Science.gov

Sample records for receptor channel gating

  1. Voltage-gated sodium channel modulation by sigma-receptors in cardiac myocytes and heterologous systems.

    PubMed

    Johannessen, Molly; Ramachandran, Subramaniam; Riemer, Logan; Ramos-Serrano, Andrea; Ruoho, Arnold E; Jackson, Meyer B

    2009-05-01

    The sigma-receptor, a broadly distributed integral membrane protein with a novel structure, is known to modulate various voltage-gated K(+) and Ca(2+) channels through a mechanism that involves neither G proteins nor phosphorylation. The present study investigated the modulation of the heart voltage-gated Na(+) channel (Na(v)1.5) by sigma-receptors. The sigma(1)-receptor ligands [SKF-10047 and (+)-pentazocine] and sigma(1)/sigma(2)-receptor ligands (haloperidol and ditolylguanidine) all reversibly inhibited Na(v)1.5 channels to varying degrees in human embryonic kidney 293 (HEK-293) cells and COS-7 cells, but the sigma(1)-receptor ligands were less effective in COS-7 cells. The same four ligands also inhibited Na(+) current in neonatal mouse cardiac myocytes. In sigma(1)-receptor knockout myocytes, the sigma(1)-receptor-specific ligands were far less effective in modulating Na(+) current, but the sigma(1)/sigma(2)-receptor ligands modulated Na(+) channels as well as in wild type. Photolabeling with the sigma(1)-receptor photoprobe [(125)I]-iodoazidococaine demonstrated that sigma(1)-receptors were abundant in heart and HEK-293 cells, but scarce in COS-7 cells. This difference was consistent with the greater efficacy of sigma(1)-receptor-specific ligands in HEK-293 cells than in COS-7 cells. sigma-Receptors modulated Na(+) channels despite the omission of GTP and ATP from the patch pipette solution. sigma-Receptor-mediated inhibition of Na(+) current had little if any voltage dependence and produced no change in channel kinetics. Na(+) channels represent a new addition to the large number of voltage-gated ion channels modulated by sigma-receptors. The modulation of Na(v)1.5 channels by sigma-receptors in the heart suggests an important pathway by which drugs can alter cardiac excitability and rhythmicity.

  2. 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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-06-07

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

  5. Principal pathway coupling agonist binding to channel gating in nicotinic receptors

    NASA Astrophysics Data System (ADS)

    Lee, Won Yong; Sine, Steven M.

    2005-11-01

    Synaptic receptors respond to neurotransmitters by opening an intrinsic ion channel in the final step in synaptic transmission. How binding of the neurotransmitter is conveyed over the long distance to the channel remains a central question in neurobiology. Here we delineate a principal pathway that links neurotransmitter binding to channel gating by using a structural model of the Torpedo acetylcholine receptor at 4-Å resolution, recordings of currents through single receptor channels and determinations of energetic coupling between pairs of residues. We show that a pair of invariant arginine and glutamate residues in each receptor α-subunit electrostatically links peripheral and inner β-sheets from the binding domain and positions them to engage with the channel. The key glutamate and flanking valine residues energetically couple to conserved proline and serine residues emerging from the top of the channel-forming α-helix, suggesting that this is the point at which the binding domain triggers opening of the channel. The series of interresidue couplings identified here constitutes a primary allosteric pathway that links neurotransmitter binding to channel gating.

  6. Allosteric modulation of ATP-gated P2X receptor channels

    PubMed Central

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

    2013-01-01

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

  7. Secondary Structure and Gating Rearrangements of Transmembrane Segments in Rat P2X4 Receptor Channels

    PubMed Central

    Silberberg, Shai D.; Chang, Tsg-Hui; Swartz, Kenton J.

    2005-01-01

    P2X receptors are cation selective channels that are activated by extracellular nucleotides. These channels are likely formed by three identical or related subunits, each having two transmembrane segments (TM1 and TM2). To identify regions that undergo rearrangement during gating and to probe their secondary structure, we performed tryptophan scanning mutagenesis on the two putative TMs of the rat P2X4 receptor channel. Mutant channels were expressed in Xenopus oocytes, concentration–response relationships constructed for ATP, and the EC50 estimated by fitting the Hill equation to the data. Of the 22 mutations in TM1 and 24 in TM2, all but one in TM1 and seven in TM2 result in functional channels. Interestingly, the majority of the functional mutants display an increased sensitivity to ATP, and in general these perturbations are more pronounced for TM2 when compared with TM1. For TM1 and for the outer half of TM2, the perturbations are consistent with these regions adopting α-helical secondary structures. In addition, the greatest perturbations in the gating equilibrium occur for mutations near the outer ends of both TM1 and TM2. Surface biotinylation experiments reveal that all the nonfunctional mutants traffic to the surface membrane at levels comparable to the WT channel, suggesting that these mutations likely disrupt ion conduction or gating. Taken together, these results suggest that the outer parts of TM1 and TM2 are helical and that they move during activation. The observation that the majority of nonconducting mutations are clustered toward the inner end of TM2 suggests a critical functional role for this region. PMID:15795310

