomistic molecular dynamics simulations of TRPV1 (without N- and C-terminal cytoplasmic domains) embedded into explicit lipid bilayer in its O- and C-states. We found that the pore domain with its neighboring loops undergoes large temperature-dependent conformational transitions in an asymmetric way, when fragments of only one monomer move with large amplitude, freeing the pore upon heating. Such an asymmetrical gating looks rather biologically relevant because it is faster and more reliable than traditionally proposed “iris-like” symmetric scheme of channel opening. Analysis of structural, dynamic, and hydrophobic organization of the pore domain revealed entropy growth upon TRPV1 gating, which is in line with current concepts of thermal sensitivity. PMID:27612191

  5. Temperature-sensitive gating of TRPV1 channel as probed by atomistic simulations of its trans- and juxtamembrane domains.

    PubMed

    Chugunov, Anton O; Volynsky, Pavel E; Krylov, Nikolay A; Nolde, Dmitry E; Efremov, Roman G

    2016-01-01

    Heat-activated transient receptor potential channel TRPV1 is one of the most studied eukaryotic proteins involved in temperature sensation. Upon heating, it exhibits rapid reversible pore gating, which depolarizes neurons and generates action potentials. Underlying molecular details of such effects in the pore region of TRPV1 is of a crucial importance to control temperature responses of the organism. Despite the spatial structure of the channel in both open (O) and closed (C) states is known, microscopic nature of channel gating and mechanism of thermal sensitivity are still poorly understood. In this work, we used unrestrained atomistic molecular dynamics simulations of TRPV1 (without N- and C-terminal cytoplasmic domains) embedded into explicit lipid bilayer in its O- and C-states. We found that the pore domain with its neighboring loops undergoes large temperature-dependent conformational transitions in an asymmetric way, when fragments of only one monomer move with large amplitude, freeing the pore upon heating. Such an asymmetrical gating looks rather biologically relevant because it is faster and more reliable than traditionally proposed "iris-like" symmetric scheme of channel opening. Analysis of structural, dynamic, and hydrophobic organization of the pore domain revealed entropy growth upon TRPV1 gating, which is in line with current concepts of thermal sensitivity. PMID:27612191

  6. Ca(2+) signals mediated by Ins(1,4,5)P(3)-gated channels in rat ureteric myocytes.

    PubMed Central

    Boittin, F X; Coussin, F; Morel, J L; Halet, G; Macrez, N; Mironneau, J

    2000-01-01

    Localized Ca(2+)-release signals (puffs) and propagated Ca(2+) waves were characterized in rat ureteric myocytes by confocal microscopy. Ca(2+) puffs were evoked by photorelease of low concentrations of Ins(1,4,5)P(3) from a caged precursor and by low concentrations of acetylcholine; they were also observed spontaneously in Ca(2+)-overloaded myocytes. Ca(2+) puffs showed some variability in amplitude, time course and spatial spread, suggesting that Ins(1,4,5)P(3)-gated channels exist in clusters containing variable numbers of channels and that within these clusters a variable number of channels can be recruited. Immunodetection of Ins(1,4,5)P(3) receptors revealed the existence of several spots of fluorescence in the confocal cell sections, supporting the existence of clusters of Ins(1,4,5)P(3) receptors. Strong Ins(1,4,5)P(3) photorelease and high concentrations of acetylcholine induced Ca(2+) waves that originated from an initiation site and propagated in the whole cell by spatial recruitment of neighbouring Ca(2+)-release sites. Both Ca(2+) puffs and Ca(2+) waves were blocked selectively by intracellular applications of heparin and an anti-Ins(1,4,5)P(3)-receptor antibody, but were unaffected by ryanodine and intracellular application of an anti-ryanodine receptor antibody. mRNAs encoding for the three subtypes of Ins(1,4,5)P(3) receptor and subtype 3 of ryanodine receptor were detected in these myocytes, and the maximal binding capacity of [(3)H]Ins(1,4,5)P(3) was 10- to 12-fold higher than that of [(3)H]ryanodine. These results suggest that Ins(1,4,5)P(3)-gated channels mediate a continuum of Ca(2+) signalling in smooth-muscle cells expressing a high level of Ins(1,4,5)P(3) receptors and no subtypes 1 and 2 of ryanodine receptors. PMID:10861244

  7. 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

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

    PubMed

    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

  9. Signal Transduction at the Domain Interface of Prokaryotic Pentameric Ligand-Gated Ion Channels

    PubMed Central

    Bertozzi, Carlo; Zimmermann, Iwan; Engeler, Sibylle; Hilf, Ricarda J. C.; Dutzler, Raimund

    2016-01-01

    Pentameric ligand-gated ion channels are activated by the binding of agonists to a site distant from the ion conduction path. These membrane proteins consist of distinct ligand-binding and pore domains that interact via an extended interface. Here, we have investigated the role of residues at this interface for channel activation to define critical interactions that couple conformational changes between the two structural units. By characterizing point mutants of the prokaryotic channels ELIC and GLIC by electrophysiology, X-ray crystallography and isothermal titration calorimetry, we have identified conserved residues that, upon mutation, apparently prevent activation but not ligand binding. The positions of nonactivating mutants cluster at a loop within the extracellular domain connecting β-strands 6 and 7 and at a loop joining the pore-forming helix M2 with M3 where they contribute to a densely packed core of the protein. An ionic interaction in the extracellular domain between the turn connecting β-strands 1 and 2 and a residue at the end of β-strand 10 stabilizes a state of the receptor with high affinity for agonists, whereas contacts of this turn to a conserved proline residue in the M2-M3 loop appear to be less important than previously anticipated. When mapping residues with strong functional phenotype on different channel structures, mutual distances are closer in conducting than in nonconducting conformations, consistent with a potential role of contacts in the stabilization of the open state. Our study has revealed a pattern of interactions that are crucial for the relay of conformational changes from the extracellular domain to the pore region of prokaryotic pentameric ligand-gated ion channels. Due to the strong conservation of the interface, these results are relevant for the entire family. PMID:26943937

  10. 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. PMID:20713704

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

    SciTech Connect

    Hattori, Motoyuki; Gouaux, Eric

    2012-10-24

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

  12. Voltage-Gated Ion Channels in Cancer Cell Proliferation

    PubMed Central

    Rao, Vidhya R.; Perez-Neut, Mathew; Kaja, Simon; Gentile, Saverio

    2015-01-01

    Changes of the electrical charges across the surface cell membrane are absolutely necessary to maintain cellular homeostasis in physiological as well as in pathological conditions. The opening of ion channels alter the charge distribution across the surface membrane as they allow the diffusion of ions such as K+, Ca++, Cl−, Na+. Traditionally, voltage-gated ion channels (VGIC) are known to play fundamental roles in controlling rapid bioelectrical signaling including action potential and/or contraction. However, several investigations have revealed that these classes of proteins can also contribute significantly to cell mitotic biochemical signaling, cell cycle progression, as well as cell volume regulation. All these functions are critically important for cancer cell proliferation. Interestingly, a variety of distinct VGICs are expressed in different cancer cell types, including metastasis but not in the tissues from which these tumors were generated. Given the increasing evidence suggesting that VGIC play a major role in cancer cell biology, in this review we discuss the role of distinct VGIC in cancer cell proliferation and possible therapeutic potential of VIGC pharmacological manipulation. PMID:26010603

  13. 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

  14. 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

    The acetylcholine receptor (AChR) from vertebrate skeletal muscle initiates voluntary movement, and its kinetics of activation are crucial for maintaining the safety margin for neuromuscular transmission. Furthermore, the kinetic mechanism of the muscle AChR serves as an archetype for understanding activation mechanisms of related receptors from the Cys-loop superfamily. Here we record currents through single muscle AChR channels with improved temporal resolution approaching half an order of magnitude over our previous best. A range of concentrations of full and partial agonists are used to elicit currents from human wild-type and gain-of-function mutant AChRs. For each agonist-receptor combination, rate constants are estimated from maximum likelihood analysis using a kinetic scheme comprised of agonist binding, priming, and channel gating steps. The kinetic scheme and rate constants are tested by stochastic simulation, followed by incorporation of the experimental step response, sampling rate, background noise, and filter bandwidth. Analyses of the simulated data confirm all rate constants except those for channel gating, which are overestimated because of the established effect of noise on the briefest dwell times. Estimates of the gating rate constants were obtained through iterative simulation followed by kinetic fitting. The results reveal that the agonist association rate constants are independent of agonist occupancy but depend on receptor state, whereas those for agonist dissociation depend on occupancy but not on state. The priming rate and equilibrium constants increase with successive agonist occupancy, and for a full agonist, the forward rate constant increases more than the equilibrium constant; for a partial agonist, the forward rate and equilibrium constants increase equally. The gating rate and equilibrium constants also increase with successive agonist occupancy, but unlike priming, the equilibrium constants increase more than the forward rate

  15. Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine

    PubMed Central

    Pan, Jianjun; Chen, Qiang; Willenbring, Dan; Yoshida, Ken; Tillman, Tommy; Kashlan, Ossama B.; Cohen, Aina; Kong, Xiang-Peng; Xu, Yan; Tang, Pei

    2012-01-01

    ELIC, the pentameric ligand-gated ion channel from Erwinia chrysanthemi, is a prototype for Cys-loop receptors. Here we show that acetylcholine is a competitive antagonist for ELIC. We determine the acetylcholine–ELIC cocrystal structure to a 2.9-Å resolution and find that acetylcholine binding to an aromatic cage at the subunit interface induces a significant contraction of loop C and other structural rearrangements in the extracellular domain. The side chain of the pore-lining residue F247 reorients and the pore size consequently enlarges, but the channel remains closed. We attribute the inability of acetylcholine to activate ELIC primarily to weak cation-π and electrostatic interactions in the pocket, because an acetylcholine derivative with a simple quaternary-to-tertiary ammonium substitution activates the channel. This study presents a compelling case for understanding the structural underpinning of the functional relationship between agonism and competitive antagonism in the Cys-loop receptors, providing a new framework for developing novel therapeutic drugs. PMID:22395605

  16. Voltage-Dependent Gating in a “Voltage Sensor-Less” Ion Channel

    PubMed Central

    Kurata, Harley T.; Rapedius, Markus; Kleinman, Marc J.; Baukrowitz, Thomas .; Nichols, Colin G.

    2010-01-01

    The voltage sensitivity of voltage-gated cation channels is primarily attributed to conformational changes of a four transmembrane segment voltage-sensing domain, conserved across many levels of biological complexity. We have identified a remarkable point mutation that confers significant voltage dependence to Kir6.2, a ligand-gated channel that lacks any canonical voltage-sensing domain. Similar to voltage-dependent Kv channels, the Kir6.2[L157E] mutant exhibits time-dependent activation upon membrane depolarization, resulting in an outwardly rectifying current-voltage relationship. This voltage dependence is convergent with the intrinsic ligand-dependent gating mechanisms of Kir6.2, since increasing the membrane PIP2 content saturates Po and eliminates voltage dependence, whereas voltage activation is more dramatic when channel Po is reduced by application of ATP or poly-lysine. These experiments thus demonstrate an inherent voltage dependence of gating in a “ligand-gated” K+ channel, and thereby provide a new view of voltage-dependent gating mechanisms in ion channels. Most interestingly, the voltage- and ligand-dependent gating of Kir6.2[L157E] is highly sensitive to intracellular [K+], indicating an interaction between ion permeation and gating. While these two key features of channel function are classically dealt with separately, the results provide a framework for understanding their interaction, which is likely to be a general, if latent, feature of the superfamily of cation channels. PMID:20208975

  17. 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

  18. High-frequency stimulation-induced synaptic potentiation in dorsal and ventral CA1 hippocampal synapses: the involvement of NMDA receptors, mGluR5, and (L-type) voltage-gated calcium channels.

    PubMed

    Papatheodoropoulos, Costas; Kouvaros, Stylianos

    2016-09-01

    The ability of the ventral hippocampus (VH) for long-lasting long-term potentiation (LTP) and the mechanisms underlying its lower ability for short-lasting LTP compared with the dorsal hippocampus (DH) are unknown. Using recordings of field excitatory postsynaptic potentials (EPSPs) from the CA1 field of adult rat hippocampal slices, we found that 200-Hz stimulation induced nondecremental LTP that was maintained for at least 7 h and was greater in the DH than in the VH. The interaction of NMDA receptors with L-type voltage-dependent calcium channels appeared to be more effective in the DH than in the VH. Furthermore, the LTP was significantly enhanced in the DH only, between 2 and 5 h post-tetanus. Furthermore, the mGluR5 contributed to the post-tetanic potentiation more in the VH than in the DH. PMID:27531836

  19. 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).

  20. PSD-95 and PKC converge in regulating NMDA receptor trafficking and gating

    PubMed Central

    Lin, Ying; Jover-Mengual, Teresa; Wong, Judy; Bennett, Michael V. L.; Zukin, R. Suzanne

    2006-01-01

    Neuronal NMDA receptors (NMDARs) colocalize with postsynaptic density protein-95 (PSD-95), a putative NMDAR anchoring protein and core component of the PSD, at excitatory synapses. PKC activation and PSD-95 expression each enhance NMDAR channel opening rate and number of functional channels at the cell surface. Here we show in Xenopus oocytes that PSD-95 and PKC potentiate NMDA gating and trafficking in a nonadditive manner. PSD-95 and PKC each enhance NMDA channel activity, with no change in single-channel conductance, reversal potential or mean open time. PSD-95 and PKC each potentiate NMDA channel opening rate (kβ) and number of functional channels at the cell surface (N), as indicated by more rapid current decay and enhanced charge transfer in the presence of the open channel blocker MK-801. PSD-95 and PKC each increase NMDAR surface expression, as indicated by immunofluorescence. PKC potentiates NMDA channel function and NMDAR surface expression to the same final absolute values in the absence or presence of PSD-95. Thus, PSD-95 partially occludes PKC potentiation. We further show that Ser-1462, a putative phosphorylation target within the PDZ-binding motif of the NR2A subunit, is required for PSD-95-induced potentiation and partial occlusion of PKC potentiation. Coimmunoprecipitation experiments with cortical neurons in culture indicate that PKC activation promotes assembly of NR2 with NR1, and that the newly assembled NMDARs are not associated with PSD-95. These findings predict that synaptic scaffolding proteins and protein kinases convergently modulate NMDAR gating and trafficking at synaptic sites. PMID:17179037

  1. The Central domain of RyR1 is the transducer for long-range allosteric gating of channel opening.

    PubMed

    Bai, Xiao-Chen; Yan, Zhen; Wu, Jianping; Li, Zhangqiang; Yan, Nieng

    2016-09-01

    The ryanodine receptors (RyRs) are intracellular calcium channels responsible for rapid release of Ca(2+) from the sarcoplasmic/endoplasmic reticulum (SR/ER) to the cytoplasm, which is essential for the excitation-contraction (E-C) coupling of cardiac and skeletal muscles. The near-atomic resolution structure of closed RyR1 revealed the molecular details of this colossal channel, while the long-range allosteric gating mechanism awaits elucidation. Here, we report the cryo-EM structures of rabbit RyR1 in three closed conformations at about 4 Å resolution and an open state at 5.7 Å. Comparison of the closed RyR1 structures shows a breathing motion of the cytoplasmic platform, while the channel domain and its contiguous Central domain remain nearly unchanged. Comparison of the open and closed structures shows a dilation of the S6 tetrahelical bundle at the cytoplasmic gate that leads to channel opening. During the pore opening, the cytoplasmic "O-ring" motif of the channel domain and the U-motif of the Central domain exhibit coupled motion, while the Central domain undergoes domain-wise displacement. These structural analyses provide important insight into the E-C coupling in skeletal muscles and identify the Central domain as the transducer that couples the conformational changes of the cytoplasmic platform to the gating of the central pore. PMID:27468892

  2. 5-Hydroxytryptamine-induced calcium-channel gating in rainbow trout (Oncorhynchus mykiss) peripheral blood lymphocytes.

    PubMed Central

    Ferriere, F; Khan, N A; Meyniel, J P; Deschaux, P

    1997-01-01

    The present study was conducted on peripheral blood lympho-cytes of rainbow trout (Oncorhynchus mykiss) to assess the role of 5-hydroxytryptamine (5-HT; 'serotonin') in calcium signalling. 5-HT-induced increases in intracellular free calcium concentrations, [Ca2+]i, and its action was mediated by 5-HT receptor subtype 3 (5-HT3), but not by 5-HT receptor subtype 1A (5-HT1A) or subtype 2 (5-HT2) in these cells. In Ca2+-containing medium (1 mM CaCl2), 5-HT and 2-methyl-5-HT (5-HT3 receptor agonist) induced increases in [Ca2+]i, whereas in Ca2+-free medium (0 Ca2+, 1 mM EGTA), these two agents failed to evoke increases in [Ca2+]i in these cells, demonstrating that 5-HT mobilizes Ca2+ from the extracellular environment. Furthermore, 5-HT-induced increases in [Ca2+]i are not contributed to by the intracellular endoplasmic reticulum (ER) pool, as thapsigargin, an agent that recruits Ca2+ from ER stores, had additive effects on 5-HT-induced [Ca2+]i responses in fish peripheral lymphocytes. 5-HT-induced increases in [Ca2+]i were mediated by 5-HT3 receptors via gating the calcium through L-type, but not N-type, calcium channels in trout lymphocytes. PMID:9173890

  3. Voltage-gated calcium and sodium channels mediate Sema3A retrograde signaling that regulates dendritic development.

    PubMed

    Yamashita, Naoya; Aoki, Reina; Chen, Sandy; Jitsuki-Takahashi, Aoi; Ohura, Shunsuke; Kamiya, Haruyuki; Goshima, Yoshio

    2016-01-15

    Growing axons rely on local signaling at the growth cone for guidance cues. Semaphorin3A (Sema3A), a secreted repulsive axon guidance molecule, regulates synapse maturation and dendritic branching. We previously showed that local Sema3A signaling in the growth cones elicits retrograde retrograde signaling via PlexinA4 (PlexA4), one component of the Sema3A receptor, thereby regulating dendritic localization of AMPA receptor GluA2 and proper dendritic development. In present study, we found that nimodipine (voltage-gated L-type Ca(2+) channel blocker) and tetrodotoxin (TTX; voltage-gated Na(+) channel blocker) suppress Sema3A-induced dendritic localization of GluA2 and dendritic branch formation in cultured hippocampal neurons. The local application of nimodipine or TTX to distal axons suppresses retrograde transport of Venus-Sema3A that has been exogenously applied to the distal axons. Sema3A facilitates axonal transport of PlexA4, which is also suppressed in neurons treated with either TTX or nimodipine. These data suggest that voltage-gated calcium and sodium channels mediate Sema3A retrograde signaling that regulates dendritic GluA2 localization and branch formation. PMID:26638837

  4. G-protein-gated potassium (GIRK) channels containing the GIRK2 subunit are control hubs for pharmacologically induced hypothermic responses.

    PubMed

    Costa, Alberto C S; Stasko, Melissa R; Stoffel, Markus; Scott-McKean, Jonah J

    2005-08-24

    Hypothermic responses of rodents to the peripheral or intraventricular injection of many individual neurotransmitter receptor agonists have been well documented. Because many hypothermia-inducing agonists are also known to activate G-protein-gated potassium (GIRK) channels, we investigated the hypothermic response to several of these agents on Girk2 null mutant mice. Core body temperatures were measured through radiotelemetry, and animals were maintained in special temperature-regulated chambers to ensure the accuracy of the measurements. The resulting data indicate that the activation of GIRK2-containing potassium channels plays a significant role in hypothermia induced by the activation of serotonergic (5-HT(1A)), GABAergic (GABA(B)), muscarinic (m2), adenosine (A1), and mu, delta, and kappa opioid receptors. These channels also are involved in the alcohol-induced hypothermic response. These results have implications for the understanding of pharmacologically induced hypothermia and thermoregulatory mechanisms. PMID:16120781

  5. 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

  6. Nuclear-localized cyclic nucleotide-gated channels mediate symbiotic calcium oscillations.

    PubMed

    Charpentier, Myriam; Sun, Jongho; Vaz Martins, Teresa; Radhakrishnan, Guru V; Findlay, Kim; Soumpourou, Eleni; Thouin, Julien; Véry, Anne-Aliénor; Sanders, Dale; Morris, Richard J; Oldroyd, Giles E D

    2016-05-27

    Nuclear-associated Ca(2+) oscillations mediate plant responses to beneficial microbial partners--namely, nitrogen-fixing rhizobial bacteria that colonize roots of legumes and arbuscular mycorrhizal fungi that colonize roots of the majority of plant species. A potassium-permeable channel is known to be required for symbiotic Ca(2+) oscillations, but the calcium channels themselves have been unknown until now. We show that three cyclic nucleotide-gated channels in Medicago truncatula are required for nuclear Ca(2+) oscillations and subsequent symbiotic responses. These cyclic nucleotide-gated channels are located at the nuclear envelope and are permeable to Ca(2+) We demonstrate that the cyclic nucleotide-gated channels form a complex with the postassium-permeable channel, which modulates nuclear Ca(2+) release. These channels, like their counterparts in animal cells, might regulate multiple nuclear Ca(2+) responses to developmental and environmental conditions. PMID:27230377

  7. Structural Basis for Pharmacology of Voltage-Gated Sodium and Calcium Channels

    PubMed Central

    Swanson, Teresa M.

    2015-01-01

    Voltage-gated sodium channels initiate action potentials in nerve, muscle, and other electrically excitable cells. Voltage-gated calcium channels are activated by depolarization during action potentials, and calcium influx through them is the key second messenger of electrical signaling, initiating secretion, contraction, neurotransmission, gene transcription, and many other intracellular processes. Drugs that block sodium channels are used in local anesthesia and the treatment of epilepsy, bipolar disorder, chronic pain, and cardiac arrhythmia. Drugs that block calcium channels are used in the treatment of epilepsy, chronic pain, and cardiovascular disorders, including hypertension, angina pectoris, and cardiac arrhythmia. The principal pore-forming subunits of voltage-gated sodium and calcium channels are structurally related and likely to have evolved from ancestral voltage-gated sodium channels that are widely expressed in prokaryotes. Determination of the structure of a bacterial ancestor of voltage-gated sodium and calcium channels at high resolution now provides a three-dimensional view of the binding sites for drugs acting on sodium and calcium channels. In this minireview, we outline the different classes of sodium and calcium channel drugs, review studies that have identified amino acid residues that are required for their binding and therapeutic actions, and illustrate how the analogs of those key amino acid residues may form drug-binding sites in three-dimensional models derived from bacterial channels. PMID:25848093

  8. 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. PMID:17981864

  9. Mechanism of Inactivation in Voltage-Gated Na(+) Channels.

    PubMed

    Gawali, V S; Todt, H

    2016-01-01

    Voltage-gated Na(+) channels (VGSCs) initiate action potentials thereby giving rise to rapid transmission of electrical signals along cell membranes and between cells. Depolarization of the cell membrane causes VGSCs to open but also gives rise to a nonconducting state termed inactivation. Inactivation of VGSCs serves a critical physiologic function as it determines the extent of excitability of neurons and of muscle cells. Depending on the time course of development and removal of inactivation both "fast-" and "slow"-inactivated states have been described. Evidence from mutagenesis studies suggests that fast inactivation is produced by a block of the internal vestibule by a tethered inactivation particle that has been mapped to the internal linker between domains III and IV. The motion of this linker may be regulated by parts of the internal C-terminus. The molecular mechanism of slow inactivation is less clear. However, aside from a high number of mutagenesis studies, the recent availability of 3D structures of crystallized prokaryotic VGSCs offers insights into the molecular motions associated with slow inactivation. One possible scenario is that slow movements of the voltage sensors are transmitted to the external vestibule giving rise to a conformational change of this region. This molecular rearrangement is transmitted to the S6 segments giving rise to collapse of the internal vestibule. PMID:27586291

  10. 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

  11. Targeting voltage-gated calcium channels for neuropathic pain management

    PubMed Central

    Perret, Danielle; Luo, Z. David

    2009-01-01

    Voltage-gated calcium channels (VGCC) play obligatory roles in diverse physiological functions. Pathological conditions leading to changes in their biophysical properties and expression levels may cause malfunctions of VGCC mediated activities, resulting in disease states. It is believed that changes in VGCC properties under pain-inducing conditions may play a causal role in the development of chronic pain, including nerve injury-induced pain, or neuropathic pain. Over the past decades, preclinical and clinical research in developing VGCC blockers or modulators for chronic pain management has been fruitful, leading to some US Food and Drug Administration approved drugs currently available for chronic pain management. However, their efficacy in pain relief is limited in some patients and their long-term use is limited by their side effect profiles. Certainly, there is room for improvement in developing more subtype specific VGCC blockers or modulators for chronic pain conditions. In this review, we summarized the most recent preclinical and clinical studies related to chronic pain medications acting on the VGCC. We also included clinical trials aiming to expand the application of approved VGCC drugs to different pain states derived from various pathological conditions, as well as drug combination therapies trying to improve the efficacies and side effect profiles of current pain medications. PMID:19789072

  12. Crystal structure of the channelrhodopsin light-gated cation channel

    PubMed Central

    Kato, Hideaki E.; Zhang, Feng; Yizhar, Ofer; Ramakrishnan, Charu; Nishizawa, Tomohiro; Hirata, Kunio; Ito, Jumpei; Aita, Yusuke; Tsukazaki, Tomoya; Hayashi, Shigehiko; Hegemann, Peter; Maturana, Andrés D.; Ishitani, Ryuichiro; Deisseroth, Karl; Nureki, Osamu

    2014-01-01

    Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3 Å resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties. PMID:22266941

  13. Comparative Study of the Gating Motif and C-type Inactivation in Prokaryotic Voltage-gated Sodium Channels*

    PubMed Central

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

    2010-01-01

    Prokaryotic voltage-gated sodium channels (NaVs) 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 NaV, as well as of three new homologues cloned from Bacillus licheniformis (NaVBacL), Shewanella putrefaciens (NaVSheP), and Roseobacter denitrificans (NaVRosD). We found that, although activated by a lower membrane potential, NaVBacL inactivates as slowly as NaChBac. NaVSheP and NaVRosD 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 NaVs, but mutations in the regions changed the inactivation rates. Mutation of the region corresponding to the glycine hinge in NaVBacL (A214G), NaVSheP (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), NaVBacL (G224A), and NaVRosD (G217A) reduced the inactivation rate. These results imply that activation gating in prokaryotic NaVs does not require gating motifs and that the residues of helix S6 affect C-type inactivation rates in these channels. PMID:19959480

  14. Functional Characterization of a Novel Family of Acetylcholine-Gated Chloride Channels in Schistosoma mansoni

    PubMed Central

    MacDonald, Kevin; Buxton, Samuel; Kimber, Michael J.; Day, Tim A.; Robertson, Alan P.; Ribeiro, Paula

    2014-01-01

    Acetylcholine is the canonical excitatory neurotransmitter of the mammalian neuromuscular system. However, in the trematode parasite Schistosoma mansoni, cholinergic stimulation leads to muscle relaxation and a flaccid paralysis, suggesting an inhibitory mode of action. Information about the pharmacological mechanism of this inhibition is lacking. Here, we used a combination of techniques to assess the role of cholinergic receptors in schistosome motor function. The neuromuscular effects of acetylcholine are typically mediated by gated cation channels of the nicotinic receptor (nAChR) family. Bioinformatics analyses identified numerous nAChR subunits in the S. mansoni genome but, interestingly, nearly half of these subunits carried a motif normally associated with chloride-selectivity. These putative schistosome acetylcholine-gated chloride channels (SmACCs) are evolutionarily divergent from those of nematodes and form a unique clade within the larger family of nAChRs. Pharmacological and RNA interference (RNAi) behavioral screens were used to assess the role of the SmACCs in larval motor function. Treatment with antagonists produced the same effect as RNAi suppression of SmACCs; both led to a hypermotile phenotype consistent with abrogation of an inhibitory neuromuscular mediator. Antibodies were then generated against two of the SmACCs for use in immunolocalization studies. SmACC-1 and SmACC-2 localize to regions of the peripheral nervous system that innervate the body wall muscles, yet neither appears to be expressed directly on the musculature. One gene, SmACC-1, was expressed in HEK-293 cells and characterized using an iodide flux assay. The results indicate that SmACC-1 formed a functional homomeric chloride channel and was activated selectively by a panel of cholinergic agonists. The results described in this study identify a novel clade of nicotinic chloride channels that act as inhibitory modulators of schistosome neuromuscular function. Additionally, the

  15. Effector Contributions to Gβγ-mediated Signaling as Revealed by Muscarinic Potassium Channel Gating

    PubMed Central

    Ivanova-Nikolova, Tatyana T.; Breitwieser, Gerda E.

    1997-01-01

    Receptor-mediated activation of heterotrimeric G proteins leading to dissociation of the Gα subunit from Gβγ is a highly conserved signaling strategy used by numerous extracellular stimuli. Although Gβγ subunits regulate a variety of effectors, including kinases, cyclases, phospholipases, and ion channels (Clapham, D.E., and E.J. Neer. 1993. Nature (Lond.). 365:403–406), few tools exist for probing instantaneous Gβγ-effector interactions, and little is known about the kinetic contributions of effectors to the signaling process. In this study, we used the atrial muscarinic K+ channel, which is activated by direct interactions with Gβγ subunits (Logothetis, D.E., Y. Kurachi, J. Galper, E.J. Neer, and D.E. Clap. 1987. Nature (Lond.). 325:321–326; Wickman, K., J.A. Iniguez-Liuhi, P.A. Davenport, R. Taussig, G.B. Krapivinsky, M.E. Linder, A.G. Gilman, and D.E. Clapham. 1994. Nature (Lond.). 366: 654–663; Huang, C.-L., P.A. Slesinger, P.J. Casey, Y.N. Jan, and L.Y. Jan. 1995. Neuron. 15:1133–1143), as a sensitive reporter of the dynamics of Gβγ-effector interactions. Muscarinic K+ channels exhibit bursting behavior upon G protein activation, shifting between three distinct functional modes, characterized by the frequency of channel openings during individual bursts. Acetylcholine concentration (and by inference, the concentration of activated Gβγ) controls the fraction of time spent in each mode without changing either the burst duration or channel gating within individual modes. The picture which emerges is of a Gβγ effector with allosteric regulation and an intrinsic “off” switch which serves to limit its own activation. These two features combine to establish exquisite channel sensitivity to changes in Gβγ concentration, and may be indicative of the factors regulating other Gβγ-modulated effectors. PMID:9041452

  16. Ion Selectivity Mechanism in a Bacterial Pentameric Ligand-Gated Ion Channel

    SciTech Connect

    Fritsch, Sebastian; Ivanov, Ivaylo; Wang, Hailong; Cheng, Xiaolin

    2010-01-01

    The proton-gated ion channel from Gloeobacter violaceus (GLIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. Recent emergence of a high-resolution crystal structure of GLIC captured in a potentially open state allowed detailed, atomic-level insight into ion conduction and selectivity mechanisms in these channels. Herein, we have examined the barriers to ion conduction and origins of ion selectivity in the GLIC channel by the construction of potential-of-mean-force profiles for sodium and chloride ions inside the transmembrane region. Our calculations reveal that the GLIC channel is open for a sodium ion to transport, but presents a 11 kcal/mol free energy barrier for a chloride ion. Our collective findings identify three distinct contributions to the observed preference for the permeant ions. First, there is a substantial contribution due to a ring of negatively charged glutamate residues (E-2 ) at the narrow intracellular end of the channel. The negative electrostatics of this region and the ability of the glutamate side chains to directly bind cations would strongly favor the passage of sodium ions while hindering translocation of chloride ions. Second, our results imply a significant hydrophobic contribution to selectivity linked to differences in the desolvation penalty for the sodium versus chloride ions in the central hydrophobic region of the pore. This hydrophobic contribution is evidenced by the large free energy barriers experienced by Cl in the middle of the pore for both GLIC and the E-2 A mutant. Finally, there is a distinct contribution arising from the overall negative electrostatics of the channel.

  17. Membrane mechanics as a probe of ion-channel gating mechanisms

    PubMed Central

    Reeves, Daniel; Ursell, Tristan; Sens, Pierre; Kondev, Jane; Phillips, Rob

    2012-01-01

    The details of conformational changes undergone by transmembrane ion channels in response to stimuli, such as electric fields and membrane tension, remain controversial. We approach this problem by considering how the conformational changes impose deformations in the lipid bilayer. We focus on the role of bilayer deformations in the context of voltage-gated channels because we hypothesize that such deformations are relevant in this case as well as for channels that are explicitly mechanosensitive. As a result of protein conformational changes, we predict that the lipid bilayer suffers deformations with a characteristic free-energy scale of 10kBT. This free energy is comparable to the voltage-dependent part of the total gating energy, and we argue that these deformations could play an important role in the overall free-energy budget of gating. As a result, channel activity will depend upon mechanical membrane parameters such as tension and leaflet thickness. We further argue that the membrane deformation around any channel can be divided into three generic classes of deformation that exhibit different mechanosensitive properties. Finally, we provide the theoretical framework that relates conformational changes during gating to tension and leaflet thickness dependence in the critical gating voltage. This line of investigation suggests experiments that could discern the dominant deformation imposed upon the membrane as a result of channel gating, thus providing clues as to the channel deformation induced by the stimulus. PMID:18999449

  18. A Dipeptidyl Aminopeptidase–like Protein Remodels Gating Charge Dynamics in Kv4.2 Channels

    PubMed Central

    Dougherty, Kevin; Covarrubias, Manuel

    2006-01-01

    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. PMID:17130523

  19. Structural basis for the inhibition of voltage-dependent K+ channel by gating modifier toxin

    PubMed Central

    Ozawa, Shin-ichiro; Kimura, Tomomi; Nozaki, Tomohiro; Harada, Hitomi; Shimada, Ichio; Osawa, Masanori

    2015-01-01

    Voltage-dependent K+ (Kv) channels play crucial roles in nerve and muscle action potentials. Voltage-sensing domains (VSDs) of Kv channels sense changes in the transmembrane potential, regulating the K+-permeability across the membrane. Gating modifier toxins, which have been used for the functional analyses of Kv channels, inhibit Kv channels by binding to VSD. However, the structural basis for the inhibition remains elusive. Here, fluorescence and NMR analyses of the interaction between VSD derived from KvAP channel and its gating modifier toxin, VSTx1, indicate that VSTx1 recognizes VSD under depolarized condition. We identified the VSD-binding residues of VSTx1 and their proximal residues of VSD by the cross-saturation (CS) and amino acid selective CS experiments, which enabled to build a docking model of the complex. These results provide structural basis for the specific binding and inhibition of Kv channels by gating modifier toxins. PMID:26382304

  20. The transient receptor potential family of ion channels.

    PubMed

    Nilius, Bernd; Owsianik, Grzegorz

    2011-01-01

    The transient receptor potential (TRP) multigene superfamily encodes integral membrane proteins that function as ion channels. Members of this family are conserved in yeast, invertebrates and vertebrates. The TRP family is subdivided into seven subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin) and TRPN (NOMPC-like); the latter is found only in invertebrates and fish. TRP ion channels are widely expressed in many different tissues and cell types, where they are involved in diverse physiological processes, such as sensation of different stimuli or ion homeostasis. Most TRPs are non-selective cation channels, only few are highly Ca2+ selective, some are even permeable for highly hydrated Mg2+ ions. This channel family shows a variety of gating mechanisms, with modes of activation ranging from ligand binding, voltage and changes in temperature to covalent modifications of nucleophilic residues. Activated TRP channels cause depolarization of the cellular membrane, which in turn activates voltage-dependent ion channels, resulting in a change of intracellular Ca2+ concentration; they serve as gatekeeper for transcellular transport of several cations (such as Ca2+ and Mg2+), and are required for the function of intracellular organelles (such as endosomes and lysosomes). Because of their function as intracellular Ca2+ release channels, they have an important regulatory role in cellular organelles. Mutations in several TRP genes have been implicated in diverse pathological states, including neurodegenerative disorders, skeletal dysplasia, kidney disorders and pain, and ongoing research may help find new therapies for treatments of related diseases. PMID:21401968

  1. The transient receptor potential family of ion channels

    PubMed Central

    2011-01-01

    Summary The transient receptor potential (TRP) multigene superfamily encodes integral membrane proteins that function as ion channels. Members of this family are conserved in yeast, invertebrates and vertebrates. The TRP family is subdivided into seven subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin) and TRPN (NOMPC-like); the latter is found only in invertebrates and fish. TRP ion channels are widely expressed in many different tissues and cell types, where they are involved in diverse physiological processes, such as sensation of different stimuli or ion homeostasis. Most TRPs are non-selective cation channels, only few are highly Ca2+ selective, some are even permeable for highly hydrated Mg2+ ions. This channel family shows a variety of gating mechanisms, with modes of activation ranging from ligand binding, voltage and changes in temperature to covalent modifications of nucleophilic residues. Activated TRP channels cause depolarization of the cellular membrane, which in turn activates voltage-dependent ion channels, resulting in a change of intracellular Ca2+ concentration; they serve as gatekeeper for transcellular transport of several cations (such as Ca2+ and Mg2+), and are required for the function of intracellular organelles (such as endosomes and lysosomes). Because of their function as intracellular Ca2+ release channels, they have an important regulatory role in cellular organelles. Mutations in several TRP genes have been implicated in diverse pathological states, including neurodegenerative disorders, skeletal dysplasia, kidney disorders and pain, and ongoing research may help find new therapies for treatments of related diseases. PMID:21401968

  2. On Application Of Langevin Dynamics In Logarithmic Potential To Model Ion Channel Gate Activity.

    PubMed

    Wawrzkiewicz-Jałowiecka, Agata; Borys, Przemysław; Grzywna, Zbigniew J

    2015-12-01

    We model the activity of an ion channel gate by Langevin dynamics in a logarithmic potential. This approach enables one to describe the power-law dwell-time distributions of the considered system, and the long-term correlations between the durations of the subsequent channel states, or fractal scaling of statistical characteristics of the gate's movement with time. Activity of an ion channel gate is described as an overdamped motion of the reaction coordinate in a confining logarithmic potential, which ensures great flexibility of the model. Depending on the chosen parameters, it allows one to reproduce many types of gate dynamics within the family of non-Markovian, anomalous conformational diffusion processes. In this study we apply the constructed model to largeconductance voltage and Ca2+-activated potassium channels (BKCa). The interpretation of model assumptions and parameters is provided in terms of this biological system. Our results show good agreement with the experimental data. PMID:26317442

  3. Role of Cyclic Nucleotide Gated Channels in Stress Management in Plants.

    PubMed

    Jha, Saroj K; Sharma, Manisha; Pandey, Girdhar K

    2016-08-01

    Tolerance of plants to a number of biotic and abiotic stresses such as pathogen and herbivore attack, drought, salinity, cold and nutritional limitations is ensued by complex multimodule signaling pathways. The outcome of this complex signaling pathways results in adaptive responses by restoring the cellular homeostasis and thus promoting survival. Functions of many plant cation transporter and channel protein families such as glutamate receptor homologs (GLRs), cyclic nucleotide-gated ion channel (CNGC) have been implicated in providing biotic and abiotic stress tolerance. Ion homeostasis regulated by several transporters and channels is one of the crucial parameters for the optimal growth, development and survival of all living organisms. The CNGC family members are known to be involved in the uptake of cations such as Na(+), K(+) and Ca(2+) and regulate plant growth and development. Detail functional genomics approaches have given an emerging picture of CNGCs wherein these protein are believed to play crucial role in pathways related to cellular ion homeostasis, development and as a 'guard' in defense against biotic and abiotic challenges. Here, we discuss the current knowledge of role of CNGCs in mediating stress management and how they aid plants in survival under adverse conditions. PMID:27499681

  4. Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic.

    PubMed

    Galzi, J L; Devillers-Thiéry, A; Hussy, N; Bertrand, S; Changeux, J P; Bertrand, D

    1992-10-01

    Introduction by site-directed mutagenesis of three amino acids from the MII segment of glycine or gamma-aminobutyric acid (GABAA) receptors into the MII segment of alpha 7 nicotinic receptor was sufficient to convert a cation-selective channel into an anion-selective channel gated by acetylcholine. A critical mutation was the insertion of an uncharged residue at the amino-terminal end of MII, stressing the importance of protein geometrical constraints on ion selectivity. PMID:1383829

  5. AKAP150-dependent cooperative TRPV4 channel gating is central to endothelium-dependent vasodilation and is disrupted in hypertension

    PubMed Central

    Sonkusare, Swapnil K.; Dalsgaard, Thomas; Bonev, Adrian D.; Hill-Eubanks, David C.; Kotlikoff, Michael I.; Scott, John D.; Santana, Luis F.; Nelson, Mark T.

    2015-01-01

    Endothelial cell dysfunction, characterized by a diminished response to endothelial cell–dependent vasodilators, is a hallmark of hypertension. TRPV4 channels play a major role in endothelial-dependent vaso-dilation, a function mediated by local Ca2+ influx through clusters of functionally coupled TRPV4 channels rather than by a global increase in endothelial cell Ca2+. We showed that stimulation of muscarinic acetylcholine receptors on endothelial cells of mouse arteries exclusively activated TRPV4 channels that were localized at myoendothelial projections (MEPs), specialized regions of endothelial cells that contact smooth muscle cells. Muscarinic receptor–mediated activation of TRPV4 depended on protein kinase C (PKC) and the PKC-anchoring protein AKAP150, which was concentrated at MEPs. Cooperative opening of clustered TRPV4 channels specifically amplified Ca2+ influx at MEPs. Cooperativity of TRPV4 channels at non-MEP sites was much lower, and cooperativity at MEPs was greatly reduced by chelation of intracellular Ca2+ or AKAP150 knockout, suggesting that Ca2+ entering through adjacent channels underlies the AKAP150-dependent potentiation of TRPV4 activity. In a mouse model of angiotensin II–induced hypertension, MEP localization of AKAP150 was disrupted, muscarinic receptor stimulation did not activate TRPV4 channels, cooperativity among TRPV4 channels at MEPs was weaker, and vasodilation in response to muscarinic receptor stimulation was reduced. Thus, endothelial-dependent dilation of resistance arteries is enabled by MEP-localized AKAP150, which ensures the proximity of PKC to TRPV4 channels and the coupled channel gating necessary for efficient communication from endothelial to smooth muscle cells in arteries. Disruption of this molecular assembly may contribute to altered blood flow in hypertension. PMID:25005230

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

    PubMed Central

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

    2015-01-01

    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. PMID:25296861

  8. Permeation and interaction of monovalent cations with the cGMP-gated channel of cone photoreceptors.

    PubMed

    Picones, A; Korenbrot, J I

    1992-10-01

    energy barriers are asymmetrically located within the membrane thickness. Comparison of the quantitative features of ion permeation and interaction between the cGMP-gated channels of rod and cone photoreceptors reveals that the ion binding sites are profoundly different in the two types of channels. This molecular difference may be particularly important in explaining the differences in the transduction signal of each receptor type. PMID:1334122

  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. Voltage-Gated Channel Mechanosensitivity: Fact or Friction?

    PubMed Central

    Morris, Catherine E.

    2011-01-01

    The heart is a continually active pulsatile fluid pump. It generates appropriate forces by precisely timed and spaced engagement of its contractile machinery. Largely, it makes its own control signals, the most crucial of which are precisely timed and spaced fluxes of ions across the sarcolemma, achieved by the timely opening and closing of diverse voltage-gated channels (VGC). VGCs have four voltage sensors around a central ion-selective pore that opens and closes under the influence of membrane voltage. Operation of any VGC is secondarily tuned by the mechanical state (i.e., structure) of the bilayer in which it is embedded. Rates of opening and closing, in other words, vary with bilayer structure. Thus, in the intensely mechanical environment of the myocardium and its vasculature, VGCs kinetics might be routinely modulated by reversible and irreversible nano-scale changes in bilayer structure. If subtle bilayer deformations are routine in the pumping heart, VGCs could be subtly transducing bilayer mechanical signals, thereby tuning cardiac rhythmicity, collectively contributing to mechano-electric feedback. Reversible bilayer deformations would be expected with changing shear flows and tissue distension, while irreversible bilayer restructuring occurs with ischemia, inflammation, membrane remodeling, etc. I suggest that tools now available could be deployed to help probe whether/how the inherent mechanosensitivity of VGCs – an attribute substantially reflecting the dependence of voltage sensor stability on bilayer structure – contributes to cardiac rhythmicity. Chief among these tools are voltage sensor toxins (whose inhibitory efficacy varies with the mechanical state of bilayer) and arrhythmia-inducing VGC mutants with distinctive mechano-phenotypes. PMID:21660289

  11. ATP-independent CFTR channel gating and allosteric modulation by phosphorylation

    PubMed Central

    Wang, Wei; Wu, Jianping; Bernard, Karen; Li, Ge; Wang, Guangyu; Bevensee, Mark O.; Kirk, Kevin L.