  8. Voltage-gated calcium channels function as Ca2+-activated signaling receptors.

    PubMed

    Atlas, Daphne

    2014-02-01

    Voltage-gated calcium channels (VGCCs) are transmembrane cell surface proteins responsible for multifunctional signals. In response to voltage, VGCCs trigger synaptic transmission, drive muscle contraction, and regulate gene expression. Voltage perturbations open VGCCs enabling Ca(2+) binding to the low affinity Ca(2+) binding site of the channel pore. Subsequent to permeation, Ca(2+) targets selective proteins to activate diverse signaling pathways. It is becoming apparent that the Ca(2+)-bound channel triggers secretion in excitable cells and drives contraction in cardiomyocytes prior to Ca(2+) permeation. Here, I highlight recent data implicating receptor-like function of the Ca(2+)-bound channel in converting external Ca(2+) into an intracellular signal. The two sequential mechanistic perspectives of VGCC function are discussed in the context of the prevailing and long-standing current models of depolarization-evoked secretion and cardiac contraction.

  9. Nicotinic receptor M3 transmembrane domain: position 8' contributes to channel gating.

    PubMed

    De Rosa, María José; Rayes, Diego; Spitzmaul, Guillermo; Bouzat, Cecilia

    2002-08-01

    The nicotinic acetylcholine receptor (nAChR) is a pentamer of homologous subunits with composition alpha(2)(beta)(epsilon)(delta) in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 8' of the M3 domain is phenylalanine in all heteromeric alpha subunits, whereas it is a hydrophobic nonaromatic residue in non-alpha subunits. Given this peculiar conservation pattern, we studied its contribution to muscle nAChR activation by combining mutagenesis with single-channel kinetic analysis. Construction of nAChRs carrying different numbers of phenylalanine residues at 8' reveals that the mean open time decreases as a function of the number of phenylalanine residues. Thus, all subunits contribute through this position independently and additively to the channel closing rate. The impairment of channel opening increases when the number of phenylalanine residues at 8' increases from two (wild-type nAChR) to five. The gating equilibrium constant of the latter mutant nAChR is 13-fold lower than that of the wild-type nAChR. The replacement of (alpha)F8', (beta)L8', (delta)L8', and (epsilon)V8' by a series of hydrophobic amino acids reveals that the structural bases of the observed kinetic effects are nonequivalent among subunits. In the alpha subunit, hydrophobic amino acids at 8' lead to prolonged channel lifetimes, whereas they lead either to normal kinetics (delta and epsilon subunits) or impaired channel gating (beta subunit) in the non-alpha subunits. The overall results indicate that 8' positions of the M3 domains of all subunits contribute to channel gating.

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

    PubMed Central

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

    2011-01-01

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

  11. Tuning the allosteric regulation of artificial muscarinic and dopaminergic ligand-gated potassium channels by protein engineering of G protein-coupled receptors

    PubMed Central

    Moreau, Christophe J.; Revilloud, Jean; Caro, Lydia N.; Dupuis, Julien P.; Trouchet, Amandine; Estrada-Mondragón, Argel; Nieścierowicz, Katarzyna; Sapay, Nicolas; Crouzy, Serge; Vivaudou, Michel

    2017-01-01

    Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications. PMID:28145461

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

    PubMed

    Gurevitz, Michael

    2012-09-15

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

  13. Sources of energy for gating by neurotransmitters in acetylcholine receptor channels.

    PubMed

    Purohit, Prasad; Bruhova, Iva; Auerbach, Anthony

    2012-06-12

    Nicotinic acetylcholine receptors (AChRs) mediate signaling in the central and peripheral nervous systems. The AChR gating conformational change is powered by a low- to high-affinity change for neurotransmitters at two transmitter binding sites. We estimated (from single-channel currents) the components of energy for gating arising from binding site aromatic residues in the α-subunit. All mutations reduced the energy (TyrC1>TrpB≈TyrC2>TyrA), with TyrC1 providing ~40% of the total. Considered one at a time, the fractional energy contributions from the aromatic rings were TrpB ~35%, TyrC1 ~28%, TyrC2 ~28%, and TyrA ~10%. Together, TrpB, TyrC1, and TyrC2 comprise an "aromatic triad" that provides much of the total energy from the transmitter for gating. Analysis of mutant pairs suggests that the energy contributions from some residues are nearly independent. Mutations of TyrC1 cause particularly large energy reductions because they remove two favorable and approximately equal interactions between the aromatic ring and the quaternary amine of the agonist and between the hydroxyl and αLysβ7.

  14. 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.

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

    PubMed Central

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

    2017-01-01

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

  16. Fundamental Gating Mechanism of Nicotinic Receptor Channel Revealed by Mutation Causing a Congenital Myasthenic Syndrome

    PubMed Central

    Wang, Hai-Long; Ohno, Kinji; Milone, Margherita; Brengman, Joan M.; Evoli, Amelia; Batocchi, Anna-Paola; Middleton, Lefkos T.; Christodoulou, Kyproula; Engel, Andrew G.; Sine, Steven M.