    2010-01-01

    Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligand-gated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization. PMID:20133716

  12. Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme.

    PubMed

    Zeltwanger, S; Wang, F; Wang, G T; Gillis, K D; Hwang, T C

    1999-04-01

    Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant (tauc) decreased with increasing [ATP] to a minimum value of approximately 0.43 s at [ATP] >1.00 mM. The open time constant (tauo) increased with increasing [ATP] with a minimal tauo of approximately 260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of approximately 250 ms is dominant at low ATP concentrations (10 microM) and a much longer open state with a time constant of approximately 3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent. PMID:10102935

  13. Odorant receptors activated by amino acids in sensory neurons of the channel catfish Ictalurus punctatus.

    PubMed

    Ivanova, T T; Caprio, J

    1993-12-01

    Odorant receptors activated by amino acids were investigated with patch-clamp techniques in olfactory receptor neurons of the channel catfish, Ictalurus punctatus. The L-isomers of alanine, norvaline, arginine, and glutamate, known to act predominantly on different olfactory receptor sites, activated nondesensitizing inward currents with amplitudes of -2.5 to -280 pA in olfactory neurons voltage-clamped at membrane potentials of -72 or -82 mV. Different amino acids were shown to induce responses in the same sensory neurons; however, the amplitude and the kinetics of the observed whole cell currents differed among the stimuli and may therefore reflect activation of different amino acid receptor types or combinations of receptor types in these cells. Amino acid-induced currents appeared to have diverse voltage dependence and could also be classified according to the amplitude of the spontaneous channel fluctuations underlying the macroscopic currents. A mean single-channel conductance (gamma) of 360 fS was estimated from small noise whole-cell currents evoked by arginine within the same olfactory neuron in which a mean gamma value of 23.6 pS was estimated from 'large noise' response to norvaline. Quiescent olfactory neurons fired bursts of action potentials in response to either amino acid stimulation or application of 8-Br-cyclic GMP (100 microM), and voltage-gated channels underlying generation of action potentials were similar in these neurons. However, in whole-cell voltage-clamp, 8-Br-cyclic GMP evoked large rectangular current pulses, and single-channel conductances of 275, 220, and 110 pS were obtained from the discrete current levels. These results suggest that in addition to the cyclic nucleotide-gated transduction channels, olfactory neurons of the channel catfish possess a variety of odor receptors coupled to different types of transduction channels. PMID:8133240

  14. Activation and Regulation of Purinergic P2X Receptor Channels

    PubMed Central

    Coddou, Claudio; Yan, Zonghe; Obsil, Tomas; Huidobro-Toro, J. Pablo

    2011-01-01

    Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions. PMID:21737531

  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. A combined coarse-grained and all-atom simulation of TRPV1 channel gating and heat activation

    PubMed Central

    Qin, Feng

    2015-01-01

    The transient receptor potential (TRP) channels act as key sensors of various chemical and physical stimuli in eukaryotic cells. Despite years of study, the molecular mechanisms of TRP channel activation remain unclear. To elucidate the structural, dynamic, and energetic basis of gating in TRPV1 (a founding member of the TRPV subfamily), we performed coarse-grained modeling and all-atom molecular dynamics (MD) simulation based on the recently solved high resolution structures of the open and closed form of TRPV1. Our coarse-grained normal mode analysis captures two key modes of collective motions involved in the TRPV1 gating transition, featuring a quaternary twist motion of the transmembrane domains (TMDs) relative to the intracellular domains (ICDs). Our transition pathway modeling predicts a sequence of structural movements that propagate from the ICDs to the TMDs via key interface domains (including the membrane proximal domain and the C-terminal domain), leading to sequential opening of the selectivity filter followed by the lower gate in the channel pore (confirmed by modeling conformational changes induced by the activation of ICDs). The above findings of coarse-grained modeling are robust to perturbation by lipids. Finally, our MD simulation of the ICD identifies key residues that contribute differently to the nonpolar energy of the open and closed state, and these residues are predicted to control the temperature sensitivity of TRPV1 gating. These computational predictions offer new insights to the mechanism for heat activation of TRPV1 gating, and will guide our future electrophysiology and mutagenesis studies. PMID:25918362

  17. Molecular Mapping of General Anesthetic Sites in a Voltage-Gated Ion Channel

    PubMed Central

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

    2011-01-01

    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. PMID:21961587

  18. Zinc inhibition of monomeric and dimeric proton channels suggests cooperative gating

    PubMed Central

    Musset, Boris; Smith, Susan M E; Rajan, Sindhu; Cherny, Vladimir V; Sujai, Sukrutha; Morgan, Deri; DeCoursey, Thomas E

    2010-01-01

    Voltage-gated proton channels are strongly inhibited by Zn2+, which binds to His residues. However, in a molecular model, the two externally accessible His are too far apart to coordinate Zn2+. We hypothesize that high-affinity Zn2+ binding occurs at the dimer interface between pairs of His residues from both monomers. Consistent with this idea, Zn2+ effects were weaker in monomeric channels. Mutation of His193 and His140 in various combinations and in tandem dimers revealed that channel opening was slowed by Zn2+ only when at least one His was present in each monomer, suggesting that in wild-type (WT) HV1, Zn2+ binding between His of both monomers inhibits channel opening. In addition, monomeric channels opened exponentially, and dimeric channels opened sigmoidally. Monomeric channel gating had weaker temperature dependence than dimeric channels. Finally, monomeric channels opened 6.6 times faster than dimeric channels. Together, these observations suggest that in the proton channel dimer, the two monomers are closely apposed and interact during a cooperative gating process. Zn2+ appears to slow opening by preventing movement of the monomers relative to each other that is prerequisite to opening. These data also suggest that the association of the monomers is tenuous and allows substantial freedom of movement. The data support the idea that native proton channels are dimeric. Finally, the idea that monomer–dimer interconversion occurs during activation of phagocytes appears to be ruled out. PMID:20231140

  19. Role of CFTR's intrinsic adenylate kinase activity in gating of the Cl(-) channel.

    PubMed

    Randak, Christoph O; Welsh, Michael J

    2007-12-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)channel in the ATP-binding cassette (ABC) transporter protein family. CFTR features the modular design characteristic of ABC transporters, which includes two membrane-spanning domains forming the channel pore, and two ABC nucleotide-binding domains that interact with ATP and contain the enzymatic activity coupled to normal gating. Like other ABC transporters CFTR is an ATPase (ATP + H(2)O --> ADP + Pi). Recent work has shown that CFTR also possesses intrinsic adenylate kinase activity (ATP + AMP left arrow over right arrow ADP + ADP). This finding raises important questions: How does AMP influence CFTR gating? Why does ADP inhibit CFTR current? Which enzymatic activity gates CFTR in vivo? Are there implications for other ABC transporters? This minireview attempts to shed light on these questions by summarizing recent advances in our understanding of the role of the CFTR adenylate kinase activity for channel gating. PMID:17965924

  20. Trafficking Mechanisms Underlying Neuronal Voltage-gated Ion Channel Localization at the Axon Initial Segment

    PubMed Central

    Vacher, Helene; Trimmer, James S.

    2012-01-01

    Summary Voltage-gated ion channels are diverse and fundamental determinants of neuronal intrinsic excitability. Voltage-gated K+ (Kv) and Na+ (Nav) channels play complex yet fundamentally important roles in determining intrinsic excitability. The Kv and Nav channels located at the axon initial segment (AIS) play a unique and especially important role in generating neuronal output in the form of anterograde axonal and backpropagating action potentials, Aberrant intrinsic excitability in individual neurons within networks contributes to synchronous neuronal activity leading to seizures. Mutations in ion channel genes gives rise to a variety of seizure-related “Channelopathies”, and many of the ion channel subunits associated with epilepsy mutations are localized at the AIS, making this a hotspot for epileptogenesis. Here we review the cellular mechanisms that underlie the trafficking of Kv and Nav channels found at the AIS, and how Kv and Nav channel mutations associated with epilepsy can alter these processes. PMID:23216576

  1. 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

  2. Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor.

    PubMed

    Revah, F; Bertrand, D; Galzi, J L; Devillers-Thiéry, A; Mulle, C; Hussy, N; Bertrand, S; Ballivet, M; Changeux, J P

    1991-10-31

    A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor. PMID:1719423

  3. 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

  4. 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.

  5. Ion Selectivity Mechanism in a Bacterial Pentameric Ligand-Gated Ion Channel

    SciTech Connect

    Fritsch, Sebastian M; Ivanov, Ivaylo N; Wang, Hailong; Cheng, Xiaolin

    2011-01-01

    The proton-gated ion channel from Gloeobacter violaceus (GLIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor (nAChR) that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. Recent emergence of a high resolution crystal structure of GLIC captured in a potentially open state allowed detailed, atomic-level insight into ion conduction and selectivity mechanisms in these channels. Herein, we have examined the barriers to ion conduction and origins of ion selectivity in the GLIC channel by the construction of potential of mean force (PMF) profiles for sodium and chloride ions inside the transmembrane region. Our calculations reveal that the GLIC channel is open for a sodium ion to transport, but presents a ~10 kcal/mol free energy barrier for a chloride ion, which arises primarily from the unfavorable interactions with a ring of negatively charged glutamate residues (E-2 ) at the intracellular end and a ring of hydrophobic residues (I9 ) in the middle of the transmembrane domain. Our collective findings further suggest that the charge selection mechanism can, to a large extent, be attributed to the narrow intracellular end and a ring of glutamate residues in this position their strong negative electrostatics and ability to bind cations. By contrast, E19 at the extracellular entrance only plays a minor role in ion selectivity of GLIC. In addition to electrostatics, both ion hydration and protein dynamics are found to be crucial for ion conduction as well, which explains why a chloride ion experiences a much greater barrier than a sodium ion in the hydrophobic region of the pore.

  6. Molecular dissection of gating in the ClC-2 chloride channel.

    PubMed

    Jordt, S E; Jentsch, T J

    1997-04-01

    The ClC-2 chloride channel is probably involved in the regulation of cell volume and of neuronal excitability. Site-directed mutagenesis was used to understand ClC-2 activation in response to cell swelling, hyperpolarization and acidic extracellular pH. Similar to equivalent mutations in ClC-0, neutralizing Lys566 at the end of the transmembrane domains results in outward rectification and a shift in voltage dependence, but leaves the basic gating mechanism, including swelling activation, intact. In contrast, mutations in the cytoplasmic loop between transmembrane domains D7 and D8 abolish all three modes of activation by constitutively opening the channel without changing its pore properties. These effects resemble those observed with deletions of an amino-terminal inactivation domain, and suggest that it may act as its receptor. Such a 'ball-and-chain' type mechanism may act as a final pathway in the activation of ClC-2 elicited by several stimuli. PMID:9130703

  7. Proton-gated Ca2+-permeable TRP channels damage myelin in conditions mimicking ischaemia

    PubMed Central

    Hamilton, Nicola B.; Kolodziejczyk, Karolina; Kougioumtzidou, Eleni; Attwell, David

    2015-01-01

    The myelin sheaths wrapped around axons by oligodendrocytes are crucial for brain function. In ischaemia myelin is damaged in a Ca2+-dependent manner, abolishing action potential propagation1,2. This has been attributed to glutamate release activating Ca2+-permeable NMDA receptors2-4. Surprisingly, we now show that NMDA does not raise [Ca2+]i in mature oligodendrocytes and that, although ischaemia evokes a glutamate-triggered membrane current4, this is generated by a rise of extracellular [K+] and decrease of membrane K+ conductance. Nevertheless, ischaemia raises oligodendrocyte [Ca2+]i, [Mg2+]i and [H+]i, and buffering intracellular pH reduces the [Ca2+]i and [Mg2+]i increases, showing that these are evoked by the rise of [H+]i. The H+-gated [Ca2+]i elevation is mediated by channels with characteristics of TRPA1, being inhibited by ruthenium red, isopentenyl pyrophosphate, HC-030031, A967079 or TRPA1 knockout. TRPA1 block reduces myelin damage in ischaemia. These data suggest TRPA1-containing ion channels as a therapeutic target in white matter ischaemia. PMID:26760212

  8. Selective expression of ligand-gated ion channels in L5 pyramidal cell axons

    PubMed Central

    Christie, Jason M.; Jahr, Craig E.

    2009-01-01

    NMDA receptor (NMDAR)-dependent strengthening of neurotransmitter release has been widely observed, including in layer 5 (L5) pyramidal cells of the visual cortex, and is attributed to the axonal expression of NMDARs. However, we failed to detect NMDAR-mediated depolarizations or Ca2+ entry in L5 pyramidal cell axons when focally stimulated with NMDAR agonists. This suggests that NMDARs are excluded from the axon. In contrast, local GABAAR activation alters axonal excitability indicating that exclusion of ligand-gated ion channels from the axon is not absolute. Because NMDARs are restricted to the dendrite, NMDARs must signal to the axon by an indirect mechanism to alter release. Although subthreshold somatic depolarizations were found to spread electrotonically hundreds of micrometers through the axon, the resulting axonal potential was insufficient to open voltage-sensitive Ca2+ channels (VSCCs). Therefore, if NMDAR-mediated facilitation of release is cell-autonomous, it may depend on voltage signaling but apparently is independent of changes in basal Ca2+. Alternatively, this facilitation may be even less direct, requiring a cascade of events that are merely triggered by NMDAR activation. PMID:19759293

  9. Structural basis of ion permeation gating in Slo2.1 K+ channels

    PubMed Central

    Garg, Priyanka; Gardner, Alison; Garg, Vivek

    2013-01-01

    The activation gate of ion channels controls the transmembrane flux of permeant ions. In voltage-gated K+ channels, the aperture formed by the S6 bundle crossing can widen to open or narrow to close the ion permeation pathway, whereas the selectivity filter gates ion flux in cyclic-nucleotide gated (CNG) and Slo1 channels. Here we explore the structural basis of the activation gate for Slo2.1, a weakly voltage-dependent K+ channel that is activated by intracellular Na+ and Cl−. Slo2.1 channels were heterologously expressed in Xenopus laevis oocytes and activated by elevated [NaCl]i or extracellular application of niflumic acid. In contrast to other voltage-gated channels, Slo2.1 was blocked by verapamil in an activation-independent manner, implying that the S6 bundle crossing does not gate the access of verapamil to its central cavity binding site. The structural basis of Slo2.1 activation was probed by Ala scanning mutagenesis of the S6 segment and by mutation of selected residues in the pore helix and S5 segment. Mutation to Ala of three S6 residues caused reduced trafficking of channels to the cell surface and partial (K256A, I263A, Q273A) or complete loss (E275A) of channel function. P271A Slo2.1 channels trafficked normally, but were nonfunctional. Further mutagenesis and intragenic rescue by second site mutations suggest that Pro271 and Glu275 maintain the inner pore in an open configuration by preventing formation of a tight S6 bundle crossing. Mutation of several residues in S6 and S5 predicted by homology modeling to contact residues in the pore helix induced a gain of channel function. Substitution of the pore helix residue Phe240 with polar residues induced constitutive channel activation. Together these findings suggest that (1) the selectivity filter and not the bundle crossing gates ion permeation and (2) dynamic coupling between the pore helix and the S5 and S6 segments mediates Slo2.1 channel activation. PMID:24166878

  10. Progesterone Inhibition of Voltage-Gated Calcium Channels is a Potential Neuroprotective Mechanism against Excitotoxicity

    PubMed Central

    Luoma, Jessie I; Kelley, Brooke G; Mermelstein, Paul G

    2011-01-01

    The therapeutic use of progesterone following traumatic brain injury has recently entered phase III clinical trials as a means of neuroprotection. Although it has been hypothesized that progesterone protects against calcium overload following excitotoxic shock, the exact mechanisms underlying the beneficial effects of progesterone have yet to be determined. We found that therapeutic concentrations of progesterone to be neuroprotective against depolarization-induced excitotoxicity in cultured striatal neurons. Through use of calcium imaging, electrophysiology and the measurement of changes in activity-dependent gene expression, progesterone was found to block calcium entry through voltage-gated calcium channels, leading to alterations in the signaling of the activity-dependent transcription factors NFAT and CREB. The effects of progesterone were highly specific to this steroid hormone, although they did not appear to be receptor mediated. In addition, progesterone did not inhibit AMPA or NMDA receptor signaling. This analysis regarding the effect of progesterone on calcium signaling provides both a putative mechanism by which progesterone acts as a neuroprotectant, as well as affords a greater appreciation for its potential far-reaching effects on cellular function. PMID:21371490

  11. The Molecular Mechanism of Opening the Helix Bundle Crossing (HBC) Gate of a Kir Channel.

    PubMed

    Meng, Xuan-Yu; Liu, Shengtang; Cui, Meng; Zhou, Ruhong; Logothetis, Diomedes E

    2016-01-01

    Inwardly rectifying K(+) (Kir) channels, serving as natural molecular nanomachines, transport potassium ions across the plasma membrane of the cell. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter (SF), the inner helix bundle crossing (HBC), and the cytosolic G loop) may serve as gates to control ion permeation. Our previous molecular dynamics simulations based on the crystal structure of a Kir3.1 chimera revealed the possible gating mechanism of the G loop gate. Here, we introduced a proline mutation in the inner helix and obtained a channel model of the open HBC gate. The open HBC gate reaches 0.6 nm in diameter, which allows partial hydrated K(+) ions to pass through. During the gating process, both the transmembrane helices TM1 and TM2 cooperatively rotate in a counterclockwise direction (viewed from the extracellular side) with the aid of the phospholipid PIP2. Only when all the transmembrane helices adopt a counterclockwise rotation, the HBC gate can be stabilized in the open state. We estimate that introduction of the proline mutation decreases the energy required to open the HBC gate by about 1.4 kcal/mol (ΔΔG). PMID:27439597

  12. The Molecular Mechanism of Opening the Helix Bundle Crossing (HBC) Gate of a Kir Channel

    PubMed Central

    Meng, Xuan-Yu; Liu, Shengtang; Cui, Meng; Zhou, Ruhong; Logothetis, Diomedes E.

    2016-01-01

    Inwardly rectifying K+ (Kir) channels, serving as natural molecular nanomachines, transport potassium ions across the plasma membrane of the cell. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter (SF), the inner helix bundle crossing (HBC), and the cytosolic G loop) may serve as gates to control ion permeation. Our previous molecular dynamics simulations based on the crystal structure of a Kir3.1 chimera revealed the possible gating mechanism of the G loop gate. Here, we introduced a proline mutation in the inner helix and obtained a channel model of the open HBC gate. The open HBC gate reaches 0.6 nm in diameter, which allows partial hydrated K+ ions to pass through. During the gating process, both the transmembrane helices TM1 and TM2 cooperatively rotate in a counterclockwise direction (viewed from the extracellular side) with the aid of the phospholipid PIP2. Only when all the transmembrane helices adopt a counterclockwise rotation, the HBC gate can be stabilized in the open state. We estimate that introduction of the proline mutation decreases the energy required to open the HBC gate by about 1.4 kcal/mol (ΔΔG). PMID:27439597

  13. 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

  14. 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. PMID:26712848

  15. 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

  16. 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

    2014-09-01

    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. PMID:25185546

  17. GlialCAM, a CLC-2 Cl- Channel Subunit, Activates the Slow Gate of CLC Chloride Channels

    PubMed Central

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

    2014-01-01

    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. PMID:25185546

  18. Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis.

    PubMed

    Gao, Qi-Fei; Gu, Li-Li; Wang, Hui-Qin; Fei, Cui-Fang; Fang, Xiang; Hussain, Jamshaid; Sun, Shu-Jing; Dong, Jing-Yun; Liu, Hongtao; Wang, Yong-Fei

    2016-03-15

    In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca(2+) gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca(2+) channels in pollen tube tips are core components that regulate Ca(2+) gradients by mediating and regulating external Ca(2+) influx. Therefore, Ca(2+) channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca(2+) channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca(2+) gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/-) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18's transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca(2+) channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca(2+) channel for pollen tube guidance in Arabidopsis. PMID:26929345

  19. Overview of the voltage-gated sodium channel family

    PubMed Central

    Yu, Frank H; Catterall, William A

    2003-01-01

    Selective permeation of sodium ions through voltage-dependent sodium channels is fundamental to the generation of action potentials in excitable cells such as neurons. These channels are large integral membrane proteins and are encoded by at least ten genes in mammals. The different sodium channels have remarkably similar functional properties, but small changes in sodium-channel function are biologically relevant, as underscored by mutations that cause several human diseases of hyperexcitability. PMID:12620097

  20. The non-hydrolytic pathway of cystic fibrosis transmembrane conductance regulator ion channel gating.

    PubMed

    Aleksandrov, A A; Chang, X; Aleksandrov, L; Riordan, J R

    2000-10-15

    It has been suggested that the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel may utilize a novel gating mechanism in which open and closed states are not in thermodynamic equilibrium. This suggestion is based on the assumption that energy of ATP hydrolysis drives the gating cycle. We demonstrate that CFTR channel gating occurs in the absence of ATP hydrolysis and hence does not depend on an input of free energy from this source. The binding of ATP or structurally related analogues that are poorly or non-hydrolysable is sufficient to induce opening. Closing occurs on dissociation of these ligands or the hydrolysis products of those that can be cleaved. Not only can channel opening occur without ATP hydrolysis but the temperature dependence of the open probability (Po.) is reversed, i.e. Po. increases as temperature is lowered whereas under hydrolytic conditions, Po. increases as temperature is elevated. This indicates that there are different rate-limiting steps in the alternate gating pathways (hydrolytic and non-hydrolytic). These observations demonstrate that phosphorylated CFTR behaves as a conventional ligand-gated channel employing cytoplasmic ATP as a readily available cytoplasmic ligand; under physiological conditions ligand hydrolysis provides efficient reversibility of channel opening. PMID:11034616

  1. The non-hydrolytic pathway of cystic fibrosis transmembrane conductance regulator ion channel gating

    PubMed Central

    Aleksandrov, Andrei A; Chang, Xiu-bao; Aleksandrov, Luba; Riordan, John R

    2000-01-01

    It has been suggested that the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel may utilize a novel gating mechanism in which open and closed states are not in thermodynamic equilibrium. This suggestion is based on the assumption that energy of ATP hydrolysis drives the gating cycle. We demonstrate that CFTR channel gating occurs in the absence of ATP hydrolysis and hence does not depend on an input of free energy from this source. The binding of ATP or structurally related analogues that are poorly or non-hydrolysable is sufficient to induce opening. Closing occurs on dissociation of these ligands or the hydrolysis products of those that can be cleaved. Not only can channel opening occur without ATP hydrolysis but the temperature dependence of the open probability (Po) is reversed, i.e. Po increases as temperature is lowered whereas under hydrolytic conditions, Po increases as temperature is elevated. This indicates that there are different rate-limiting steps in the alternate gating pathways (hydrolytic and non-hydrolytic). These observations demonstrate that phosphorylated CFTR behaves as a conventional ligand-gated channel employing cytoplasmic ATP as a readily available cytoplasmic ligand; under physiological conditions ligand hydrolysis provides efficient reversibility of channel opening. PMID:11034616

  2. Single-Channel Kinetic Analysis for Activation and Desensitization of Homomeric 5-HT3A Receptors

    PubMed Central

    Corradi, Jeremías; Gumilar, Fernanda; Bouzat, Cecilia

    2009-01-01

    Abstract The 5-HT3A receptor is a member of the Cys-loop family of ligand-gated ion channels. To perform kinetic analysis, we mutated the 5-HT3A subunit to obtain a high-conductance form so that single-channel currents can be detected. At all 5-HT concentrations (>0.1 μM), channel activity appears as openings in quick succession that form bursts, which coalesce into clusters. By combining single-channel and macroscopic data, we generated a kinetic model that perfectly describes activation, deactivation, and desensitization. The model shows that full activation arises from receptors with three molecules of agonist bound. It reveals an earlier conformational change of the fully liganded receptor that occurs while the channel is still closed. From this pre-open closed state, the receptor enters into an open-closed cycle involving three open states, which form the cluster whose duration parallels the time constant of desensitization. A similar model lacking the pre-open closed state can describe the data only if the opening rates are fixed to account for the slow activation rate. The application of the model to M4 mutant receptors shows that position 10′ contributes to channel opening and closing rates. Thus, our kinetic model provides a foundation for understanding structural bases of activation and drug action. PMID:19720021

  3. Ion channels and receptor as targets for the control of parasitic nematodes

    PubMed Central

    Wolstenholme, Adrian J.

    2011-01-01

    Many of the anthelmintic drugs in use today act on the nematode nervous system. Ion channel targets have some obvious advantages. They tend to act quickly, which means that they will clear many infections rapidly. They produce very obvious effects on the worms, typically paralyzing them, and these effects are suitable for use in rapid and high-throughput assays. Many of the ion channels and enzymes targeted can also be incorporated into such assays. The macrocyclic lactones bind to an allosteric site on glutamate-gated chloride channels, either directly activating the channel or enhancing the effect of the normal agonist, glutamate. Many old and new anthelmintics, including tribendimidine and the amino-acetonitrile derivatives, act as agonists at nicotinic acetylcholine receptors; derquantel is an antagonist at these receptors. Nematodes express many different types of nicotinic receptor and this diversity means that they are likely to remain important targets for the foreseeable future. Emodepside may have multiple effects, affecting both a potassium channel and a pre-synaptic G protein-coupled receptor; although few other current drugs act at such targets, this example indicates that they may be more important in the future. The nematode nervous system contains many other ion channels and receptors that have not so far been exploited in worm control but which should be explored in the development of effective new compounds. PMID:24533259

  4. Gating characteristics control glutamate receptor distribution and trafficking in vivo.

    PubMed

    Petzoldt, Astrid G; Lee, Yü-Hien; Khorramshahi, Omid; Reynolds, Eric; Plested, Andrew J R; Herzel, Hanspeter; Sigrist, Stephan J

    2014-09-01

    Glutamate-releasing synapses dominate excitatory release in the brain. Mechanisms governing their assembly are of major importance for circuit development and long-term plasticity underlying learning and memory. AMPA/Kainate-type glutamate receptors (GluRs) are tetrameric ligand-gated ion channels that open their ion-conducting pores in response to binding of the neurotransmitter. Changes in subunit composition of postsynaptic GluRs are highly relevant for plasticity and development of glutamatergic synapses [1-4]. To date, posttranslational modifications, mostly operating via the intracellular C-terminal domains (CTDs) of GluRs, are presumed to be the major regulator of trafficking [5]. In recent years, structural and electrophysiological analyses have improved our understanding of GluR gating mechanism [6-11]. However, whether conformational changes subsequent to glutamate binding may per se be able to influence GluR trafficking has remained an unaddressed question. Using a Drosophila system allowing for extended visualization of GluR trafficking in vivo, we here provide evidence that mutations changing the gating behavior alter GluR distribution and trafficking. GluR mutants associated with reduced charge transfer segregated from coexpressed wild-type GluRs on the level of individual postsynaptic densities. Segregation was lost upon blocking of evoked glutamate release. Photobleaching experiments suggested increased mobility of mutants with reduced charge transfer, which accumulated prematurely during early steps of synapse assembly, but failed to further increase their level in accordance with assembly of the presynaptic scaffold. In summary, gating characteristics seem to be a new variable for the understanding of GluR trafficking relevant to both development and plasticity. PMID:25131677

  5. Dynamic aspects of functional regulation of the ATP receptor channel P2X2.

    PubMed

    Kubo, Yoshihiro; Fujiwara, Yuichiro; Keceli, Batu; Nakajo, Koichi

    2009-11-15

    The P2X(2) channel is a ligand-gated channel activated by ATP. Functional features that reflect the dynamic flexibility of the channel include time-dependent pore dilatation following ATP application and direct inhibitory interaction with activated nicotinic acetylcholine receptors on the membrane. We have been studying the mechanisms by which P2X(2) channel functionality is dynamically regulated. Using a Xenopus oocyte expression system, we observed that the pore properties, including ion selectivity and rectification, depend on the open channel density on the membrane. Pore dilatation was apparent when the open channel density was high and inward rectification was modest. We also observed that P2X(2) channels show voltage dependence, despite the absence of a canonical voltage sensor. At a semi-steady state after ATP application, P2X(2) channels were activated upon membrane hyperpolarization. This voltage-dependent activation was also [ATP] dependent. With increases in [ATP], the speed of hyperpolarization-induced activation was increased and the conductance-voltage relationship was shifted towards depolarized potentials. Based on analyses of experimental data and various simulations, we propose that these phenomena can be explained by assuming a fast ATP binding step and a rate-limiting voltage-dependent gating step. Complete elucidation of these regulatory mechanisms awaits dynamic imaging of functioning P2X(2) channels. PMID:19752115

  6. M2 pore mutations convert the glycine receptor channel from being anion- to cation-selective.

    PubMed Central

    Keramidas, A; Moorhouse, A J; French, C R; Schofield, P R; Barry, P H

    2000-01-01

    Three mutations in the M2 transmembrane domains of the chloride-conducting alpha1 homomeric glycine receptor (P250Delta, A251E, and T265V), which normally mediate fast inhibitory neurotransmission, produced a cation-selective channel with P(Cl)/P(Na), = 0.27 (wild-type P(Cl)/P(Na) = 25), a permeability sequence P(Cs) > P(K) > P(Na) > P(Li), an impermeability to Ca(2+), and a reduced glycine sensitivity. Outside-out patch measurements indicated reversed and accentuated rectification with extremely low mean single channel conductances of 3 pS (inward current) and 11 pS (outward current). The three inverse mutations, to those analyzed in this study, have previously been shown to make the alpha7 acetylcholine receptor channel anion-selective, indicating a common location for determinants of charge selectivity of inhibitory and excitatory ligand-gated ion channels. PMID:10866951

  7. Main ion channels and receptors associated with visceral hypersensitivity in irritable bowel syndrome

    PubMed Central

    de Carvalho Rocha, Heraldo Arcela; Dantas, Bruna Priscilla Vasconcelos; Rolim, Thaísa Leite; Costa, Bagnólia Araújo; de Medeiros, Arnaldo Correia

    2014-01-01

    Irritable bowel syndrome (IBS) is a very frequent functional gastrointestinal disorder characterized by recurrent abdominal pain or discomfort and alteration of bowel habits. The IBS physiopathology is extremely complex. Visceral hypersensitivity plays an important role in the pathogenesis of abdominal pain in both in vitro and in vivo models of this functional disorder. In order to obtain a general view of the participation of the main ion channels and receptors regarding the visceral hypersensitivity in the IBS and to describe their chemical structure, a literature review was carried out. A bibliographical research in the following electronic databases: Pubmed and Virtual Library in Health (BVS) was fulfilled by using the search terms “ion channels” “or” “receptors” “and” “visceral hypersensitivity” “or” “visceral nociception” “and” “irritable bowel syndrome”. Original and review articles were considered for data acquisition. The activation of the ATP ion-gated channels, voltage-gated sodium (Nav) and calcium (Cav) channels, as well as the activation of protease-activated receptors (PAR2), transient receptor potential vanilloide-1, serotonin, cannabinoids and cholecystokinin are involved in the genesis of visceral hypersensitivity in IBS. The involvement of ion channels and receptors concerning visceral hypersensitivity is noteworthy in IBS models. PMID:24976114

  8. A Transient Receptor Potential Ion Channel in Chlamydomonas Shares Key Features with Sensory Transduction-Associated TRP Channels in Mammals

    PubMed Central

    Arias-Darraz, Luis; Cabezas, Deny; Colenso, Charlotte K.; Alegría-Arcos, Melissa; Bravo-Moraga, Felipe; Varas-Concha, Ignacio; Almonacid, Daniel E.; Madrid, Rodolfo; Brauchi, Sebastian

    2015-01-01

    Sensory modalities are essential for navigating through an ever-changing environment. From insects to mammals, transient receptor potential (TRP) channels are known mediators for cellular sensing. Chlamydomonas reinhardtii is a motile single-celled freshwater green alga that is guided by photosensory, mechanosensory, and chemosensory cues. In this type of alga, sensory input is first detected by membrane receptors located in the cell body and then transduced to the beating cilia by membrane depolarization. Although TRP channels seem to be absent in plants, C. reinhardtii possesses genomic sequences encoding TRP proteins. Here, we describe the cloning and characterization of a C. reinhardtii version of a TRP channel sharing key features present in mammalian TRP channels associated with sensory transduction. In silico sequence-structure analysis unveiled the modular design of TRP channels, and electrophysiological experiments conducted on Human Embryonic Kidney-293T cells expressing the Cr-TRP1 clone showed that many of the core functional features of metazoan TRP channels are present in Cr-TRP1, suggesting that basic TRP channel gating characteristics evolved early in the history of eukaryotes. PMID:25595824

  9. Ligand-gated ion channel interacting proteins and their role in neuroprotection

    PubMed Central

    Li, Shupeng; Wong, Albert H. C.; Liu, Fang

    2014-01-01

    Ion channel receptors are a vital component of nervous system signaling, allowing rapid and direct conversion of a chemical neurotransmitter message to an electrical current. In recent decades, it has become apparent that ionotropic receptors are regulated by protein-protein interactions with other ion channels, G-protein coupled receptors and intracellular proteins. These other proteins can also be modulated by these interactions with ion channel receptors. This bidirectional functional cross-talk is important for critical cellular functions such as excitotoxicity in pathological and disease states like stroke, and for the basic dynamics of activity-dependent synaptic plasticity. Protein interactions with ion channel receptors can therefore increase the computational capacity of neuronal signaling cascades and also represent a novel target for therapeutic intervention in neuropsychiatric disease. This review will highlight some examples of ion channel receptor interactions and their potential clinical utility for neuroprotection. PMID:24847210

  10. Transformation of the plasmon spectrum in a grating-gate transistor structure with spatially modulated two-dimensional electron channel

    SciTech Connect

    Fateev, D. V. Popov, V. V.; Shur, M. S.

    2010-11-15

    We present the theory of plasmon excitation in a grating-gate transistor structure with spatially modulated 2D electron channel. The plasmon spectrum varies depending on the electron density modulation in the transistor channel. We report on the frequency ranges of plasmon mode excitation in the gated and ungated regions of the channel and on the interaction of these different types of plasmon modes. We show that a constructive influence of the ungated regions of the electron channel considerably increases the intensity of the gated plasmon resonances and reduces the plasmon-resonance linewidth in the grating-gated transistor structure.

  11. 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. PMID:26356684

  12. 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

  13. 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.

  14. Chimeric constructs endow the human CFTR Cl− channel with the gating behavior of murine CFTR

    PubMed Central

    Scott-Ward, Toby S.; Cai, Zhiwei; Dawson, Elizabeth S.; Doherty, Ann; Carina Da Paula, Ana; Davidson, Heather; Porteous, David J.; Wainwright, Brandon J.; Amaral, Margarida D.; Sheppard, David N.; Boyd, A. Christopher

    2007-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl− channel gated by ATP-driven nucleotide-binding domain (NBD) dimerization. Here we exploit species differences between human and murine CFTR to investigate CFTR channel gating. Using homologous recombination, we constructed human-murine CFTR (hmCFTR) chimeras with sequences from NBD1, NBD2, or the regulatory domain (RD) of human CFTR replaced by the equivalent regions of murine CFTR. The gating behavior of hmRD and human CFTR were indistinguishable, whereas hmNBD1 and hmNBD2 had subtle effects on channel gating, prolonging both burst duration and interburst interval. By contrast, hmNBD1+2, containing both NBDs of murine CFTR, reproduced the gating behavior of the subconductance state of murine CFTR, which has dramatically prolonged channel openings. The CFTR potentiator pyrophosphate (PPi) enhanced human, hmRD, and hmNBD1 CFTR Cl− currents, but not those of hmNBD2, hmNBD1+2, and murine CFTR. By analyzing the rate-equilibrium free-energy relationships of chimeric channels, we obtained snapshots of the conformation of the NBDs during ATP-driven dimerization. Our data demonstrate that the conformation of NBD1 changes before that of NBD2 during channel opening. This finding suggests that NBD dimerization does not proceed by a symmetric tweezer-like motion, but instead in an asymmetric fashion led by NBD1. We conclude that the NBDs of murine CFTR determine the unique gating behavior of its subconductance state, whereas NBD2 controls channel potentiation by PPi. PMID:17913891

  15. 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

  16. 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. PMID:26621774

  17. Mechanisms of gain control by voltage-gated channels in intrinsically-firing neurons.

    PubMed

    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

  18. Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

    PubMed

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

    2011-01-01

    Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage. PMID:22039534

  19. Modulation of Cardiac Ryanodine Receptor Channels by Alkaline Earth Cations

    PubMed Central

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

    2011-01-01

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

  20. Consequences of Phosphate-Arginine Complexes in Voltage-Gated Ion Channels

    SciTech Connect

    Green, Michael E.

    2008-11-01

    There are two reasons for suspecting that phosphate complexes of arginine make it very difficult to derive gating charge in voltage gated potassium (and presumably sodium) channels from the motion of charged arginines. For one thing, the arginines should be complexed with phosphate, thereby neutralizing the charge, at least partially. Second, Li et al.(1) have shown that there is a large energy penalty for putting a charged arginine into a membrane. on channel gating current is generally attributed to S4 motion, in that the S4 segment of the voltage sensing domain (VSD) of these channels contains arginines, some of which are not (or at least not obviously) salt bridged, or otherwise charge compensated. There is, however, good reason to expect that there should be a complex of these arginines with phosphate, very probably from lipid headgroups. This has consequences for gating current; the complexed arginines, if they moved, would carry too much of the membrane along. This leads to the suggestion that an alternative to S4 physical motion, H+ transport, should be considered as a possible resolution of the apparent paradox. The consequences for a gating model that was proposed in our earlier work are discussed; there is one major difference in the model in the present form (a conformational change), but the proton cascade as gating current and the role of water in the closed state are reinforced.

  1. Genetic Reconstitution of Functional Acetylcholine Receptor Channels in Mouse Fibroblasts

    NASA Astrophysics Data System (ADS)

    Claudio, Toni; Green, W. N.; Hartman, Deborah S.; Hayden, Deborah; Paulson, Henry L.; Sigworth, F. J.; Sine, Steven M.; Swedlund, Anne

    1987-12-01

    Foreign genes can be stably integrated into the genome of a cell by means of DNA-mediated gene transfer techniques, and large quantities of homogenous cells that continuously express these gene products can then be isolated. Such an expression system can be used to study the functional consequences of introducing specific mutations into genes and to study the expressed protein in the absence of cellular components with which it is normally in contact. All four Torpedo acetylcholine receptor (AChR) subunit complementary DNA's were introduced into the genome of a mouse fibroblast cell by DNA-mediated gene transfer. A clonal cell line that stably produced high concentrations of correctly assembled cell surface AChR's and formed proper ligand-gated ion channels was isolated. With this new expression system, recombinant DNA, biochemical, pharmacological, and electrophysiological techniques were combined to study Torpedo AChR's in a single intact system. The physiological and pharmacological profiles of Torpedo AChR's expressed in mouse fibroblast cells differ in some details from those described earlier, and may provide a more accurate reflection of the properties of this receptor in its natural environment.

  2. Structural basis for alcohol modulation of a pentameric ligand-gated ion channel.

    PubMed

    Howard, Rebecca J; Murail, Samuel; Ondricek, Kathryn E; Corringer, Pierre-Jean; Lindahl, Erik; Trudell, James R; Harris, R Adron

    2011-07-19

    Despite its long history of use and abuse in human culture, the molecular basis for alcohol action in the brain is poorly understood. The recent determination of the atomic-scale structure of GLIC, a prokaryotic member of the pentameric ligand-gated ion channel (pLGIC) family, provides a unique opportunity to characterize the structural basis for modulation of these channels, many of which are alcohol targets in brain. We observed that GLIC recapitulates bimodal modulation by n-alcohols, similar to some eukaryotic pLGICs: methanol and ethanol weakly potentiated proton-activated currents in GLIC, whereas n-alcohols larger than ethanol inhibited them. Mapping of residues important to alcohol modulation of ionotropic receptors for glycine, γ-aminobutyric acid, and acetylcholine onto GLIC revealed their proximity to transmembrane cavities that may accommodate one or more alcohol molecules. Site-directed mutations in the pore-lining M2 helix allowed the identification of four residues that influence alcohol potentiation, with the direction of their effects reflecting α-helical structure. At one of the potentiation-enhancing residues, decreased side chain volume converted GLIC into a highly ethanol-sensitive channel, comparable to its eukaryotic relatives. Covalent labeling of M2 positions with an alcohol analog, a methanethiosulfonate reagent, further implicated residues at the extracellular end of the helix in alcohol binding. Molecular dynamics simulations elucidated the structural consequences of a potentiation-enhancing mutation and suggested a structural mechanism for alcohol potentiation via interaction with a transmembrane cavity previously termed the "linking tunnel." These results provide a unique structural model for independent potentiating and inhibitory interactions of n-alcohols with a pLGIC family member. PMID:21730162

  3. In vivo potency of different ligands on voltage-gated sodium channels.

    PubMed

    Safrany-Fark, Arpad; Petrovszki, Zita; Kekesi, Gabriella; Liszli, Peter; Benedek, Gyorgy; Keresztes, Csilla; Horvath, Gyongyi

    2015-09-01

    The Ranvier nodes of thick myelinated nerve fibers contain almost exclusively voltage-gated sodium channels (Navs), while the unmyelinated fibers have several receptors (e.g., cannabinoid, transient receptor potential vanilloid receptor 1), too. Therefore, a nerve which contains only motor fibers can be an appropriate in vivo model for selective influence of Navs. The goals were to evaluate the potency of local anesthetic drugs on such a nerve in vivo; furthermore, to investigate the effects of ligands with different structures (arachidonic acid, anandamide, capsaicin and nisoxetine) that were proved to inhibit Navs in vitro with antinociceptive properties. The marginal mandibular branch of the facial nerve was explored in anesthetized Wistar rats; after its stimulation, the electrical activity of the vibrissae muscles was registered following the perineural injection of different drugs. Lidocaine, bupivacaine and ropivacaine evoked dose-dependent decrease in electromyographic activity, i.e., lidocaine had lower potency than bupivacaine or ropivacaine. QX-314 did not cause any effect by itself, but its co-application with lidocaine produced a prolonged inhibition. Nisoxetine had a very low potency. While anandamide and capsaicin in high doses caused about 50% decrease in the amplitude of action potential, arachidonic acid did not influence the responses. We proved that the classical local anesthetics have high potency on motor nerves, suggesting that this method might be a reliable model for selective targeting of Navs in vivo circumstances. It is proposed that the effects of these endogenous lipids and capsaicin on sensory fibers are not primarily mediated by Navs. PMID:26033207

  4. Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics

    PubMed Central

    Govorunova, Elena G.; Sineshchekov, Oleg A.; Janz, Roger; Liu, Xiaoqin; Spudich, John L.

    2016-01-01

    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. PMID:26113638

  5. 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

    2015-08-01

    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. PMID:26113638

  6. Molecular characterisation of a pH-gated chloride channel from Sarcoptes scabiei.

    PubMed

    Mounsey, Kate E; Dent, Joseph A; Holt, Deborah C; McCarthy, James; Currie, Bart J; Walton, Shelley F

    2007-09-01

    Reports of ivermectin resistance in scabies mites raise concerns regarding the sustainability of mass intervention programs for scabies worldwide and for the treatment of crusted scabies. Ligand gated ion channels (LGICs) are the primary targets of ivermectin in invertebrates. We report the molecular characterisation of SsCl--a novel LGIC from Sarcoptes scabiei var. hominis. While SsCl shows sequence similarity to other LGICs, phylogenetic analysis does not suggest strong homology to conventional glutamate, histamine or GABA gated channels. Instead, it is most similar to Drosophila pH-sensitive and group 1 clades. When expressed in Xenopus oocytes, SsCl forms a homomeric, pH-gated chloride channel that is irreversibly activated by ivermectin. These results provide the first confirmation that this group of LGIC exists in arachnids, and suggest that SsCl may be an in vivo target of ivermectin in S. scabiei. PMID:17602250

  7. Direct deposition of aluminum oxide gate dielectric on graphene channel using nitrogen plasma treatment

    SciTech Connect

    Lim, Taekyung; Kim, Dongchool; Ju, Sanghyun

    2013-07-01

    Deposition of high-quality dielectric on a graphene channel is an essential technology to overcome structural constraints for the development of nano-electronic devices. In this study, we investigated a method for directly depositing aluminum oxide (Al{sub 2}O{sub 3}) on a graphene channel through nitrogen plasma treatment. The deposited Al{sub 2}O{sub 3} thin film on graphene demonstrated excellent dielectric properties with negligible charge trapping and de-trapping in the gate insulator. A top-gate-structural graphene transistor was fabricated using Al{sub 2}O{sub 3} as the gate dielectric with nitrogen plasma treatment on graphene channel region, and exhibited p-type transistor characteristics.