    2000-01-01

    We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation εA411P in the amphipathic helix of the acetylcholine receptor (AChR) ε subunit. Myasthenic patients from three unrelated families are either homozygous for εA411P or are heterozygous and harbor a null mutation in the second ε allele, indicating that εA411P is recessive. We expressed human AChRs containing wild-type or A411P ε subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by εA411P. Prolines engineered into positions flanking residue 411 of the ε subunit greatly increase the range of activation kinetics similar to εA411P, whereas prolines engineered into positions equivalent to εA411 in β and δ subunits are without effect. Thus, the amphipathic helix of the ε subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well. PMID:10962020

  17. Antagonist action of progesterone at σ-receptors in the modulation of voltage-gated sodium channels.

    PubMed

    Johannessen, Molly; Fontanilla, Dominique; Mavlyutov, Timur; Ruoho, Arnold E; Jackson, Meyer B

    2011-02-01

    σ-Receptors are integral membrane proteins that have been implicated in a number of biological functions, many of which involve the modulation of ion channels. A wide range of synthetic ligands activate σ-receptors, but endogenous σ-receptor ligands have proven elusive. One endogenous ligand, dimethyltryptamine (DMT), has been shown to act as a σ-receptor agonist. Progesterone and other steroids bind σ-receptors, but the functional consequences of these interactions are unclear. Here we investigated progesterone binding to σ(1)- and σ(2)-receptors and evaluated its effect on σ-receptor-mediated modulation of voltage-gated Na(+) channels. Progesterone binds both σ-receptor subtypes in liver membranes with comparable affinities and blocks photolabeling of both subtypes in human embryonic kidney 293 cells that stably express the human cardiac Na(+) channel Na(v)1.5. Patch-clamp recording in this cell line tested Na(+) current modulation by the σ-receptor ligands ditolylguanidine, PB28, (+)SKF10047, and DMT. Progesterone inhibited the action of these ligands to varying degrees, and some of these actions were reduced by σ(1)-receptor knockdown with small interfering RNA. Progesterone inhibition of channel modulation by drugs was consistent with stronger antagonism of σ(2)-receptors. By contrast, progesterone inhibition of channel modulation by DMT was consistent with stronger antagonism of σ(1)-receptors. Progesterone binding to σ-receptors blocks σ-receptor-mediated modulation of a voltage-gated ion channel, and this novel membrane action of progesterone may be relevant to changes in brain and cardiovascular function during endocrine transitions.

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

    PubMed Central

    Gavazzo, P; Picco, C; Menini, A

    1997-01-01

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

  19. 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

  20. Energy for wild-type acetylcholine receptor channel gating from different choline derivatives.

    PubMed

    Bruhova, Iva; Gregg, Timothy; Auerbach, Anthony

    2013-02-05

    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 (ΔG(B)). To understand the distinct actions of structurally related agonist molecules, we measured ΔG(B) 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 ΔG(B) 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.

  1. Cross-talk and co-trafficking between rho1/GABA receptors and ATP-gated channels.

    PubMed

    Boué-Grabot, Eric; Emerit, Michel B; Toulmé, Estelle; Séguéla, Philippe; Garret, Maurice

    2004-02-20

    Gamma-aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functionally different classes of neurotransmitter-gated channels involved in fast synaptic transmission. We demonstrate here that, when the inhibitory rho1/GABA and the excitatory P2X2 receptor channels are co-expressed in Xenopus oocytes, activation of one channel reduces the currents mediated by the other one. This reciprocal inhibitory cross-talk is a receptor-mediated phenomenon independent of agonist cross-modulation, membrane potential, direction of ionic flux, or channel densities. Functional interaction is disrupted when the cytoplasmic C-terminal domain of P2X2 is deleted or in competition experiments with minigenes coding for the C-terminal domain of P2X2 or the main intracellular loop of rho1 subunits. We also show a physical interaction between P2X2 and rho1 receptors expressed in oocytes and the co-clustering of these receptors in transfected hippocampal neurons. Co-expression with P2X2 induces retargeting and recruitment of mainly intracellular rho1/GABA receptors to surface clusters. Therefore, molecular and functional cross-talk between inhibitory and excitatory ligand-gated channels may regulate synaptic strength both by activity-dependent current occlusion and synaptic receptors co-trafficking.

  2. Role of pairwise interactions between M1 and M2 domains of the nicotinic receptor in channel gating.

    PubMed

    Corradi, Jeremías; Spitzmaul, Guillermo; De Rosa, María José; Costabel, Marcelo; Bouzat, Cecilia