  8. The role of thermosensitive TRP (transient receptor potential) channels in insulin secretion.

    PubMed

    Uchida, Kunitoshi; Tominaga, Makoto

    2011-01-01

    Insulin secretion from pancreatic β-cells is the only efficient means to decrease blood glucose concentrations. Glucose is the principal stimulator of insulin secretion with the ATP-sensitive K+ channel-voltage-gated Ca2+ channel-mediated pathway being the primary one involved in glucose-stimulated insulin secretion. Recently, several reports demonstrated that some transient receptor potential (TRP) channels are expressed in pancreatic β-cells and contribute to pancreatic β-cell functions. Interestingly, six of them (TRPM2, TRPM4, TRPM5, TRPV1, TRPV2 and TRPV4) are thermosensitive TRP channels. Thermosensitive TRP channels in pancreatic β-cells can function as multimodal receptors and cause Ca2+ influx and membrane depolarization at physiological body temperature. TRPM channels (TRPM2, TRPM4 and TRPM5) control insulin secretion levels by sensing intracellular Ca2+ increase, NAD metabolites, or hormone receptor activation. TRPV2 is involved not only in insulin secretion but also cell proliferation, and is regulated by the autocrine effects of insulin. TRPV1 expressed in sensory neurons is involved in β-cell stress and islet inflammation by controlling neuropeptide release levels. It is thus clear that thermosensitive TRP channels play important roles in pancreatic β-cell functions, and future analyses of TRP channel function will lead to better understanding of the complicated mechanisms involved in insulin secretion and diabetes pathogenesis. PMID:21785227

  9. 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

  10. Stochastic resonance in an ion channel following the non-Arrhenius gating rate

    NASA Astrophysics Data System (ADS)

    Parc, Yong Woon; Koh, Duk-Su; Sung, Wokyung

    2009-05-01

    Stochastic resonance (SR) is a novel cooperative phenomenon occurring in nonlinear systems due to coupling of an ambient noise and an external signal. Biological systems may use SR mechanism to detect the signal efficiently from an external environment. A number of studies have addressed the SR in artificial ion channels considering external voltages as noises. More important than these external noises is the internal, thermal noise which changes the channel conformations essential for biological functions. In this work, we consider that the channel gating rates follow a non-Arrhenius temperature dependence derived from experimental data of a real biological channel. Using the Monte-Carlo simulations, we find that in this channel SR occurs near a physiological temperature in a very distinctive manner compared with that for the Arrhenius gating model.

  11. Single-Channel Current Through Nicotinic Receptor Produced by Closure of Binding Site C-Loop

    SciTech Connect

    Wang, Hailong; Cheng, Xiaolin; McCammon, Jonathan

    2009-01-01

    We investigated the initial coupling of agonist binding to channel gating of the nicotinic acetylcholine receptor using targeted molecular-dynamics (TMD) simulation. After TMD simulation to accelerate closure of the C-loops at the agonist binding sites, the region of the pore that passes through the cell membrane expands. To determine whether the structural changes in the pore result in ion conduction, we used a coarse-grained ion conduction simulator, Biology Boltzmann transport Monte Carlo, and applied it to two structural frames taken before and after TMD simulation. The structural model before TMD simulation represents the channel in the proposed resting state, whereas the model after TMD simulation represents the channel in the proposed active state. Under external voltage biases, the channel in the active state was permeable to cations. Our simulated ion conductance approaches that obtained experimentally and recapitulates several functional properties characteristic of the nicotinic acetylcholine receptor. Thus, closure of the C-loop triggers a structural change in the channel sufficient to account for the open channel current. This approach of applying Biology Boltzmann transport Monte Carlo simulation can be used to further investigate the binding to gating transduction mechanism and the structural bases for ion selection and translocation.

  12. A mutation in the pore of the sodium channel alters gating.

    PubMed Central

    Tomaselli, G F; Chiamvimonvat, N; Nuss, H B; Balser, J R; Pérez-García, M T; Xu, R H; Orias, D W; Backx, P H; Marban, E

    1995-01-01

    Ion permeation and channel gating are classically considered independent processes, but site-specific mutagenesis studies in K channels suggest that residues in or near the ion-selective pore of the channel can influence activation and inactivation. We describe a mutation in the pore of the skeletal muscle Na channel that alters gating. This mutation, I-W53C (residue 402 in the mu 1 sequence), decreases the sensitivity to block by tetrodotoxin and increases the sensitivity to block by externally applied Cd2+ relative to the wild-type channel, placing this residue within the pore near the external mouth. Based on contemporary models of the structure of the channel, this residue is remote from the regions of the channel known to be involved in gating, yet this mutation abbreviates the time to peak and accelerates the decay of the macroscopic Na current. At the single-channel level we observe a shortening of the latency to first opening and a reduction in the mean open time compared with the wild-type channel. The acceleration of macroscopic current kinetics in the mutant channels can be simulated by changing only the activation and deactivation rate constants while constraining the microscopic inactivation rate constants to the values used to fit the wild-type currents. We conclude that the tryptophan at position 53 in the domain IP-loop may act as a linchpin in the pore that limits the opening transition rate. This effect could reflect an interaction of I-W53 with the activation voltage sensors or a more global gating-induced change in pore structure. Images FIGURE 1 PMID:7612823

  13. Analytical Model for Direct Tunneling Gate Current in Long-Channel Undoped Cylindrical Surrounding Gate Metal-Oxide-Semiconductor Field Effect Transistors

    NASA Astrophysics Data System (ADS)

    Han, Ru; Li, Cong

    2013-02-01

    In this study, an analytical direct tunneling gate current model for long-channel undoped cylindrical surrounding gate (CSG) MOSFETs is developed. On the basis of an analytical model, the direct tunneling gate current in CSG MOSFETs is investigated. It is found that direct tunneling gate current is a strong function of gate oxide thickness, but less affected by the change in channel radius. It is also revealed that considering the influence of the source and drain, as the length of the underlap region decreases to zero, the direct tunneling gate current drastically increases. 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.

  14. Psychiatric presentation of voltage-gated potassium channel antibody-associated encephalopathy

    PubMed Central

    PARTHASARATHI, U. D.; HARROWER, T.; TEMPEST, M.; HODGES, J. R.; WALSH, C.; McKENNA, P. J.; FLETCHER, P.C.

    2012-01-01

    Summary 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. PMID:16880491

  15. Apoptotic proteins Reaper and Grim induce stable inactivation in voltage-gated K+ channels

    PubMed Central

    Avdonin, V.; Kasuya, J.; Ciorba, M. A.; Kaplan, B.; Hoshi, T.; Iverson, L.

    1998-01-01

    Drosophila genes reaper, grim, and head-involution-defective (hid) induce apoptosis in several cellular contexts. N-terminal sequences of these proteins are highly conserved and are similar to N-terminal inactivation domains of voltage-gated potassium (K+) channels. Synthetic Reaper and Grim N terminus peptides induced fast inactivation of Shaker-type K+ channels when applied to the cytoplasmic side of the channel that was qualitatively similar to the inactivation produced by other K+ channel inactivation particles. Mutations that reduce the apoptotic activity of Reaper also reduced the synthetic peptide’s ability to induce channel inactivation, indicating that K+ channel inactivation correlated with apoptotic activity. Coexpression of Reaper RNA or direct injection of full length Reaper protein caused near irreversible block of the K+ channels. These results suggest that Reaper and Grim may participate in initiating apoptosis by stably blocking K+ channels. PMID:9751729

  16. Liposome reconstitution and modulation of recombinant N-methyl-d-aspartate receptor channels by membrane stretch

    PubMed Central

    Kloda, Anna; Lua, Linda; Hall, Rhonda; Adams, David J.; Martinac, Boris

    2007-01-01

    In this study, the heteromeric N-methyl-d-aspartate (NMDA) receptor channels composed of NR1a and NR2A subunits were expressed, purified, reconstituted into liposomes, and characterized by using the patch clamp technique. The protein exhibited the expected electrophysiological profile of activation by glutamate and glycine and internal Mg2+ blockade. We demonstrated that the mechanical energy transmitted to membrane-bound NMDA receptor channels can be exerted directly by tension developed in the lipid bilayer. Membrane stretch and application of arachidonic acid potentiated currents through NMDA receptor channels in the presence of intracellular Mg2+. The correlation of membrane tension induced by either mechanical or chemical stimuli with the physiological Mg2+ block of the channel suggests that the synaptic transmission can be altered if NMDA receptor complexes experience local changes in bilayer thickness caused by dynamic targeting to lipid microdomains, electrocompression, or chemical modification of the cell membranes. The ability to study gating properties of NMDA receptor channels in artificial bilayers should prove useful in further study of structure–function relationships and facilitate discoveries of new therapeutic agents for treatment of glutamate-mediated excitotoxicity or analgesic therapies. PMID:17242368

  17. 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

  18. Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons.

    PubMed

    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

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

    PubMed Central

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

    2008-01-01

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

  20. [Molecular dynamics simulations of migration of ions and molecules through the acetylcholine receptor channel].

    PubMed

    Shaĭtan, K V; Li, A; Tershkina, K B; Kirpichnikov, M P

    2007-01-01

    A dynamic model of the channel of an acetylcholine receptor in a closed state has been proposed. The channel is formed by five a-helices of subunit M2 and stabilized by the cyclic hydrocarbon (CH2)105. The migration of charged and unchanged van der Waals particles with a diameter of 7.72 A equivalent to the diameter of a hydrated sodium ion has been studied. The migration occurred by the action of external force applied to the complex along the channel axis. In the closed state, the inhibition of ions is due to two components: electrostatic interaction and steric constraints. The van der Waals channel gate is formed by residues 13'-A-Val255, B-Val261, C-Val269, D-Val255, and E-Ile264, and the negatively changed residues occurring in the upper part of the channel have a great effect on ion selectivity. PMID:17633536

  1. Modulation of BKCa channel gating by endogenous signaling molecules

    PubMed Central

    Hou, Shangwei; Heinemann, Stefan H.; Hoshi, Toshinori

    2010-01-01

    Large-conductance Ca2+- and voltage-activated K+ (BKCa, 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 BKCa 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 BKCa 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 BKCa channels. PMID:19196649

  2. Surface dynamics of voltage-gated ion channels.

    PubMed

    Heine, Martin; Ciuraszkiewicz, Anna; Voigt, Andreas; Heck, Jennifer; Bikbaev, Arthur

    2016-07-01

    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

  3. Open-State Occupancy Prevents Gating Charge Relaxation of N-type (CaV2.2) Calcium Channels

    PubMed Central

    Yarotskyy, Viktor; Elmslie, Keith S.

    2009-01-01

    N-type and L-type channels have significant gating differences, and we wondered whether some of these differences are linked to the relationship between charge movement and channel opening. The time constants for N-channel closing (τDeact) and Off-gating charge movement (τQOff) were compared over a range of voltages. τQOff was significantly larger than τDeact at voltages < −10 mV, and the voltage dependence of the τQOff was less steep than that for τDeact, which suggests that gating charge relaxation does not limit channel closing. Roscovitine, a drug that slows N-channel closing by holding the channel in a high open-probability state, was found to slow both τQOff and τDeact, and thus the time courses of channel closing and gating charge relaxation were similar. Our gating current results were reproduced with the addition of a voltage-independent, closed-closed transition to our previously published two-open-state N-channel model. This work suggests that, like L-type channels, there is a voltage-independent transition along the N-channel activation/deactivation pathway, but this transition occurs between closed states instead of the closed-open states of the L-channel. Also unlike L-type channels, the gating charge appears to be locked into the activated position by the N-channel open state. PMID:19883587

  4. Ionotropic receptors and ion channels in ischemic neuronal death and dysfunction

    PubMed Central

    Weilinger, Nicholas L; Maslieieva, Valentyna; Bialecki, Jennifer; Sridharan, Sarup S; Tang, Peter L; Thompson, Roger J

    2013-01-01

    Loss of energy supply to neurons during stroke induces a rapid loss of membrane potential that is called the anoxic depolarization. Anoxic depolarizations result in tremendous physiological stress on the neurons because of the dysregulation of ionic fluxes and the loss of ATP to drive ion pumps that maintain electrochemical gradients. In this review, we present an overview of some of the ionotropic receptors and ion channels that are thought to contribute to the anoxic depolarization of neurons and subsequently, to cell death. The ionotropic receptors for glutamate and ATP that function as ligand-gated cation channels are critical in the death and dysfunction of neurons. Interestingly, two of these receptors (P2X7 and NMDAR) have been shown to couple to the pannexin-1 (Panx1) ion channel. We also discuss the important roles of transient receptor potential (TRP) channels and acid-sensing ion channels (ASICs) in responses to ischemia. The central challenge that emerges from our current understanding of the anoxic depolarization is the need to elucidate the mechanistic and temporal interrelations of these ion channels to fully appreciate their impact on neurons during stroke. PMID:22864302

  5. Interaction of androsterone and progesterone with inhibitory ligand-gated ion channels: a patch clamp study.

    PubMed

    Ziegler, Elke; Bodusch, M; Song, Y; Jahn, K; Wolfes, H; Steinlechner, S; Dengler, R; Bufler, J; Krampfl, K

    2009-10-01

    Gamma-aminobutyric acid receptor type A (GABA(A)) receptor channels mediate fast inhibitory neurotransmission throughout the central nervous system while the expression of ionotropic glycine receptors is mainly restricted to the spinal cord and brain stem. Neuroactive steroids are well known as positive allosteric modulators of GABA(A) receptor function. Furthermore, there have been hints for an interaction of neuroactive steroids with ionotropic glycine receptors. The aim of the study was to characterize the effect of androsterone and progesterone on alpha(1) and alpha(1)beta glycine receptor and alpha(1)beta(2)gamma(2) GABA(A) receptor channels and to examine the molecular interactions between ligands and receptors. Electrophysiological recordings were performed on HEK 293 cells using the patch clamp technique in combination with an ultrafast perfusion system. A direct activation of inhibitory ionotropic receptors was observed for androsterone at GABA(A) receptor channels. A coactivation of currents elicited by nonsaturating agonist concentrations was observed with androsterone and progesterone at glycine and GABA(A) receptor channels. We could show that association of beta subunits with alpha subunits affects the sensitivity of glycine receptors to androsterone. In contrast to previous reports in which recombinant glycine receptors were inhibited by progesterone, a potentiating effect was revealed by our experiments. At concentrations of 0.1 mM and higher, there were also hints to a channel block-like mechanism. In conclusion, different molecular mechanisms of interaction between neuroactive steroids and GABA as well as glycine receptors could be identified and quantitatively described. Our data clarify the role of steroid compounds in the modulation of inhibitory receptor channel function. PMID:19705103

  6. Cornichons modify channel properties of recombinant and glial AMPA receptors

    PubMed Central

    Coombs, Ian D.; Soto, David; Zonouzi, Marzieh; Renzi, Massimiliano; Shelley, Chris; Farrant, Mark; Cull-Candy, Stuart G.

    2012-01-01

    Ionotropic glutamate receptors, which underlie a majority of excitatory synaptic transmission in the CNS, associate with transmembrane proteins that modify their intracellular trafficking and channel gating. For AMPA-type glutamate receptors (AMPARs), significant advances have been made in our understanding of their regulation by transmembrane AMPAR regulatory proteins (TARPs). Less is known about the functional influence of cornichons – unrelated AMPAR-interacting proteins, identified by proteomic analysis. Here we confirm that cornichon homologs 2 and 3 (CNIH-2 and CNIH-3), but not CNIH-1, slow the deactivation and desensitization of both GluA2-containing calcium-impermeable (CI-) and GluA2-lacking calcium-permeable (CP-) AMPARs expressed in tsA201 cells. CNIH-2 and -3 also enhanced the glutamate sensitivity, single-channel conductance and calcium permeability of CP-AMPARs, while decreasing their block by intracellular polyamines. We examined the potential effects of CNIHs on native AMPARs by recording from rat optic nerve oligodendrocyte precursor cells (OPCs), known to express a significant population of CP-AMPARs. These glial cells exhibited surface labelling with an anti-CNIH-2/3 antibody. Two features of their AMPAR-mediated currents – the relative efficacy of the partial agonist kainate (IKA/IGlu ratio 0.4), and a greater than five-fold potentiation of kainate responses by cyclothiazide – suggest AMPAR association with CNIHs. Additionally, overexpression of CNIH-3 in OPCs markedly slowed AMPAR desensitization. Together, our experiments support the view that CNIHs are capable of altering key properties of AMPARs and suggest that they may do so in glia. PMID:22815494

  7. Synthesis of photoreactive ivermectin B1a derivatives and their actions on Haemonchus and Bombyx glutamate-gated chloride channels.

    PubMed

    Fuse, Toshinori; Ikeda, Izumi; Kita, Tomo; Furutani, Shogo; Nakajima, Hiromitsu; Matsuda, Kazuhiko; Ozoe, Fumiyo; Ozoe, Yoshihisa

    2015-05-01

    Glutamate-gated chloride channels (GluCls) are inhibitory neurotransmitter receptors that are present only in invertebrates such as nematodes and insects. These channels are important targets of insecticidal, acaricidal, and anthelmintic macrolides such as avermectins, ivermectin (IVM), and milbemycins. To identify the amino acid residues that interact with IVM in GluCls, three IVM B1a derivatives with different photoreactive substitutions at C-13 were synthesized in the present study. These derivatives displayed low- or subnanomolar affinity for parasitic nematode (Haemonchus contortus) and silkworm (Bombyx mori) GluCls expressed in COS-1 cells. The derivatives also activated homomeric H. contortus GluCls expressed in Xenopus oocytes. The results indicate that synthesized photoreactive IVM B1a derivatives have superior affinity and functionality for chemically labeling the macrolide-binding site in GluCls. . PMID:25987225

  8. Betaine acts on a ligand-gated ion channel in the nervous system of the nematode C. elegans

    PubMed Central

    Peden, Aude S.; Mac, Patrick; Fei, You-Jun; Castro, Cecilia; Jiang, Guoliang; Murfitt, Kenneth J.; Miska, Eric A.; Griffin, Julian L.; Ganapathy, Vadivel; Jorgensen, Erik M.

    2014-01-01

    Prior to the advent of synthetic nematocides, natural products such as seaweed were used to control nematode infestations. The nematocidal agent in seaweed is betaine, an amino acid that functions as an osmolyte and methyl donor. However, the molecular mechanisms of betaine toxicity are unknown. Here, we identify the betaine transporter SNF-3 and a betaine receptor ACR-23 in the nematode C. elegans. Mutating snf-3 in a sensitized background causes the animals to be hypercontracted and paralyzed, presumably because of excess extracellular betaine. These behavioral defects are suppressed by mutations in acr-23, which encodes a ligand-gated cation channel of the cys-loop family. ACR-23 is activated by betaine and functions in the mechanosensory neurons to maintain basal levels of locomotion. However, overactivation of the receptor by excess betaine or by the allosteric modulator monepantel causes hypercontraction and death of the nematode. Thus, monepantel targets a betaine signaling pathway in nematodes. PMID:24212673

  9. 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. PMID:24671572

  10. Genotype phenotype associations across the voltage-gated sodium channel family.

    PubMed

    Brunklaus, Andreas; Ellis, Rachael; Reavey, Eleanor; Semsarian, Christopher; Zuberi, Sameer M

    2014-10-01

    Mutations in genes encoding voltage-gated sodium channels have emerged as the most clinically relevant genes associated with epilepsy, cardiac conduction defects, skeletal muscle channelopathies and peripheral pain disorders. Geneticists in partnership with neurologists and cardiologists are often asked to comment on the clinical significance of specific mutations. We have reviewed the evidence relating to genotype phenotype associations among the best known voltage-gated sodium channel related disorders. Comparing over 1300 sodium channel mutations in central and peripheral nervous system, heart and muscle, we have identified many similarities in the genetic and clinical characteristics across the voltage-gated sodium channel family. There is evidence, that the level of impairment a specific mutation causes can be anticipated by the underlying physico-chemical property change of that mutation. Across missense mutations those with higher Grantham scores are associated with more severe phenotypes and truncating mutations underlie the most severe phenotypes. Missense mutations are clustered in specific areas and are associated with distinct phenotypes according to their position in the protein. Inherited mutations tend to be less severe than de novo mutations which are usually associated with greater physico-chemical difference. These findings should lead to a better understanding of the clinical significance of specific voltage-gated sodium channel mutations, aiding geneticists and physicians in the interpretation of genetic variants and counselling individuals and their families. PMID:25163687

  11. 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

    2014-01-01

    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. PMID:25385530

  12. 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

  13. 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. PMID:27032955

  14. Reprint of "Mechanisms of lipid regulation and lipid gating in TRPC channels".

    PubMed

    Svobodova, Barbora; Groschner, Klaus

    2016-08-01

    TRPC proteins form cation channels that integrate and relay cellular signals by mechanisms involving lipid recognition and lipid-dependent gating. The lipohilic/amphiphilic molecules that function as cellular activators or modulators of TRPC proteins span a wide range of chemical structures. In this context, cellular redox balance is likely linked to the lipid recognition/gating features of TRPC channels. Both classical ligand-protein interactions as well as indirect and promiscuous sensory mechanisms have been proposed. Some of the recognition processes are suggested to involve ancillary lipid-binding scaffolds or regulators as well as dynamic protein-protein interactions determined by bilayer architecture. A complex interplay of protein-protein and protein-lipid interactions is likely to govern the gating and/or plasma membrane recruitment of TRPC channels, thereby providing a distinguished platform for signal integration and coincident signal detection. Both the primary molecular event(s) of lipid recognition by TRPC channels as well as the transformation of these events into distinct gating movements is poorly understood at the molecular level, and it remains elusive whether lipid sensing in TRPCs is conferred to a distinct sensor domain. Recent structural information on the molecular action of lipophilic activators in distantly related members of the TRP superfamily encourages speculations on TRPC gating mechanisms involved in lipid recognition/gating. This review aims to provide an update on the current understanding of the lipid-dependent control of TRPC channels with focus on the TRPC lipid sensing, signal-integration hub and a short discussion of potential links to redox signaling. PMID:27431463