    2007-01-01

    The adult form of the nicotinic acetylcholine receptor (AChR) consists of five subunits (alpha(2)betaepsilondelta), each having four transmembrane domains (M1-M4). The atomic model of the nicotinic acetylcholine receptor shows that the pore-lining M2 domains make no extensive contacts with the rest of the transmembrane domains. However, there are several sites where close appositions between segments occur. It has been suggested that the pair alphaM1-F15' and alphaM2-L11' is one of the potential interactions between segments. To determine experimentally if these residues are interacting and to explore if this interhelical interaction is essential for channel gating, we combined mutagenesis with single-channel kinetic analysis. Mutations in alphaM1-F15' lead to profound changes in the opening rate and slighter changes in the closing rate. Channel gating is impaired as the volume of the residue increases. Rate-equilibrium linear free-energy relationship analysis reveals an approximately 70% open-state-like environment for alphaM1-F15' at the transition state of the gating reaction, suggesting that it moves early during the gating process. Replacing the residue at alphaM1-15' by that at alphaM2-11' and vice versa profoundly alters gating, but the combination of the two mutations restores gating to near normal, indicating that alphaM1-F15' and alphaM2-L11' are interchangeable. Double-mutant cycle analysis shows that these residues are energetically coupled. Thus, the interaction between M1 and M2 plays a key role in channel gating.

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

    PubMed

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

    2017-03-27

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

  4. Channel gating of the glycine receptor changes accessibility to residues implicated in receptor potentiation by alcohols and anesthetics.

    PubMed

    Lobo, Ingrid A; Mascia, Maria Paola; Trudell, James R; Harris, R Adron

    2004-08-06

    The glycine receptor is a target for both alcohols and anesthetics, and certain amino acids in the alpha1 subunit transmembrane segments (TM) are critical for drug effects. Introducing larger amino acids at these positions increases the potency of glycine, suggesting that introducing larger residues, or drug molecules, into the drug-binding cavity facilitates channel opening. A possible mechanism for these actions is that the volume of the cavity expands and contracts during channel opening and closing. To investigate this hypothesis, mutations for amino acids in TM1 (I229C) and TM2 (G256C, T259C, V260C, M263C, T264C, S267C, S270C) and TM3 (A288C) were individually expressed in Xenopus laevis oocytes. The ability of sulfhydryl-specific alkyl methanethiosulfonate (MTS) compounds of different lengths to covalently react with introduced cysteines in both the closed and open states of the receptor was determined. S267C was accessible to short chain (C3-C8) MTS in both open and closed states, but was only accessible to longer chain (C10-C16) MTS compounds in the open state. Reaction with S267C was faster in the open state. I229C and A288C showed state-dependent reaction with MTS only in the presence of agonist. M263C and S270C were also accessible to MTS labeling. Mutated residues more intracellular than M263C did not react, indicating a floor of the cavity. These data demonstrate that the conformational changes accompanying channel gating increase accessibility to amino acids critical for drug action in TM1, TM2, and TM3, which may provide a mechanism by which alcohols and anesthetics can act on glycine (and likely other) receptors.

  5. Insights into the gating mechanism of the ryanodine-modified human cardiac Ca2+-release channel (ryanodine receptor 2).

    PubMed

    Mukherjee, Saptarshi; Thomas, N Lowri; Williams, Alan J

    2014-09-01

    Ryanodine receptors (RyRs) are intracellular membrane channels playing key roles in many Ca(2+) signaling pathways and, as such, are emerging novel therapeutic and insecticidal targets. RyRs are so named because they bind the plant alkaloid ryanodine with high affinity and although it is established that ryanodine produces profound changes in all aspects of function, our understanding of the mechanisms underlying altered gating is minimal. We address this issue using detailed single-channel gating analysis, mathematical modeling, and energetic evaluation of state transitions establishing that, with ryanodine bound, the RyR pore adopts an extremely stable open conformation. We demonstrate that stability of this state is influenced by interaction of divalent cations with both activating and inhibitory cytosolic sites and, in the absence of activating Ca(2+), trans-membrane voltage. Comparison of the conformational stability of ryanodine- and Imperatoxin A-modified channels identifies significant differences in the mechanisms of action of these qualitatively similar ligands.

  6. Cooperative gating between ion channels.

    PubMed

    Choi, Kee-Hyun

    2014-01-01

    Cooperative gating between ion channels, i.e. the gating of one channel directly coupled to the gating of neighboring channels, has been observed in diverse channel types at the single-channel level. Positively coupled gating could enhance channel-mediated signaling while negative coupling may effectively reduce channel gating noise. Indeed, the physiological significance of cooperative channel gating in signal transduction has been recognized in several in vivo studies. Moreover, coupled gating of ion channels was reported to be associated with some human disease states. In this review, physiological roles for channel cooperativity and channel clustering observed in vitro and in vivo are introduced, and stimulation-induced channel clustering and direct channel cross linking are suggested as the physical mechanisms of channel assembly. Along with physical clustering, several molecular mechanisms proposed as the molecular basis for functional coupling of neighboring channels are covered: permeant ions as a channel coupling mediator, concerted channel activation through the membrane, and allosteric mechanisms. Also, single-channel analysis methods for cooperative gating such as the binomial analysis, the variance analysis, the conditional dwell time density analysis, and the maximum likelihood fitting analysis are reviewed and discussed.