  15. Ca2+ controls gating of voltage-gated calcium channels by releasing the β2e subunit from the plasma membrane.

    PubMed

    Kim, Dong-Il; Kweon, Hae-Jin; Park, Yongsoo; Jang, Deok-Jin; Suh, Byung-Chang

    2016-01-01

    Voltage-gated calcium (Cav) channels, which are regulated by membrane potential, cytosolic Ca(2+), phosphorylation, and membrane phospholipids, govern Ca(2+) entry into excitable cells. Cav channels contain a pore-forming α1 subunit, an auxiliary α2δ subunit, and a regulatory β subunit, each encoded by several genes in mammals. In addition to a domain that interacts with the α1 subunit, β2e and β2a also interact with the cytoplasmic face of the plasma membrane through an electrostatic interaction for β2e and posttranslational acylation for β2a. We found that an increase in cytosolic Ca(2+) promoted the release of β2e from the membrane without requiring substantial depletion of the anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) from the plasma membrane. Experiments with liposomes indicated that Ca(2+) disrupted the interaction of the β2e amino-terminal peptide with membranes containing PIP2 Ca(2+) binding to calmodulin (CaM) leads to CaM-mediated inactivation of Cav currents. Although Cav2.2 coexpressed with β2a required Ca(2+)-dependent activation of CaM for Ca(2+)-mediated reduction in channel activity, Cav2.2 coexpressed with β2e exhibited Ca(2+)-dependent inactivation of the channel even in the presence of Ca(2+)-insensitive CaM. Inducible depletion of PIP2 reduced Cav2.2 currents, and in cells coexpressing β2e, but not a form that lacks the polybasic region, increased intracellular Ca(2+) further reduced Cav2.2 currents. Many hormone- or neurotransmitter-activated receptors stimulate PIP2 hydrolysis and increase cytosolic Ca(2+); thus, our findings suggest that β2e may integrate such receptor-mediated signals to limit Cav activity. PMID:27382026

  16. Molecular determinants of prokaryotic voltage-gated sodium channels for recognition of local anesthetics.

    PubMed

    Shimomura, Takushi; Irie, Katsumasa; Fujiyoshi, Yoshinori

    2016-08-01

    Local anesthetics (LAs) inhibit mammalian voltage-gated Na(+) channels (Navs) and are thus clinically important. LAs also inhibit prokaryotic Navs (BacNavs), which have a simpler structure than mammalian Navs. To elucidate the detailed mechanisms of LA inhibition to BacNavs, we used NavBh, a BacNav from Bacillus halodurans, to analyze the interactions of several LAs and quaternary ammoniums (QAs). Based on the chemical similarity of QA with the tertiary-alkylamine (TAA) group of LAs, QAs were used to determine the residues required for the recognition of TAA by NavBh. We confirmed that two residues, Thr220 and Phe227, are important for LA binding; a methyl group of Thr220 is important for recognizing both QAs and LAs, whereas Phe227 is involved in holding blockers at the binding site. In addition, we found that NavBh holds blockers in a closed state, consistent with the large inner cavity observed in the crystal structures of BacNavs. These findings reveal the inhibition mechanism of LAs in NavBh, where the methyl group of Thr220 provides the main receptor site for the TAA group and the bulky phenyl group of Phe227 holds the blockers inside the large inner cavity. These two residues correspond to the two LA recognition residues in mammalian Navs, which suggests the relevance of the LA recognition between BacNavs and mammalian Navs. PMID:27273848

  17. Gating of the CFTR Cl- channel by ATP-driven nucleotide-binding domain dimerisation.

    PubMed

    Hwang, Tzyh-Chang; Sheppard, David N

    2009-05-15

    The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl- channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating. PMID:19332488

  18. Gating of the CFTR Cl− channel by ATP-driven nucleotide-binding domain dimerisation

    PubMed Central

    Hwang, Tzyh-Chang; Sheppard, David N

    2009-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl− channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating. PMID:19332488

  19. 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.

  20. 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.

  1. 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.

  2. Structural dynamics of potassium channel gating revealed by single molecule FRET

    PubMed Central

    Borschel, William F.; Ha, Taekjip; Nichols, Colin G.

    2016-01-01

    Crystallography has provided invaluable insights to ion channel selectivity and gating, but to advance understanding to a new level, dynamic views of channel structures within membranes are essential. We labeled tetrameric KirBac1.1 potassium channels with single donor and acceptor fluorophores at different sites, and examined structural dynamics within lipid membranes by single molecule FRET. We found that the extracellular region is structurally rigid in both closed and open states, whereas the N-terminal slide helix undergoes marked conformational fluctuations. The cytoplasmic C-terminal domain fluctuates between two major structural states both of which become less dynamic and move away from the pore axis and away from the membrane in closed channels. Our results reveal mobile and rigid conformations of functionally relevant KirBac1.1 channel motifs, implying similar dynamics for similar motifs in eukaryotic Kir channels and for cation channels in general. PMID:26641713

  3. 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

  4. A role of the sulfonylurea receptor 1 in endocytic trafficking of ATP-sensitive potassium channels

    PubMed Central

    Bruederle, Cathrin E.; Gay, Joel; Shyng, Show-Ling

    2011-01-01

    The ATP-sensitive potassium (KATP) channel consisting of sulfonylurea receptor 1 (SUR1) and inward rectifier potassium channel 6.2 (Kir6.2) has a well-established role in insulin secretion. Mutations in either subunit can lead to disease due to aberrant channel gating, altered channel density at the cell surface or a combination of both. Endocytic trafficking of channels at the plasma membrane is one way to influence surface channel numbers. It has been previously reported that channel endocytosis is dependent on a tyrosine-based motif in Kir6.2 while SUR1 alone is unable to internalize. In this study, we followed endocytic trafficking of surface channels in real time by live cell imaging of channel subunits tagged with an extracellular minimal α-bungarotoxin binding peptide labeled with a fluorescent dye. We demonstrate that SUR1 undergoes endocytosis independent of Kir6.2. Moreover, mutations in the putative endocytosis motif of Kir6.2, Y330C, Y330A and F333I are unable to prevent channel endocytosis. These findings challenge the notion that Kir6.2 bears the sole endocytic signal for KATP channels and support a role of SUR1 in this trafficking process. PMID:21649805

  5. Excessive activation of cyclic nucleotide-gated channels contributes to neuronal degeneration of photoreceptors.

    PubMed

    Vallazza-Deschamps, Géraldine; Cia, David; Gong, Jie; Jellali, Abdeljelil; Duboc, Agnès; Forster, Valérie; Sahel, Jose A; Tessier, Luc-Henri; Picaud, Serge

    2005-09-01

    In different animal models, photoreceptor degeneration was correlated to an abnormal increase in cGMP concentration. The cGMP-induced photoreceptor toxicity was demonstrated by applying the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine on retinal explants. To assess the role of cGMP-gated channels in this cGMP toxicity, the Ca(2+) channel blockers verapamil and L- and D-diltiazem, which block cGMP-gated channels with different efficacies, were applied to in vitro animal models of photoreceptor degeneration. These models included: (i) adult rat retinal explants incubated with zaprinast, a more specific inhibitor of the rod phosphodiesterase than 3-isobutyl-1-methylxanthine and (ii) rd mouse retinal explants. Photoreceptor apoptosis was assessed by terminal dUTP nick end labelling and caspase 3 activation. Effects of the blockers on the synaptic rod Ca(2+) channels were measured by patch-clamp recording. In the zaprinast-induced photoreceptor degeneration model, both diltiazem isomers rescued photoreceptors whereas verapamil had no influence. Their neuroprotective efficacy was correlated to their inhibition of cGMP-gated channels (l-diltiazem>d-diltiazem>verapamil=0). In contrast, all three Ca(2+) channel blockers suppressed rod Ca(2+) channel currents similarly. This suppression of the currents by the diltiazem isomers was very weak (16.5%) at the neuroprotective concentration (10 microm). In rd retinal explants, both diltiazem isomers also slowed down rod degeneration in contrast to verapamil. L-diltiazem exhibited this effect at concentrations ranging from 1 to 20 microm. This study further supports the photoreceptor neuroprotection by diltiazem particularly in the rd mouse retina, whereas the absence of neuroprotection by verapamil further suggests the role of cGMP-gated channel activation in the induction of photoreceptor degeneration. PMID:16176343

  6. Maxi-K(Ca), a Unique Member of the Voltage-Gated K Channel Superfamily.

    PubMed

    Toro, L.; Wallner, M.; Meera, P.; Tanaka, Y.

    1998-06-01

    Large-conductance, voltage-, and Ca(2+)-sensitive K(+) (maxi-K(Ca)) channels regulate neuronal and smooth muscle excitability. Their pore-forming alpha-subunit shows similarities with voltage-gated channels and indeed can open in the practical absence of Ca(2+). The NH(2) terminus is unique, with a seventh transmembrane segment involved in beta-subunit modulation. The long COOH terminus is implied in Ca(2+) modulation. PMID:11390773

  7. Stoichiometry of altered hERG1 channel gating by small molecule activators.

    PubMed

    Wu, Wei; Sachse, Frank B; Gardner, Alison; Sanguinetti, Michael C

    2014-04-01

    Voltage-gated K(+) channels are tetramers formed by coassembly of four identical or highly related subunits. All four subunits contribute to formation of the selectivity filter, the narrowest region of the channel pore which determines K(+) selective conductance. In some K(+) channels, the selectivity filter can undergo a conformational change to reduce K(+) flux by a mechanism called C-type inactivation. In human ether-a-go-go-related gene 1 (hERG1) K(+) channels, C-type inactivation is allosterically inhibited by ICA-105574, a substituted benzamide. PD-118057, a 2-(phenylamino) benzoic acid, alters selectivity filter gating to enhance open probability of channels. Both compounds bind to a hydrophobic pocket located between adjacent hERG1 subunits. Accordingly, a homotetrameric channel contains four identical activator binding sites. Here we determine the number of binding sites required for maximal drug effect and determine the role of subunit interactions in the modulation of hERG1 gating by these compounds. Concatenated tetramers were constructed to contain a variable number (zero to four) of wild-type and mutant hERG1 subunits, either L646E to inhibit PD-118057 binding or F557L to inhibit ICA-105574 binding. Enhancement of hERG1 channel current magnitude by PD-118057 and attenuated inactivation by ICA-105574 were mediated by cooperative subunit interactions. Maximal effects of the both compounds required the presence of all four binding sites. Understanding how hERG1 agonists allosterically modify channel gating may facilitate mechanism-based drug design of novel agents for treatment of long QT syndrome. PMID:24638994

  8. Stoichiometry of altered hERG1 channel gating by small molecule activators

    PubMed Central

    Wu, Wei; Sachse, Frank B.; Gardner, Alison

    2014-01-01

    Voltage-gated K+ channels are tetramers formed by coassembly of four identical or highly related subunits. All four subunits contribute to formation of the selectivity filter, the narrowest region of the channel pore which determines K+ selective conductance. In some K+ channels, the selectivity filter can undergo a conformational change to reduce K+ flux by a mechanism called C-type inactivation. In human ether-a-go-go–related gene 1 (hERG1) K+ channels, C-type inactivation is allosterically inhibited by ICA-105574, a substituted benzamide. PD-118057, a 2-(phenylamino) benzoic acid, alters selectivity filter gating to enhance open probability of channels. Both compounds bind to a hydrophobic pocket located between adjacent hERG1 subunits. Accordingly, a homotetrameric channel contains four identical activator binding sites. Here we determine the number of binding sites required for maximal drug effect and determine the role of subunit interactions in the modulation of hERG1 gating by these compounds. Concatenated tetramers were constructed to contain a variable number (zero to four) of wild-type and mutant hERG1 subunits, either L646E to inhibit PD-118057 binding or F557L to inhibit ICA-105574 binding. Enhancement of hERG1 channel current magnitude by PD-118057 and attenuated inactivation by ICA-105574 were mediated by cooperative subunit interactions. Maximal effects of the both compounds required the presence of all four binding sites. Understanding how hERG1 agonists allosterically modify channel gating may facilitate mechanism-based drug design of novel agents for treatment of long QT syndrome. PMID:24638994

  9. Molecular mechanisms of Cys-loop ion channel receptor modulation by ivermectin

    PubMed Central

    Lynagh, Timothy; Lynch, Joseph W.

    2012-01-01

    Ivermectin is an anthelmintic drug that works by inhibiting neuronal activity and muscular contractility in arthropods and nematodes. It works by activating glutamate-gated chloride channels (GluClRs) at nanomolar concentrations. These receptors, found exclusively in invertebrates, belong to the pentameric Cys-loop receptor family of ligand-gated ion channels (LGICs). Higher (micromolar) concentrations of ivermectin also activate or modulate vertebrate Cys-loop receptors, including the excitatory nicotinic and the inhibitory GABA type-A and glycine receptors (GlyRs). An X-ray crystal structure of ivermectin complexed with the C. elegans α GluClR demonstrated that ivermectin binds to the transmembrane domain in a cleft at the interface of adjacent subunits. It also identified three hydrogen bonds thought to attach ivermectin to its site. Site-directed mutagenesis and voltage-clamp electrophysiology have also been employed to probe the binding site for ivermectin in α1 GlyRs. These have raised doubts as to whether the hydrogen bonds are essential for high ivermectin potency. Due to its lipophilic nature, it is likely that ivermectin accumulates in the membrane and binds reversibly (i.e., weakly) to its site. Several lines of evidence suggest that ivermectin opens the channel pore via a structural change distinct from that induced by the neurotransmitter agonist. Conformational changes occurring at locations distant from the pore can be probed using voltage-clamp fluorometry (VCF), a technique which involves quantitating agonist-induced fluorescence changes from environmentally sensitive fluorophores covalently attached to receptor domains of interest. This technique has demonstrated that ivermectin induces a global conformational change that propagates from the transmembrane domain to the neurotransmitter binding site, thus suggesting a mechanism by which ivermectin potentiates neurotransmitter-gated currents. Together, this information provides new insights into

  10. 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

  11. Cysteines control the N- and C-linker-dependent gating of KCNH1 potassium channels.

    PubMed

    Sahoo, Nirakar; Schönherr, Roland; Hoshi, Toshinori; Heinemann, Stefan H

    2012-05-01

    KCNH1 (EAG1) is a member of the Kv family of voltage-gated potassium channels. However, KCNH1 channels also show some amino-acid sequence similarity to cyclic-nucleotide-regulated channels: they harbor an N-terminal PAS domain, a C-terminal cyclic nucleotide binding homology domain (cNBHD), and N- and C-terminal binding sites for calmodulin. Another notable feature is the channels' high sensitivity toward oxidative modification. Using human KCNH1 expressed in Xenopus oocytes and HEK 293 cells we investigated how oxidative modification alters channel function. Intracellular application of H(2)O(2) or cysteine-specific modifiers potently inhibited KCNH1 channels in two phases. Our systematic cysteine mutagenesis study showed that the rapid and dominant phase was attributed to a right-shift in the voltage dependence of activation, caused by chemical modification of residues C145 and C214. The slow component depended on the C-terminal residues C532 and C562. The cysteine pairs are situated at structural elements linking the transmembrane S1 segment with the PAS domain (N-linker) and the transmembrane channel gate S6 with the cNBH domain (C-linker), respectively. The functional state of KCNH1 channels is determined by the oxidative status of these linkers that provide an additional dimension of channel regulation. PMID:22310694

  12. THE VOLTAGE DEPENDENCE OF GATING CURRENTS OF THE NEURONAL CAV3.3 CHANNEL IS DETERMINED BY THE GATING BRAKE IN THE I-II LOOP

    PubMed Central

    Karmažínová, Mária; Baumgart, Joel; Perez-Reyes, Edward; Lacinová, L'ubica

    2012-01-01

    Low-voltage activated CaV3 Ca2+ channels have an activation threshold around −60 mV, which is lower than the activation threshold of other voltage-dependent calcium channels (VDCC). The kinetics of their activation at membrane voltages just above the activation threshold is much slower than the activation kinetics of other VDCCs. It was demonstrated recently that the intracellular loop connecting repeats I and II of all three CaV3 channels contains a so-called “gating brake.” Disruption of this brake yields channels that activate at even more hyperpolarized potentials with significantly accelerated kinetics. We have compared gating of a wild type CaV3.3 channel and a mutated ID12 channel, in which the putative gating brake at the proximal part of the I-II loop was removed. Voltage dependence of the gating current activation was shifted by 34.6 mV towards more hyperpolarized potentials in ID12 channel. Kinetics of the on-charge activation was significantly accelerated, while kinetics of the off-charge was not altered. We conclude that the putative gating brake in I-II loop hinders not only the opening of the conducting pore but also the activating movement of voltage sensing S4 segments, stabilizing the channel in its closed state. PMID:21340458

  13. Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel

    PubMed Central

    Di Maio, Danilo; Chandramouli, Balasubramanian; Brancato, Giuseppe

    2015-01-01

    Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested. PMID:26465896

  14. Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel.

    PubMed

    Di Maio, Danilo; Chandramouli, Balasubramanian; Brancato, Giuseppe

    2015-01-01

    Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested. PMID:26465896

  15. Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels

    PubMed Central

    Ihara, Makoto; Ling, Yun; Yang, Xinling; Kai, Kenji; Hayashi, Hideo; Matsuda, Kazuhiko

    2015-01-01

    Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs. PMID:25902139

  16. Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels.

    PubMed

    Xu, Yan; Furutani, Shogo; Ihara, Makoto; Ling, Yun; Yang, Xinling; Kai, Kenji; Hayashi, Hideo; Matsuda, Kazuhiko

    2015-01-01

    Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to 10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs. PMID:25902139

  17. The Drosophila Sodium Channel 1 (DSC1): The founding member of a new family of voltage-gated cation channels.

    PubMed

    Dong, Ke; Du, Yuzhe; Rinkevich, Frank; Wang, Lingxin; Xu, Peng

    2015-05-01

    It has been nearly three decades since the identification of the Drosophila Sodium Channel 1 (DSC1) gene from Drosophila melanogaster. The orthologs of the DSC1 gene have now been identified in other insect species including BSC1 from Blattella germanica. Functional analyses of DSC1/BSC1 channels in Xenopus oocytes reveal that DSC1 and BSC1 encode voltage-gated cation channels that are more permeable to Ca(2+) than to Na(+). Genetic and electrophysiological analyses show that knockout of the DSC1 gene in D. melanogaster causes behavioral and neurological modifications. In this review, we summarize major findings from recent studies and highlight a unique role of the DSC1 channel, distinct from that of the sodium channel, in regulating membrane excitability and modulating toxicity of pyrethroid insecticides. PMID:25987218

  18. 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

    2013-12-01

    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. PMID:24113522

  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. A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels.

    PubMed

    Schewe, Marcus; Nematian-Ardestani, Ehsan; Sun, Han; Musinszki, Marianne; Cordeiro, Sönke; Bucci, Giovanna; de Groot, Bert L; Tucker, Stephen J; Rapedius, Markus; Baukrowitz, Thomas

    2016-02-25

    Two-pore domain (K2P) K(+) channels are major regulators of excitability that endow cells with an outwardly rectifying background "leak" conductance. In some K2P channels, strong voltage-dependent activation has been observed, but the mechanism remains unresolved because they lack a canonical voltage-sensing domain. Here, we show voltage-dependent gating is common to most K2P channels and that this voltage sensitivity originates from the movement of three to four ions into the high electric field of an inactive selectivity filter. Overall, this ion-flux gating mechanism generates a one-way "check valve" within the filter because outward movement of K(+) induces filter opening, whereas inward movement promotes inactivation. Furthermore, many physiological stimuli switch off this flux gating mode to convert K2P channels into a leak conductance. These findings provide insight into the functional plasticity of a K(+)-selective filter and also refine our understanding of K2P channels and the mechanisms by which ion channels can sense voltage. PMID:26919430

  1. State-Dependent Network Connectivity Determines Gating in a K+ Channel

    PubMed Central

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

    2014-01-01

    Summary X-ray crystallography has provided tremendous insight into the different structural states of membrane proteins and, in particular, of ion channels. However, the molecular forces that determine the thermodynamic stability of a particular state are poorly understood. Here we analyze the different X-ray structures of an inwardly rectifying potassium channel (Kir1.1) in relation to functional data we obtained for over 190 mutants in Kir1.1. This mutagenic perturbation analysis uncovered an extensive, state-dependent network of physically interacting residues that stabilizes the pre-open and open states of the channel, but fragments upon channel closure. We demonstrate that this gating network is an important structural determinant of the thermodynamic stability of these different gating states and determines the impact of individual mutations on channel function. These results have important implications for our understanding of not only K+ channel gating but also the more general nature of conformational transitions that occur in other allosteric proteins. PMID:24980796

  2. State-dependent network connectivity determines gating in a K+ channel.

    PubMed

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

    2014-07-01

    X-ray crystallography has provided tremendous insight into the different structural states of membrane proteins and, in particular, of ion channels. However, the molecular forces that determine the thermodynamic stability of a particular state are poorly understood. Here we analyze the different X-ray structures of an inwardly rectifying potassium channel (Kir1.1) in relation to functional data we obtained for over 190 mutants in Kir1.1. This mutagenic perturbation analysis uncovered an extensive, state-dependent network of physically interacting residues that stabilizes the pre-open and open states of the channel, but fragments upon channel closure. We demonstrate that this gating network is an important structural determinant of the thermodynamic stability of these different gating states and determines the impact of individual mutations on channel function. These results have important implications for our understanding of not only K+ channel gating but also the more general nature of conformational transitions that occur in other allosteric proteins. PMID:24980796

  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

    2014-09-01

    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. PMID:25136136

  4. 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

  5. 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. PMID:23589829

  6. A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K+ Channels

    PubMed Central

    Schewe, Marcus; Nematian-Ardestani, Ehsan; Sun, Han; Musinszki, Marianne; Cordeiro, Sönke; Bucci, Giovanna; de Groot, Bert L.; Tucker, Stephen J.; Rapedius, Markus; Baukrowitz, Thomas

    2016-01-01

    Summary Two-pore domain (K2P) K+ channels are major regulators of excitability that endow cells with an outwardly rectifying background “leak” conductance. In some K2P channels, strong voltage-dependent activation has been observed, but the mechanism remains unresolved because they lack a canonical voltage-sensing domain. Here, we show voltage-dependent gating is common to most K2P channels and that this voltage sensitivity originates from the movement of three to four ions into the high electric field of an inactive selectivity filter. Overall, this ion-flux gating mechanism generates a one-way “check valve” within the filter because outward movement of K+ induces filter opening, whereas inward movement promotes inactivation. Furthermore, many physiological stimuli switch off this flux gating mode to convert K2P channels into a leak conductance. These findings provide insight into the functional plasticity of a K+-selective filter and also refine our understanding of K2P channels and the mechanisms by which ion channels can sense voltage. PMID:26919430

  7. Simulation Studies of Ion Permeation and Selectivity in Voltage-Gated Sodium Channels.

    PubMed

    Ing, C; Pomès, R

    2016-01-01

    Voltage-gated ion channels are responsible for the generation and propagation of action potentials in electrically excitable cells. Molecular dynamics simulations have become a useful tool to study the molecular basis of ion transport in atomistic models of voltage-gated ion channels. The elucidation of several three-dimensional structures of bacterial voltage-gated sodium channels (Nav) in 2011 and 2012 opened the way to detailed computational investigations of this important class of membrane proteins. Here we review the numerous simulation studies of Na(+) permeation and selectivity in bacterial Nav channels published in the past 5years. These studies use a variety of simulation methodologies differing in force field parameters, molecular models, sampling algorithms, and simulation times. Although results disagree on the details of ion permeation mechanisms, they concur in the presence of two primary Na(+) binding sites in the selectivity filter and support a loosely coupled knock-on mechanism of Na(+) permeation. Comparative studies of Na(+), K(+), and Ca(2+) permeation reveal sites within Nav channels that are Na(+) selective, yet a consensus model of selectivity has not been established. We discuss the agreement between simulation and experimental results and propose strategies that may be used to resolve discrepancies between simulation studies in order to improve future computational studies of permeation and selectivity in ion channels. PMID:27586286

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

    PubMed

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

    1999-08-01

    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

  9. 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

  10. L-type Voltage-Gated Calcium Channels in Conditioned Fear: A Genetic and Pharmacological Analysis

    ERIC Educational Resources Information Center

    McKinney, Brandon C.; Sze, Wilson; White, Jessica A.; Murphy, Geoffrey G.