  7. Altered Channel Conductance States and Gating of GABAA Receptors by a Pore Mutation Linked to Dravet Syndrome

    PubMed Central

    Hernandez, Ciria C.; Kong, Weijing; Zhang, Yujia; Jackson, Laurel; Liu, Xiaoyan; Jiang, Yuwu

    2017-01-01

    Abstract We identified a de novo missense mutation, P302L, in the γ-aminobutyric acid type A (GABAA) receptor γ2 subunit gene GABRG2 in a patient with Dravet syndrome using targeted next-generation sequencing. The mutation was in the cytoplasmic portion of the transmembrane segment M2 of the γ2 subunit that faces the pore lumen. GABAA receptor α1 and β3 subunits were coexpressed with wild-type (wt) γ2L or mutant γ2L(P302L) subunits in HEK 293T cells and cultured mouse cortical neurons. We measured currents using whole-cell and single-channel patch clamp techniques, surface and total expression levels using surface biotinylation and Western blotting, and potential structural perturbations in mutant GABAA receptors using structural modeling. The γ2(P302L) subunit mutation produced an ∼90% reduction of whole-cell current by increasing macroscopic desensitization and reducing GABA potency, which resulted in a profound reduction of GABAA receptor-mediated miniature IPSCs (mIPSCs). The conductance of the receptor channel was reduced to 24% of control conductance by shifting the relative contribution of the conductance states from high- to low-conductance levels with only slight changes in receptor surface expression. Structural modeling of the GABAA receptor in the closed, open, and desensitized states showed that the mutation was positioned to slow activation, enhance desensitization, and shift channels to a low-conductance state by reshaping the hour-glass-like pore cavity during transitions between closed, open, and desensitized states. Our study revealed a novel γ2 subunit missense mutation (P302L) that has a novel pathogenic mechanism to cause defects in the conductance and gating of GABAA receptors, which results in hyperexcitability and contributes to the pathogenesis of the genetic epilepsy Dravet syndrome. PMID:28197552

  8. Analysis of macroscopic ionic currents mediated by GABAρ1 receptors during lanthanide modulation predicts novel states controlling channel gating

    PubMed Central

    Goutman, Juan D; Escobar, Ariel L; Calvo, Daniel J

    2005-01-01

    Lanthanide-induced modulation of GABAC receptors expressed in Xenopus oocytes was studied. We obtained two-electrode voltage-clamp recordings of ionic currents mediated by recombinant homomeric GABAρ1 receptors and performed numerical simulations of kinetic models of the macroscopic ionic currents. GABA-evoked chloride currents were potentiated by La3+, Lu3+ and Gd3+ in the micromolar range. Lanthanide effects were rapid, reversible and voltage independent. The degree of potentiation was reduced by increasing GABA concentration. Lu3+ also induced receptor desensitization and decreased the deactivation rate of GABAρ1 currents. In the presence of 300 μM Lu3+, dose–response curves for GABA-evoked currents showed a significant enhancement of the maximum amplitude and an increase of the apparent affinity. The rate of onset of TPMPA and picrotoxin antagonism of GABAρ1 receptors was modulated by Lu3+. These results suggest that the potentiation of the anionic current was the result of a direct lanthanide–receptor interaction at a site capable of allosterically modulating channel properties. Based on kinetic schemes, which included a second open state and a nonconducting desensitized state that closely reproduced the experimental results, two nonexclusive probable models of GABAρ1 channels gating are proposed. PMID:16231008

  9. Phenylalanine in the Pore of the Erwinia Ligand-Gated Ion Channel Modulates Picrotoxinin Potency but Not Receptor Function

    PubMed Central

    2014-01-01

    The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16′A-, F16′D-, F16′S-, and F16′T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16′ is small. T6′S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16′S, indicating that the inhibitor binds at position 6′, as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16′ is taken into consideration. PMID:25238029

  10. A data-driven model of a modal gated ion channel: the inositol 1,4,5-trisphosphate receptor in insect Sf9 cells.

    PubMed

    Ullah, Ghanim; Mak, Don-On Daniel; Pearson, John E

    2012-08-01

    The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) channel is crucial for the generation and modulation of intracellular Ca(2+) signals in animal cells. To gain insight into the complicated ligand regulation of this ubiquitous channel, we constructed a simple quantitative continuous-time Markov-chain model from the data. Our model accounts for most experimentally observed gating behaviors of single native IP(3)R channels from insect Sf9 cells. Ligand (Ca(2+) and IP(3)) dependencies of channel activity established six main ligand-bound channel complexes, where a complex consists of one or more states with the same ligand stoichiometry and open or closed conformation. Channel gating in three distinct modes added one complex and indicated that three complexes gate in multiple modes. This also restricted the connectivity between channel complexes. Finally, latencies of channel responses to abrupt ligand concentration changes defined a model with specific network topology between 9 closed and 3 open states. The model with 28 parameters can closely reproduce the equilibrium gating statistics for all three gating modes over a broad range of ligand concentrations. It also captures the major features of channel response latency distributions. The model can generate falsifiable predictions of IP(3)R channel gating behaviors and provide insights to both guide future experiment development and improve IP(3)R channel gating analysis. Maximum likelihood estimates of the model parameters and of the parameters in the De Young-Keizer model yield strong statistical evidence in favor of our model. Our method is simple and easily applicable to the dynamics of other ion channels and molecules.