    2008-01-01

    Using pharmacological approaches, others have suggested that L-type voltage-gated calcium channels (L-VGCCs) mediate both consolidation and extinction of conditioned fear. In the absence of L-VGCC isoform-specific antagonists, we have begun to investigate the subtype-specific role of LVGCCs in consolidation and extinction of conditioned fear…

  11. A specialized molecular motion opens the Hv1 voltage-gated proton channel

    PubMed Central

    Isacoff, Ehud Y.

    2015-01-01

    Summary The Hv1 proton channel is unique among voltage-gated channels for containing the pore and gate within its voltage-sensing domain (VSD). Opening of the pore has been proposed to include assembly of the selectivity filter between the third arginine (R3) of S4 and an aspartate of S1 (D1). We asked whether gating involves a motion of S1 using Ciona intestinalis Hv1. We find that channel opening is concomitant with solution access from the cytoplasm deep into the pore-lining face of S1. Voltage- and patch-clamp fluorometry show that this involves a motion of S1 relative to its surround. S1 motion, and the S4 motion that precedes it, are each influenced by residues on the other helix, suggesting a dynamic interaction between S1 and S4. Our findings suggest that the S1 of Hv1 has specialized to function as part of the channel's gate. PMID:25730777

  12. Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation.

    PubMed

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

    2016-02-25

    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 (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) 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 BacNa(V) 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 BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel. PMID:26919429

  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. PMID:22234285

  14. Diacylglycerol mediates regulation of TASK potassium channels by Gq-coupled receptors.

    PubMed

    Wilke, Bettina U; Lindner, Moritz; Greifenberg, Lea; Albus, Alexandra; Kronimus, Yannick; Bünemann, Moritz; Leitner, Michael G; Oliver, Dominik

    2014-01-01

    The two-pore domain potassium (K2P) channels TASK-1 (KCNK3) and TASK-3 (KCNK9) are important determinants of background K(+) conductance and membrane potential. TASK-1/3 activity is regulated by hormones and transmitters that act through G protein-coupled receptors (GPCR) signalling via G proteins of the Gαq/11 subclass. How the receptors inhibit channel activity has remained unclear. Here, we show that TASK-1 and -3 channels are gated by diacylglycerol (DAG). Receptor-initiated inhibition of TASK required the activity of phospholipase C, but neither depletion of the PLC substrate PI(4,5)P2 nor release of the downstream messengers IP3 and Ca(2+). Attenuation of cellular DAG transients by DAG kinase or lipase suppressed receptor-dependent inhibition, showing that the increase in cellular DAG-but not in downstream lipid metabolites-mediates channel inhibition. The findings identify DAG as the signal regulating TASK channels downstream of GPCRs and define a novel role for DAG that directly links cellular DAG dynamics to excitability. PMID:25420509

  15. Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel

    PubMed Central

    Nomura, Yoshiko; Satar, Gul; Hu, Zhaonong; Nauen, Ralf; He, Sheng Yang; Zhorov, Boris S.; Dong, Ke

    2013-01-01

    Pyrethroid insecticides are widely used as one of the most effective control measures in the global fight against agricultural arthropod pests and mosquito-borne diseases, including malaria and dengue. They exert toxic effects by altering the function of voltage-gated sodium channels, which are essential for proper electrical signaling in the nervous system. A major threat to the sustained use of pyrethroids for vector control is the emergence of mosquito resistance to pyrethroids worldwide. Here, we report the successful expression of a sodium channel, AaNav1–1, from Aedes aegypti in Xenopus oocytes, and the functional examination of nine sodium channel mutations that are associated with pyrethroid resistance in various Ae. aegypti and Anopheles gambiae populations around the world. Our analysis shows that five of the nine mutations reduce AaNav1–1 sensitivity to pyrethroids. Computer modeling and further mutational analysis revealed a surprising finding: Although two of the five confirmed mutations map to a previously proposed pyrethroid-receptor site in the house fly sodium channel, the other three mutations are mapped to a second receptor site. Discovery of this second putative receptor site provides a dual-receptor paradigm that could explain much of the molecular mechanisms of pyrethroid action and resistance as well as the high selectivity of pyrethroids on insect vs. mammalian sodium channels. Results from this study could impact future prediction and monitoring of pyrethroid resistance in mosquitoes and other arthropod pests and disease vectors. PMID:23821746

  16. Slow Inactivation in Voltage Gated Potassium Channels Is Insensitive to the Binding of Pore Occluding Peptide Toxins

    PubMed Central

    Oliva, Carolina; González, Vivian; Naranjo, David

    2005-01-01

    Voltage gated potassium channels open and inactivate in response to changes of the voltage across the membrane. After removal of the fast N-type inactivation, voltage gated Shaker K-channels (Shaker-IR) are still able to inactivate through a poorly understood closure of the ion conduction pore. This, usually slower, inactivation shares with binding of pore occluding peptide toxin two important features: i), both are sensitive to the occupancy of the pore by permeant ions or tetraethylammonium, and ii), both are critically affected by point mutations in the external vestibule. Thus, mutual interference between these two processes is expected. To explore the extent of the conformational change involved in Shaker slow inactivation, we estimated the energetic impact of such interference. We used κ−conotoxin-PVIIA (κ−PVIIA) and charybdotoxin (CTX) peptides that occlude the pore of Shaker K-channels with a simple 1:1 stoichiometry and with kinetics 100-fold faster than that of slow inactivation. Because inactivation appears functionally different between outside-out patches and whole oocytes, we also compared the toxin effect on inactivation with these two techniques. Surprisingly, the rate of macroscopic inactivation and the rate of recovery, regardless of the technique used, were toxin insensitive. We also found that the fraction of inactivated channels at equilibrium remained unchanged at saturating κ−PVIIA. This lack of interference with toxin suggests that during slow inactivation the toxin receptor site remains unaffected, placing a strong geometry-conservative constraint on the possible structural configurations of a slow inactivated K-channel. Such a constraint could be fulfilled by a concerted rotation of the external vestibule. PMID:15923220

  17. Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis.

    PubMed

    Ikuma, M; Welsh, M J

    2000-07-18

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg(2+) prevents hydrolysis. We found that even in the absence of Mg(2+), ATP could support channel activity, albeit at a reduced level compared with the presence of Mg(2+). Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg(2+) further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg(2+), divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel. PMID:10880569

  18. Regulation of CFTR Cl− channel gating by ATP binding and hydrolysis

    PubMed Central

    Ikuma, Mutsuhiro; Welsh, Michael J.

    2000-01-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg2+ prevents hydrolysis. We found that even in the absence of Mg2+, ATP could support channel activity, albeit at a reduced level compared with the presence of Mg2+. Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg2+ further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg2+, divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel. PMID:10880569

  19. Crystal structure of a heterotetrameric NMDA receptor ion channel

    PubMed Central

    Karakas, Erkan; Furukawa, Hiro

    2014-01-01

    N -methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors, which mediate most excitatory synaptic transmission in mammalian brains. Calcium permeation triggered by activation of NMDA receptors is the pivotal event for initiation of neuronal plasticity. Here we show the crystal structure of the intact heterotetrameric GluN1/GluN2B NMDA receptor ion channel at 4 Å. The NMDA receptors are arranged as a dimer of GluN1-GluN2B heterodimers with the two-fold symmetry axis running through the entire molecule composed of an amino terminal domain (ATD), a ligand-binding domain (LBD), and a transmembrane domain (TMD). The ATD and LBD are much more highly packed in the NMDA receptors than non-NMDA receptors, which may explain why ATD regulates ion channel activity in NMDA receptors but not in non-NMDA receptors. PMID:24876489

  20. Voltage-gated calcium channel and antisense oligonucleotides thereto

    NASA Technical Reports Server (NTRS)

    Hruska, Keith A. (Inventor); Friedman, Peter A. (Inventor); Barry, Elizabeth L. R. (Inventor); Duncan, Randall L. (Inventor)

    1998-01-01

    An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

  1. Voltage-Gated Sodium Channels: Mechanistic Insights From Atomistic Molecular Dynamics Simulations.

    PubMed

    Oakes, V; Furini, S; Domene, C

    2016-01-01

    The permeation of ions and other molecules across biological membranes is an inherent requirement of all cellular organisms. Ion channels, in particular, are responsible for the conduction of charged species, hence modulating the propagation of electrical signals. Despite the universal physiological implications of this property, the molecular functioning of ion channels remains ambiguous. The combination of atomistic structural data with computational methodologies, such as molecular dynamics (MD) simulations, is now considered routine to investigate structure-function relationships in biological systems. A fuller understanding of conduction, selectivity, and gating, therefore, is steadily emerging due to the applicability of these techniques to ion channels. However, because their structure is known at atomic resolution, studies have consistently been biased toward K(+) channels, thus the molecular determinants of ionic selectivity, activation, and drug blockage in Na(+) channels are often overlooked. The recent increase of available crystallographic data has eminently encouraged the investigation of voltage-gated sodium (NaV) channels via computational methods. Here, we present an overview of simulation studies that have contributed to our understanding of key principles that underlie ionic conduction and selectivity in Na(+) channels, in comparison to the K(+) channel analogs. PMID:27586285

  2. 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. PMID:24799674

  3. Aromatic Residues {epsilon}Trp-55 and {delta}Trp-57 and the Activation of Acetylcholine Receptor Channels.

    PubMed

    Bafna, Pallavi A; Jha, Archana; Auerbach, Anthony

    2009-03-27

    The two transmitter binding sites of the neuromuscular acetylcholine (ACh) receptor channel contain several aromatic residues, including a tryptophan located on the complementary, negative face of each binding pocket. These two residues, Trp-55 in the epsilon subunit and Trp-57 in the delta subunit, were mutated (AEFHILRVY), and for most constructs the rate constants for acetylcholine binding and channel gating were estimated by using single channel kinetic analyses. The rate constants for unliganded channel opening and closing were also estimated for some mutants. From these measurements we calculated all of the equilibrium constants of the "allosteric" cycle as follows: diliganded gating, unliganded gating, dissociation from the C(losed) conformation, and dissociation from the O(pen) conformation. The results indicate the following. (i) These aromatic side chains play a relatively minor role in ACh receptor channel activation. (ii) The main consequence of mutations is to reduce the affinity of the O conformation of the binding site for ACh, with the effect being greater at the epsilon subunit. (iii) In epsilon (but not delta) the aromatic nature of the side chain is important in determining affinity, to a slightly greater degree in the O conformation. Phi value analyses (of both tryptophan residues) show Phi approximately 1 for both the ACh binding and diliganded gating reactions. (iv) This suggests that the structural boundaries of the dynamic elements of the gating conformational change may not be subunit-delimited, and (v) the mutated tryptophan residues experience energy changes that occur relatively early in both the ligand-binding and channel-gating reactions. PMID:19171937

  4. Different channel properties of Torpedo acetylcholine receptor monomers and dimers reconstituted in planar membranes.

    PubMed Central

    Schindler, H; Spillecke, F; Neumann, E

    1984-01-01

    It is demonstrated that the monomeric and dimeric structures of the nicotinic acetylcholine receptor of Torpedo californica electric tissue, reconstituted in planar lipid bilayers, are functionally different. The native dimer D of Mr 500,000 (heavy-form) exhibits a "single" channel conductance about twice as large as that of the monomer M of Mr 250,000 (light form). Under conditions where monomers aggregate, the conductance changes from the level of the monomer M to that of dimers M2. The dimer conductances (D and M2) seem to result from synchronous opening and closing of the two channels in the dimer, giving the impression of "single channel" activity. This channel cooperativity is apparently mediated by noncovalent interactions between the two monomers, since it requires no disulfide linkage between monomers. Both the monomers M and the dimers D and M2 show at least one substate of lower conductivity. The relative population of the two conductance levels depends on the ion type (Na+ and K+), indicating ion-specific channel states. Since the channel conductance of isolated dimers resembles those obtained from unextracted microsacs, the dimer with two synchronized channels appears to be the in vivo predominant gating unit. In the linear association of dimers, observed in the native membrane, channel synchronization may extend to more than two channels as suggested by oligomeric channel cooperativity in associations of monomers and dimers. PMID:6091143

  5. Towards a unified theory of calmodulin regulation (calmodulation) of voltage-gated calcium and sodium channels

    PubMed Central

    Yue, David T.

    2016-01-01

    Voltage-gated Na and Ca2+ channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though long-sought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge. Here, we review recent advancements in the understanding of calmodulation of Ca2+ and Na channels that suggest a remarkable similarity in their regulatory scheme. This interrelation between the two channel families now paves the way towards a unified mechanistic framework to understand vital calmodulin-dependent feedback and offers shared principles to approach related channelopathic diseases. An exciting era of synergistic study now looms. PMID:25966688

  6. Artificial phosphorylation sites modulate the activity of a voltage-gated potassium channel

    NASA Astrophysics Data System (ADS)

    Ariyaratne, Amila; Zocchi, Giovanni

    2015-03-01

    The KvAP potassium channel is representative of a family of voltage-gated ion channels where the membrane potential is sensed by a transmembrane helix containing several positively charged arginines. Previous work by Wang and Zocchi [A. Wang and G. Zocchi, PLoS ONE 6, e18598 (2011), 10.1371/journal.pone.0018598] showed how a negatively charged polyelectrolyte attached in proximity to the voltage sensing element can bias the opening probability of the channel. Here we introduce three phosphorylation sites at the same location and show that the response curve of the channel shifts by about 20 mV upon phosphorylation, while other characteristics such as the single-channel conductance are unaffected. In summary, we construct an artificial phosphorylation site which confers allosteric regulation to the channel.

  7. Control of Neuronal Voltage-Gated Calcium Ion Channels From RNA to Protein

    PubMed Central

    Lipscombe, Diane; Allen, Summer E; Toro, Cecilia P.

    2013-01-01

    Voltage-gated calcium (CaV) ion channels convert neuronal activity into rapid intracellular calcium signals to trigger a myriad of cellular responses. Their involvement in major neurological and psychiatric diseases, and importance as therapeutic targets, has propelled interest in subcellular-specific mechanisms that align CaV channel activity to specific tasks. Here we highlight recent studies that delineate mechanisms controlling the expression of CaV channels at the level of RNA and protein. We discuss the roles of RNA editing and alternative pre-mRNA splicing in generating CaV channel isoforms with activities specific to the demands of individual cells; the roles of ubiquitination and accessory proteins in regulating CaV channel expression; and the specific binding partners which contribute to both pre- and post- synaptic CaV channel function. PMID:23907011

  8. Control of neuronal voltage-gated calcium ion channels from RNA to protein.

    PubMed

    Lipscombe, Diane; Allen, Summer E; Toro, Cecilia P

    2013-10-01

    Voltage-gated calcium ion (CaV) channels convert neuronal activity into rapid intracellular calcium signals to trigger a myriad of cellular responses. Their involvement in major neurological and psychiatric diseases, and importance as therapeutic targets, has propelled interest in subcellular-specific mechanisms that align CaV channel activity to specific tasks. Here, we highlight recent studies that delineate mechanisms controlling the expression of CaV channels at the level of RNA and protein. We discuss the roles of RNA editing and alternative pre-mRNA splicing in generating CaV channel isoforms with activities specific to the demands of individual cells; the roles of ubiquitination and accessory proteins in regulating CaV channel expression; and the specific binding partners that contribute to both pre- and postsynaptic CaV channel function. PMID:23907011

  9. Molecular basis of ion permeability in a voltage-gated sodium channel.

    PubMed

    Naylor, Claire E; Bagnéris, Claire; DeCaen, Paul G; Sula, Altin; Scaglione, Antonella; Clapham, David E; Wallace, B A

    2016-04-15

    Voltage-gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na(+) ≈ Li(+) ≫ K(+), Ca(2+), Mg(2+)) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones. PMID:26873592

  10. Post-translational regulation of P2X receptor channels: modulation by phospholipids

    PubMed Central

    Bernier, Louis-Philippe; Ase, Ariel R.; Séguéla, Philippe

    2013-01-01

    P2X receptor channels mediate fast excitatory signaling by ATP and play major roles in sensory transduction, neuro-immune communication and inflammatory response. P2X receptors constitute a gene family of calcium-permeable ATP-gated cation channels therefore the regulation of P2X signaling is critical for both membrane potential and intracellular calcium homeostasis. Phosphoinositides (PIPn) are anionic signaling phospholipids that act as functional regulators of many types of ion channels. Direct PIPn binding was demonstrated for several ligand- or voltage-gated ion channels, however no generic motif emerged to accurately predict lipid-protein binding sites. This review presents what is currently known about the modulation of the different P2X subtypes by phospholipids and about critical determinants underlying their sensitivity to PIPn levels in the plasma membrane. All functional mammalian P2X subtypes tested, with the notable exception of P2X5, have been shown to be positively modulated by PIPn, i.e., homomeric P2X1, P2X2, P2X3, P2X4, and P2X7, as well as heteromeric P2X1/5 and P2X2/3 receptors. Based on various results reported on the aforementioned subtypes including mutagenesis of the prototypical PIPn-sensitive P2X4 and PIPn-insensitive P2X5 receptor subtypes, an increasing amount of functional, biochemical and structural evidence converges on the modulatory role of a short polybasic domain located in the proximal C-terminus of P2X subunits. This linear motif, semi-conserved in the P2X family, seems necessary and sufficient for encoding direct modulation of ATP-gated channels by PIPn. Furthermore, the physiological impact of the regulation of ionotropic purinergic responses by phospholipids on pain pathways was recently revealed in the context of native crosstalks between phospholipase C (PLC)-linked metabotropic receptors and P2X receptor channels in dorsal root ganglion sensory neurons and microglia. PMID:24324400

  11. Molecular basis for the toxin insensitivity of scorpion voltage-gated potassium channel MmKv1.

    PubMed

    Zhang, Chuangeng; Xie, Zili; Li, Xinxin; Chen, Jing; Feng, Jing; Lang, Yange; Yang, Weishan; Li, Wenxin; Chen, Zongyun; Yao, Jing; Cao, Zhijian; Wu, Yingliang

    2016-05-01

    Scorpions are insensitive to their own venoms, which contain various neurotoxins specific for mammalian or insect ion channels, whose molecular mechanism remains unsolved. Using MmKv1, a potassium channel identified from the genome of the scorpion Mesobuthus martensii, channel kinetic experiments showed that MmKv1 was a classical voltage-gated potassium channel with a voltage-dependent fast activation and slow inactivation. Compared with the human Kv1.3 channel (hKv1.3), the MmKv1 channel exhibited a remarkable insensitivity to both scorpion venom and toxin. The chimaeric channels of MmKv1 and hKv1.3 revealed that both turret and filter regions of the MmKv1 channel were critical for the toxin insensitivity of MmKv1. Furthermore, mutagenesis of the chimaeric channel indicated that two basic residues (Arg(399) and Lys(403)) in the MmKv1 turret region and Arg(425) in the MmKv1 filter region significantly affected its toxin insensitivity. Moreover, when these three basic residues of MmKv1 were simultaneously substituted with the corresponding residues from hKv1.3, the MmKv1-R399T/K403S/R425H mutant channels exhibited similar sensitivity to both scorpion venom and toxin to hKv1.3, which revealed the determining role of these three basic residues in the toxin insensitivity of the MmKv1 channel. More strikingly, a similar triad sequence structure is present in all Shaker-like channels from venomous invertebrates, which suggested a possible convergent functional evolution of these channels to enable them to resist their own venoms. Together, these findings first illustrate the mechanism by which scorpions are insensitive to their own venoms at the ion channel receptor level and enrich our knowledge of the insensitivity of scorpions and other venomous animals to their own venoms. PMID:26951716