  11. Universality of receptor channel responses.

    PubMed

    Kardos, J; Nyikos, L

    2001-12-01

    Rate parameters estimated for neurotransmitter-gated receptor channel opening and receptor desensitization are classified according to their dependence on the temporal resolution of the techniques applied in the measurements. Because allosteric proteins constituting receptor channels impose restrictions on the types of model suitable to describe the dynamic response of channels to neurotransmitters, Markovian, non-linear or fractal dynamic models and their possible extension to receptor channel response in excitable membranes are discussed.

  12. 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

  13. Sigma-1 receptor alters the kinetics of Kv1.3 voltage gated potassium channels but not the sensitivity to receptor ligands.

    PubMed

    Kinoshita, Maho; Matsuoka, Yoshikazu; Suzuki, Takeshi; Mirrielees, Jennifer; Yang, Jay

    2012-05-03

    Sigma1 receptors (Sigma1R) are intracellular chaperone proteins that bind psychotropic drugs and also clinically used drugs such as ketamine and haloperidol. Co-expression of the Sigma1R has been reported to enhance the sensitivity of several voltage-gated ion channels to Sigma1R ligands. Kv1.3 is the predominant voltage-gated potassium channel expressed in T lymphocytes with a documented role in immune activation. To gain a better understanding of Sigma1R modulation of Kv ion channels, we investigated the effects of Sigma1R co-expression on Kv1.3 physiology and pharmacology in ion channels expressed in Xenopus oocytes. We also explored the protein domains of Kv1.3 necessary for protein:protein interaction between Kv1.3 and Sigma1R through co-immunoprecipitation studies. Slowly inactivating outward-going currents consistent with Kv1.3 expression were elicited on step depolarizations. The current characterized by E(rev), V(1/2), and slope factor remained unchanged when co-expressed with Sigma1R. Analysis of inactivation time constant revealed a faster Kv1.3 current decay when co-expressed with Sigma1R. However the sensitivity to Sigma1R ligands remained unaltered when co-expressed with the Sigma1R in contrast to the previously reported modulation of ligand sensitivity in closely related Kv1.4 and Kv1.5 voltage gated potassium channels. Co-immunoprecipitation assays of various Kv1.3 truncation constructs indicated that the transmembrane domain of the Kv1.3 protein was responsible for the protein:protein interaction with the Sigma1R. Sigma1R likely interacts with different domains of Kv ion channel family proteins resulting in distinct modulation of different channels.

  14. Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating.

    PubMed

    De Rosa, María José; Corradi, Jeremías; Bouzat, Cecilia

    2008-02-01

    The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of alpha and non-alpha subunits, or homo-pentameric, composed of alpha7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between alpha1 and epsilon or alpha7 subunits. The replacement of M3 in alpha1 by epsilonM3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse epsilon chimeric subunit. The duration of the open state decreases with the increase in the number of alpha1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each alpha1M3 segment decreases the energy barrier of the closing process by approximately 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of alpha1 sequence by alpha7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.

  15. GABA(A) receptor M2-M3 loop secondary structure and changes in accessibility during channel gating.

    PubMed

    Bera, Amal K; Chatav, Maya; Akabas, Myles H

    2002-11-08

    The gamma-aminobutyric acid type A (GABA(A)) receptor M2-M3 loop structure and its role in gating were investigated using the substituted cysteine accessibility method. Residues from alpha(1)Arg-273 to alpha(1)Ile-289 were mutated to cysteine, one at a time. MTSET(+) or MTSES(-) reacted with all mutants from alpha(1)R273C to alpha(1)Y281C, except alpha(1)P277C, in the absence and presence of GABA. The MTSET(+) closed-state reaction rate was >1000 liters/mol-s at alpha(1)N274C, alpha(1)S275C, alpha(1)K278C, and alpha(1)Y281C and was <300 liters/mol-s at alpha(1)R273C, alpha(1)L276C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C. These two groups of residues lie on opposite sides of an alpha-helix. The fast reacting group lies on a continuation of the M2 segment channel-lining helix face. This suggests that the M2 segment alpha-helix extends about two helical turns beyond alpha(1)N274 (20'), aligned with the extracellular ring of charge. At alpha(1)S275C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C the reaction rate was faster in the presence of GABA. The reagents had no functional effect on the mutants from alpha(1)A282C to alpha(1)I289C, except alpha(1)A284C. Access may be sterically hindered possibly by close interaction with the extracellular domain. We suggest that the M2 segment alpha-helix extends beyond the predicted extracellular end of the M2 segment and that gating induces a conformational change in and/or around the N-terminal half of the M2-M3 loop. Implications for coupling ligand-evoked conformational changes in the extracellular domain to channel gating in the membrane-spanning domain are discussed.