  12. CFTR gating I: Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (DeltaR-CFTR).

    PubMed

    Bompadre, Silvia G; Ai, Tomohiko; Cho, Jeong Han; Wang, Xiaohui; Sohma, Yoshiro; Li, Min; Hwang, Tzyh-Chang

    2005-04-01

    The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed DeltaR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the DeltaR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal DeltaR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of DeltaR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and DeltaR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the DeltaR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing. PMID:15767295

  13. The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels

    PubMed Central

    Bavi, Navid; Cortes, D. Marien; Cox, Charles D.; Rohde, Paul R.; Liu, Weihong; Deitmer, Joachim W.; Bavi, Omid; Strop, Pavel; Hill, Adam P.; Rees, Douglas; Corry, Ben; Perozo, Eduardo; Martinac, Boris

    2016-01-01

    The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics. PMID:27329693

  14. The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels.

    PubMed

    Bavi, Navid; Cortes, D Marien; Cox, Charles D; Rohde, Paul R; Liu, Weihong; Deitmer, Joachim W; Bavi, Omid; Strop, Pavel; Hill, Adam P; Rees, Douglas; Corry, Ben; Perozo, Eduardo; Martinac, Boris

    2016-01-01

    The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics. PMID:27329693

  15. Molecular and functional characterization of voltage-gated sodium channels in human sperm

    PubMed Central

    Pinto, Francisco M; Ravina, Cristina G; Fernández-Sánchez, Manuel; Gallardo-Castro, Manuel; Cejudo-Román, Antonio; Candenas, Luz

    2009-01-01

    Background We have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility. Methods Freshly ejaculated semen was collected from thirty normozoospermic human donors, with each donor supplying 2 different samples. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence techniques were used to detect the mRNAs and proteins of interest. Sperm motility was measured by a computer-assisted sperm analysis system (CASA). Cytosolic free calcium was determined by fluorimetry in cells loaded with the fluorescent calcium indicator Fura-2. Results The mRNAs that encode the different Nav alpha subunits (Nav1.1-1.9) were all expressed in capacitated human spermatozoa. The mRNAs of the auxiliary subunits beta1, beta3 and beta4 were also present. Immunofluorescence studies showed that, with the exception of Nav1.1 and Nav1.3, the Nav channel proteins were present in sperm cells and show specific and different sites of localization. Veratridine, a voltage-gated sodium channel activator, caused time- and concentration-dependent increases in progressive sperm motility. In sperm suspensions loaded with Fura-2, veratridine did not modify intracellular free calcium levels. Conclusion This research shows the presence of voltage-gated sodium channels in human sperm and supports a role for these channels in the regulation of mature sperm function. PMID:19607678

  16. Cellular defibrillation: interaction of micro-scale electric fields with voltage-gated ion channels.

    PubMed

    Kargol, Armin; Malkinski, Leszek; Eskandari, Rahmatollah; Carter, Maya; Livingston, Daniel

    2015-09-01

    We study the effect of micro-scale electric fields on voltage-gated ion channels in mammalian cell membranes. Such micro- and nano-scale electric fields mimic the effects of multiferroic nanoparticles that were recently proposed [1] as a novel way of controlling the function of voltage-sensing biomolecules such as ion channels. This article describes experimental procedures and initial results that reveal the effect of the electric field, in close proximity of cells, on the ion transport through voltage-gated ion channels. We present two configurations of the whole-cell patch-clamping apparatus that were used to detect the effect of external stimulation on ionic currents and discuss preliminary results that indicate modulation of the ionic currents consistent with the applied stimulus. PMID:26067055

  17. Elimination of Rapid Potassium Channel Inactivation by Phosphorylation of the Inactivation Gate

    PubMed Central

    Covarrubias, Manuel; Wei, Aguan; Salkoff, Lawrence; Vyas, Tapan B.

    2008-01-01

    Summary The effect of protein kinase C (PKC) on rapid N-type inactivation of K+ channels has not been reported previously. We found that PKC specifically eliminates rapid inactivation of a cloned human A-type K+ channel (hKv3.4), converting this channel from a rapidly inactivating A type to a noninactivating delayed rectifier type. Biochemical analysis showed that the N-terminal domain of hKv3.4 is phosphorylated in vitro by PKC, and mutagenesis experiments revealed that two serines within the inactivation gate at the N-terminus are sites of direct PKC action. Moreover, mutating one of these serines to aspartic acid mimics the action of PKC. Serine phosphorylation may thus prevent rapid inactivation by shielding basic residues known to be critical to the function of the inactivation gate. The regulatory mechanism reported here may have substantial effects on signal coding in the nervous system. PMID:7993631

  18. Gating of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels by Adenosine Triphosphate Hydrolysis

    PubMed Central

    Zeltwanger, Shawn; Wang, Fei; Wang, Guo-Tang; Gillis, Kevin D.; Hwang, Tzyh-Chang

    1999-01-01

    Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant (τc) decreased with increasing [ATP] to a minimum value of ∼0.43 s at [ATP] >1.00 mM. The open time constant (τo) increased with increasing [ATP] with a minimal τo of ∼260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of ∼250 ms is dominant at low ATP concentrations (10 μM) and a much longer open state with a time constant of ∼3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent. PMID:10102935

  19. Nonlinearity of a Voltage-Gated Potassium Channel Revealed by the Mechanical Susceptibility

    NASA Astrophysics Data System (ADS)

    Ariyaratne, Amila; Zocchi, Giovanni

    2013-01-01

    The voltage-gated potassium channel from Aeropyrum pernix operates by coupling the voltage-driven motion of a charged group of amino acids to the opening and closing of the pore. In this experiment, we drive this charged group with an ac field and observe the effect on the gating. The measurements for different frequencies and amplitudes of the forcing reveal an essential nonlinearity in the mechanical behavior of the molecule. Within a continuum-mechanics description, we extract the effective dissipation parameter γ for this conformational motion and find γ≈0.2g/s, similar to recent nanorheology measurements on the conformational motion of an enzyme.

  20. Computational Structural Pharmacology and Toxicology of Voltage-Gated Sodium Channels.

    PubMed

    Zhorov, B S; Tikhonov, D B

    2016-01-01

    Voltage-gated sodium channels are targets for many toxins and medically important drugs. Despite decades of intensive studies in industry and academia, atomic mechanisms of action are still not completely understood. The major cause is a lack of high-resolution structures of eukaryotic channels and their complexes with ligands. In these circumstances a useful approach is homology modeling that employs as templates X-ray structures of potassium channels and prokaryotic sodium channels. On one hand, due to inherent limitations of this approach, results should be treated with caution. In particular, models should be tested against relevant experimental data. On the other hand, docking of drugs and toxins in homology models provides a unique possibility to integrate diverse experimental data provided by mutational analysis, electrophysiology, and studies of structure-activity relations. Here we describe how homology modeling advanced our understanding of mechanisms of several classes of ligands. These include tetrodotoxins and mu-conotoxins that block the outer pore, local anesthetics that block of the inner pore, batrachotoxin that binds in the inner pore but, paradoxically, activates the channel, pyrethroid insecticides that activate the channel by binding at lipid-exposed repeat interfaces, and scorpion alpha and beta-toxins, which bind between the pore and voltage-sensing domains and modify the channel gating. We emphasize importance of experimental data for elaborating the models. PMID:27586283

  1. Voltage-gated proton (H(v)1) channels, a singular voltage sensing domain.

    PubMed

    Castillo, Karen; Pupo, Amaury; Baez-Nieto, David; Contreras, Gustavo F; Morera, Francisco J; Neely, Alan; Latorre, Ramon; Gonzalez, Carlos

    2015-11-14

    The main role of voltage-gated proton channels (Hv1) is to extrude protons from the intracellular milieu when, mediated by different cellular processes, the H(+) concentration increases. Hv1 are exquisitely selective for protons and their structure is homologous to the voltage sensing domain (VSD) of other voltage-gated ion channels like sodium, potassium, and calcium channels. In clear contrast to the classical voltage-dependent channels, Hv1 lacks a pore domain and thus permeation necessarily occurs through the voltage sensing domain. Hv1 channels are activated by depolarizing voltages, and increases in internal proton concentration. It has been proposed that local conformational changes of the transmembrane segment S4, driven by depolarization, trigger the molecular rearrangements that open Hv1. However, it is still unclear how the electromechanical coupling is achieved between the VSD and the potential pore, allowing the proton flux from the intracellular to the extracellular side. Here we provide a revised view of voltage activation in Hv1 channels, offering a comparative scenario with other voltage sensing channels domains. PMID:26296320

  2. KCNE1 and KCNE3: The yin and yang of voltage-gated K(+) channel regulation.

    PubMed

    Abbott, Geoffrey W

    2016-01-15

    The human KCNE gene family comprises five genes encoding single transmembrane-spanning ion channel regulatory subunits. The primary function of KCNE subunits appears to be regulation of voltage-gated potassium (Kv) channels, and the best-understood KCNE complexes are with the KCNQ1 Kv α subunit. Here, we review the often opposite effects of KCNE1 and KCNE3 on Kv channel biology, with an emphasis on regulation of KCNQ1. Slow-activating IKs channel complexes formed by KCNQ1 and KCNE1 are essential for human ventricular myocyte repolarization, while constitutively active KCNQ1-KCNE3 channels are important in the intestine. Inherited sequence variants in human KCNE1 and KCNE3 cause cardiac arrhythmias but by different mechanisms, and each is important for hearing in unique ways. Because of their contrasting effects on KCNQ1 function, KCNE1 and KCNE3 have proved invaluable tools in the mechanistic understanding of how channel gating can be manipulated, and each may also provide a window into novel insights and new therapeutic opportunities in K(+) channel pharmacology. Finally, findings from studies of Kcne1(-/-) and Kcne3(-/-) mouse lines serve to illustrate the complexity of KCNE biology and KCNE-linked disease states. PMID:26410412

  3. STIM1 gates the store-operated calcium channel ORAI1 in vitro.

    PubMed

    Zhou, Yubin; Meraner, Paul; Kwon, Hyoung T; Machnes, Danya; Oh-hora, Masatsugu; Zimmer, Jochen; Huang, Yun; Stura, Antonio; Rao, Anjana; Hogan, Patrick G

    2010-01-01

    Store-operated Ca(2+) entry through the plasma membrane Ca(2+) release-activated Ca(2+) (CRAC) channel in mammalian T cells and mast cells depends on the sensor protein stromal interaction molecule 1 (STIM1) and the channel subunit ORAI1. To study STIM1-ORAI1 signaling in vitro, we have expressed human ORAI1 in a sec6-4 strain of the yeast Saccharomyces cerevisiae and isolated sealed membrane vesicles carrying ORAI1 from the Golgi compartment to the plasma membrane. We show by in vitro Ca(2+) flux assays that bacterially expressed recombinant STIM1 opens wild-type ORAI1 channels but not channels assembled from the ORAI1 pore mutant E106Q or the ORAI1 severe combined immunodeficiency (SCID) mutant R91W. These experiments show that the STIM1-ORAI1 interaction is sufficient to gate recombinant human ORAI1 channels in the absence of other proteins of the human ORAI1 channel complex, and they set the stage for further biochemical and biophysical dissection of ORAI1 channel gating. PMID:20037597

  4. Solution-Based Single-Molecule FRET Studies of K(+) Channel Gating in a Lipid Bilayer.

    PubMed

    Sadler, Emma E; Kapanidis, Achillefs N; Tucker, Stephen J

    2016-06-21

    Ion channels are dynamic multimeric proteins that often undergo multiple unsynchronized structural movements as they switch between their open and closed states. Such structural changes are difficult to measure within the context of a native lipid bilayer and have often been monitored via macroscopic changes in Förster resonance energy transfer (FRET) between probes attached to different parts of the protein. However, the resolution of this approach is limited by ensemble averaging of structurally heterogeneous subpopulations. These problems can be overcome by measurement of FRET in single molecules, but this presents many challenges, in particular the ability to control labeling of subunits within a multimeric protein with acceptor and donor fluorophores, as well as the requirement to image large numbers of individual molecules in a membrane environment. To address these challenges, we randomly labeled tetrameric KirBac1.1 potassium channels, reconstituted them into lipid nanodiscs, and performed single-molecule FRET confocal microscopy with alternating-laser excitation as the channels diffused in solution. These solution-based single-molecule FRET measurements of a multimeric ion channel in a lipid bilayer have allowed us to probe the structural changes that occur upon channel activation and inhibition. Our results provide direct evidence of the twist-to-shrink movement of the helix bundle crossing during channel gating and demonstrate how this method might be applied to real-time structural studies of ion channel gating. PMID:27332124

  5. Enhanced activation of an amino-terminally truncated isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells

    PubMed Central

    Hondares, Elayne; Brown, Mark Adrian; Musset, Boris; Morgan, Deri; Cherny, Vladimir V.; Taubert, Christina; Bhamrah, Mandeep K.; Coe, David; Marelli-Berg, Federica; Gribben, John G.; Dyer, Martin J. S.; DeCoursey, Thomas E.; Capasso, Melania

    2014-01-01

    HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1L, Thr29. Furthermore, the association of HVCN1S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies. PMID:25425665

  6. Relationship between nucleotide binding and ion channel gating in cystic fibrosis transmembrane conductance regulator.

    PubMed

    Aleksandrov, Andrei A; Cui, Liying; Riordan, John R

    2009-06-15

    We have employed rate-equilibrium free energy relationship (REFER) analysis to characterize the dynamic events involved in the allosteric regulation of cystic fibrosis transmembrane conductance regulator (CFTR) function. A wide range of different hydrolysable and poorly hydrolysable nucleoside triphosphates were used to elucidate the role of ATP hydrolysis in CFTR function. The linearity of the REFER plots and Phi values near unity for all ligands tested implies that CFTR channel gating is a reversible thermally driven process with all structural reorganization in the binding site(s) completed prior to channel opening. This is consistent with the requirement for nucleotide binding for channel opening. However, the channel structural transition from the open to the closed state occurs independently of any events in the binding sites. Similar results were obtained on substitution of amino acids at coupling joints between both nucleotide binding domains (NBD) and cytoplasmic loops (CL) in opposite halves of the protein, indicating that any structural reorganization there also had occurred in the channel closed state. The fact that fractional Phi values were not observed in either of these distant sites suggests that there may not be a deterministic 'lever-arm' mechanism acting between nucleotide binding sites and the channel gate. These findings favour a stochastic coupling between binding and gating in which all structural transitions are thermally driven processes. We speculate that increase of channel open state probability is due to reduction of the number of the closed state configurations available after physical interaction between ligand bound NBDs and the channel. PMID:19403599

  7. Relationship between nucleotide binding and ion channel gating in cystic fibrosis transmembrane conductance regulator

    PubMed Central

    Aleksandrov, Andrei A; Cui, Liying; Riordan, John R

    2009-01-01

    We have employed rate-equilibrium free energy relationship (REFER) analysis to characterize the dynamic events involved in the allosteric regulation of cystic fibrosis transmembrane conductance regulator (CFTR) function. A wide range of different hydrolysable and poorly hydrolysable nucleoside triphosphates were used to elucidate the role of ATP hydrolysis in CFTR function. The linearity of the REFER plots and Φ values near unity for all ligands tested implies that CFTR channel gating is a reversible thermally driven process with all structural reorganization in the binding site(s) completed prior to channel opening. This is consistent with the requirement for nucleotide binding for channel opening. However, the channel structural transition from the open to the closed state occurs independently of any events in the binding sites. Similar results were obtained on substitution of amino acids at coupling joints between both nucleotide binding domains (NBD) and cytoplasmic loops (CL) in opposite halves of the protein, indicating that any structural reorganization there also had occurred in the channel closed state. The fact that fractional Φ values were not observed in either of these distant sites suggests that there may not be a deterministic ‘lever-arm’ mechanism acting between nucleotide binding sites and the channel gate. These findings favour a stochastic coupling between binding and gating in which all structural transitions are thermally driven processes. We speculate that increase of channel open state probability is due to reduction of the number of the closed state configurations available after physical interaction between ligand bound NBDs and the channel. PMID:19403599

  8. Neurotoxins and their binding areas on voltage-gated sodium channels.

    PubMed

    Stevens, Marijke; Peigneur, Steve; Tytgat, Jan

    2011-01-01

    Voltage-gated sodium channels (VGSCs) are large transmembrane proteins that conduct sodium ions across the membrane and by doing so they generate signals of communication between many kinds of tissues. They are responsible for the generation and propagation of action potentials in excitable cells, in close collaboration with other channels like potassium channels. Therefore, genetic defects in sodium channel genes can cause a wide variety of diseases, generally called "channelopathies." The first insights into the mechanism of action potentials and the involvement of sodium channels originated from Hodgkin and Huxley for which they were awarded the Nobel Prize in 1963. These concepts still form the basis for understanding the function of VGSCs. When VGSCs sense a sufficient change in membrane potential, they are activated and consequently generate a massive influx of sodium ions. Immediately after, channels will start to inactivate and currents decrease. In the inactivated state, channels stay refractory for new stimuli and they must return to the closed state before being susceptible to a new depolarization. On the other hand, studies with neurotoxins like tetrodotoxin (TTX) and saxitoxin (STX) also contributed largely to our today's understanding of the structure and function of ion channels and of VGSCs specifically. Moreover, neurotoxins acting on ion channels turned out to be valuable lead compounds in the development of new drugs for the enormous range of diseases in which ion channels are involved. A recent example of a synthetic neurotoxin that made it to the market is ziconotide (Prialt(®), Elan). The original peptide, ω-MVIIA, is derived from the cone snail Conus magus and now FDA/EMA-approved for the management of severe chronic pain by blocking the N-type voltage-gated calcium channels in pain fibers. This review focuses on the current status of research on neurotoxins acting on VGSC, their contribution to further unravel the structure and function of

  9. Neurotoxins and Their Binding Areas on Voltage-Gated Sodium Channels

    PubMed Central

    Stevens, Marijke; Peigneur, Steve; Tytgat, Jan

    2011-01-01

    Voltage-gated sodium channels (VGSCs) are large transmembrane proteins that conduct sodium ions across the membrane and by doing so they generate signals of communication between many kinds of tissues. They are responsible for the generation and propagation of action potentials in excitable cells, in close collaboration with other channels like potassium channels. Therefore, genetic defects in sodium channel genes can cause a wide variety of diseases, generally called “channelopathies.” The first insights into the mechanism of action potentials and the involvement of sodium channels originated from Hodgkin and Huxley for which they were awarded the Nobel Prize in 1963. These concepts still form the basis for understanding the function of VGSCs. When VGSCs sense a sufficient change in membrane potential, they are activated and consequently generate a massive influx of sodium ions. Immediately after, channels will start to inactivate and currents decrease. In the inactivated state, channels stay refractory for new stimuli and they must return to the closed state before being susceptible to a new depolarization. On the other hand, studies with neurotoxins like tetrodotoxin (TTX) and saxitoxin (STX) also contributed largely to our today’s understanding of the structure and function of ion channels and of VGSCs specifically. Moreover, neurotoxins acting on ion channels turned out to be valuable lead compounds in the development of new drugs for the enormous range of diseases in which ion channels are involved. A recent example of a synthetic neurotoxin that made it to the market is ziconotide (Prialt®, Elan). The original peptide, ω-MVIIA, is derived from the cone snail Conus magus and now FDA/EMA-approved for the management of severe chronic pain by blocking the N-type voltage-gated calcium channels in pain fibers. This review focuses on the current status of research on neurotoxins acting on VGSC, their contribution to further unravel the structure and

  10. Temperature and voltage coupling to channel opening in transient receptor potential melastatin 8 (TRPM8).

    PubMed

    Raddatz, Natalia; Castillo, Juan P; Gonzalez, Carlos; Alvarez, Osvaldo; Latorre, Ramon

    2014-12-19

    Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca(2+)-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol(-1). The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening. PMID:25352597

  11. Temperature and Voltage Coupling to Channel Opening in Transient Receptor Potential Melastatin 8 (TRPM8)*♦

    PubMed Central

    Raddatz, Natalia; Castillo, Juan P.; Gonzalez, Carlos; Alvarez, Osvaldo; Latorre, Ramon

    2014-01-01

    Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca2+-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol−1. The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening. PMID:25352597

  12. Dendritic NMDA receptors activate axonal calcium channels

    PubMed Central

    Christie, Jason M.; Jahr, Craig E.

    2008-01-01

    Summary NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca2+ and monovalent cations, they could alter release directly by increasing presynaptic Ca2+ or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca2+ channels (VSCCs). Using two-photon microscopy to measure Ca2+ excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca2+ transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca2+ entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca2+-dependent forms of presynaptic plasticity and release. PMID:18957221

  13. Mechanism of anion permeation through channels gated by glycine and gamma-aminobutyric acid in mouse cultured spinal neurones.

    PubMed Central

    Bormann, J; Hamill, O P; Sakmann, B

    1987-01-01

    1. The ion-selective and ion transport properties of glycine receptor (GlyR) and gamma-aminobutyric acid receptor (GABAR) channels in the soma membrane of mouse spinal cord neurones were investigated using the whole-cell, cell-attached and outside-out patch versions of the patch-clamp technique. 2. Current-voltage (I-V) relations of transmitter-activated currents obtained from whole-cell measurements with 145 mM-Cl- intracellularly and extracellularly, showed outward rectification. In voltage-jump experiments, the instantaneous I-V relations were linear, and the steady-state I-V relations were rectifying outwardly indicating that the gating of GlyR and GABAR channels is voltage sensitive. 3. The reversal potential of whole-cell currents shifted 56 mV per tenfold change in internal Cl- activity indicating activation of Cl(-)-selective channels. The permeability ratio of K+ to Cl- (PK/PCl) was smaller than 0.05 for both channels. 4. The permeability sequence for large polyatomic anions was formate greater than bicarbonate greater than acetate greater than phosphate greater than propionate for GABAR channels; phosphate and propionate were not measurably permeant in GlyR channels. This indicates that open GlyR and GABAR channels have effective pore diameters of 5.2 and 5.6 A, respectively. The sequence of relative permeabilities for small anions was SCN- greater than I- greater than Br- greater than Cl- greater than F- for both channels. 5. GlyR and GABAR channels are multi-conductance-state channels. In cell-attached patches the single-channel slope conductances close to 0 mV membrane potential were 29, 18 and 10 pS for glycine, and 28, 17 and 10 pS for GABA-activated