  16. The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily*

    PubMed Central

    Changeux, Jean-Pierre

    2012-01-01

    A critical event in the history of biological chemistry was the chemical identification of the first neurotransmitter receptor, the nicotinic acetylcholine receptor. Disciplines as diverse as electrophysiology, pharmacology, and biochemistry joined together in a unified and rational manner with the common goal of successfully identifying the molecular device that converts a chemical signal into an electrical one in the nervous system. The nicotinic receptor has become the founding father of a broad family of pentameric membrane receptors, paving the way for their identification, including that of the GABAA receptors. PMID:23038257

  17. 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.

  18. The sigma-1 receptor binds to the Nav1.5 voltage-gated Na+ channel with 4-fold symmetry.

    PubMed

    Balasuriya, Dilshan; Stewart, Andrew P; Crottès, David; Borgese, Franck; Soriani, Olivier; Edwardson, J Michael

    2012-10-26

    The sigma-1 receptor (Sig1R) is up-regulated in many human tumors and plays a role in the control of cancer cell proliferation and invasiveness. At the molecular level, the Sig1R modulates the activity of various ion channels, apparently through a direct interaction. We have previously shown using atomic force microscopy imaging that the Sig1R binds to the trimeric acid-sensing ion channel 1A with 3-fold symmetry. Here, we investigated the interaction between the Sig1R and the Nav1.5 voltage-gated Na(+) channel, which has also been implicated in promoting the invasiveness of cancer cells. We show that the Sig1R and Nav1.5 can be co-isolated from co-transfected cells, consistent with an intimate association between the two proteins. Atomic force microscopy imaging of the co-isolated proteins revealed complexes in which Nav1.5 was decorated by Sig1Rs. Frequency distributions of angles between pairs of bound Sig1Rs had two peaks, at ∼90° and ∼180°, and the 90° peak was about twice the size of the 180° peak. These results demonstrate that the Sig1R binds to Nav1.5 with 4-fold symmetry. Hence, each set of six transmembrane regions in Nav1.5 likely constitutes a Sig1R binding site, suggesting that the Sig1R interacts with the transmembrane regions of its partners. Interestingly, two known Sig1R ligands, haloperidol and (+)-pentazocine, disrupted the Nav1.5/Sig1R interaction both in vitro and in living cells. Finally, we show that endogenously expressed Sig1R and Nav1.5 also functionally interact.

  19. 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.

  20. 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

  1. Role of amino-terminal half of the S4-S5 linker in type 1 ryanodine receptor (RyR1) channel gating.

    PubMed

    Murayama, Takashi; Kurebayashi, Nagomi; Oba, Toshiharu; Oyamada, Hideto; Oguchi, Katsuji; Sakurai, Takashi; Ogawa, Yasuo

    2011-10-14

    The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.

  2. Functional properties of internalization-deficient P2X4 receptors reveal a novel mechanism of ligand-gated channel facilitation by ivermectin.

    PubMed

    Toulmé, Estelle; Soto, Florentina; Garret, Maurice; Boué-Grabot, Eric

    2006-02-01

    Although P2X receptors within the central nervous system mediate excitatory ATP synaptic transmission, the identity of central ATP-gated channels has not yet been elucidated. P2X(4), the most widely expressed subunit in the brain, was previously shown to undergo clathrin-dependent constitutive internalization by direct interaction between activator protein (AP)2 adaptors and a tyrosine-based sorting signal specifically present in the cytosolic C-terminal tail of mammalian P2X(4) sequences. In this study, we first used internalization-deficient P2X(4) receptor mutants to show that suppression of the endocytosis motif significantly increased the apparent sensitivity to ATP and the ionic permeability of P2X(4) channels. These unique properties, observed at low channel density, suggest that interactions with AP2 complexes may modulate the function of P2X(4) receptors. In addition, ivermectin, an allosteric modulator of several receptor channels, including mammalian P2X(4), did not potentiate the maximal current of internalization-deficient rat or human P2X(4) receptors. We demonstrated that binding of ivermectin onto wild-type P2X(4) channels increased the fraction of plasma membrane P2X(4) receptors, whereas surface expression of internalization-deficient P2X(4) receptors remained unchanged. Disruption of the clathrin-mediated endocytosis with the dominant-negative mutants Eps15 or AP-50 abolished the ivermectin potentiation of wild-type P2X(4) channel currents. Likewise, ivermectin increased the membrane fraction of nicotinic alpha7 acetylcholine (nalpha7ACh) receptors and the potentiation of acetylcholine current by ivermectin was suppressed when the same dominant-negative mutants were expressed. These data showed that potentiation by ivermectin of both P2X(4) and nalpha7ACh receptors was primarily caused by an increase in the number of cell surface receptors resulting from a mechanism dependent on clathrin/AP2-mediated endocytosis.

  3. Non-charged amino acids from three different domains contribute to link agonist binding to channel gating in alpha7 nicotinic acetylcholine receptors.

    PubMed

    Aldea, Marcos; Mulet, José; Sala, Salvador; Sala, Francisco; Criado, Manuel

    2007-10-01

    Binding of agonists to nicotinic acetylcholine receptors results in channel opening. Previously, we have shown that several charged residues at three different domains of the alpha7 nicotinic receptor are involved in coupling binding and gating, probably through a network of electrostatic interactions. This network, however, could also be integrated by other residues. To test this hypothesis, non-charged amino acids were mutated and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Asn47 and Gln48 (loop 2), Ile130, Trp134, and Gln140 (loop 7), and Thr264 (M2-M3 linker) showed poor or null functional responses, despite significant membrane expression. By contrast, mutants F137A and S265A exhibited a gain of function effect. In all cases, changes in dose-response relationships were small, EC(50) values being between threefold smaller and fivefold larger, arguing against large modifications of agonist binding. Peak currents decayed at the same rate in all receptors except two, excluding large effects on desensitization. Thus, the observed changes could be mostly caused by alterations of the gating characteristics. Moreover, analysis of double mutants showed an interconnection between some residues in these domains, especially Gln48 with Ile130, suggesting a potential coupling between agonist binding and channel gating through these amino acids.

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

    PubMed Central

    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. Images Fig. 4. PMID:6315420

  5. Molecular determinants of agonist selectivity in glutamate-gated chloride channels which likely explain the agonist selectivity of the vertebrate glycine and GABAA-ρ receptors.

    PubMed

    Blarre, Thomas; Bertrand, Hugues-Olivier; Acher, Francine C; Kehoe, JacSue

    2014-01-01

    Orthologous Cys-loop glutamate-gated chloride channels (GluClR's) have been cloned and described electrophysiologically and pharmacologically in arthropods and nematodes (both members of the invertebrate ecdysozoan superphylum). Recently, GluClR's from Aplysia californica (a mollusc from the lophotrochozoan superphylum) have been cloned and similarly studied. In spite of sharing a common function, the ecdysozoan and lophotrochozoan receptors have been shown by phylogenetic analyses to have evolved independently. The recent crystallization of the GluClR from C. elegans revealed the binding pocket of the nematode receptor. An alignment of the protein sequences of the nematode and molluscan GluClRs showed that the Aplysia receptor does not contain all of the residues defining the binding mode of the ecdysozoan receptor. That the two receptors have slightly different binding modes is not surprising since earlier electrophysiological and pharmacological experiments had suggested that they were differentially responsive to certain agonists. Knowledge of the structure of the C. elegans GluClR has permitted us to generate a homology model of the binding pocket of the Aplysia receptor. We have analyzed the differences between the two binding modes and evaluated the relative significance of their non-common residues. We have compared the GluClRs electrophysiologically and pharmacologically and we have used site-directed mutagenesis on both receptor types to test predictions made from the model. Finally, we propose an explanation derived from the model for why the nematode receptors are gated only by glutamate, whereas the molluscan receptors can also be activated by β-alanine, GABA and taurine. Like the Aplysia receptor, the vertebrate glycine and GABAA-ρ receptors also respond to these other agonists. An alignment of the sequences of the molluscan and vertebrate receptors shows that the reasons we have given for the ability of the other agonists to activate the Aplysia

  6. 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

  7. Signal-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate without activation of phospholipase C: implications on gating of Drosophila TRPL (transient receptor potential-like) channel.

    PubMed

    Lev, Shaya; Katz, Ben; Tzarfaty, Vered; Minke, Baruch

    2012-01-06

    In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P(2) in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P(2) was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P(2) is not an inhibitor of TRPL channel activation. PI(4,5)P(2) hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P(2) levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P(2) is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.

  8. Hydrophobic gating in ion channels.

    PubMed

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

    2015-01-16

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

  9. Gating of two pore domain potassium channels.

    PubMed

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

    2010-09-01

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

  10. Gating of two pore domain potassium channels

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

    Bicknell, Brendan A.

    2016-01-01

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

  12. Neurosteroids shift partial agonist activation of GABA(A) receptor channels from low- to high-efficacy gating patterns.

    PubMed

    Bianchi, Matt T; Macdonald, Robert L

    2003-11-26

    Although GABA activates synaptic (alphabetagamma) GABA(A) receptors with high efficacy, partial agonist activation of alphabetagamma isoforms and GABA activation of the primary extrasynaptic (alphabetadelta) GABA(A) receptors are limited to low-efficacy activity, characterized by minimal desensitization and brief openings. The unusual sensitivity of alphabetadelta receptor channels to neurosteroid modulation prompted investigation of whether this high sensitivity was dependent on the delta subunit or the low-efficacy channel function that it confers. We show that the isoform specificity (alphabetadelta > alphabetagamma) of neurosteroid modulation could be reversed by conditions that reversed isoform-specific activity modes, including the use of beta-alanine to achieve increased efficacy with alphabetadelta receptors and taurine to render alphabetagamma receptors low efficacy. We suggest that neurosteroids preferentially enhance low-efficacy GABA(A) receptor activity independent of subunit composition. Allosteric conversion of partial to full agonism may be a general mechanism for reversibly scaling the efficacy of GABA(A) receptors to endogenous partial agonists.

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

    PubMed

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

    2009-04-01

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

  14. Neurotrophin modulation of voltage-gated potassium channels in rat through TrkB receptors is time and sensory experience dependent

    PubMed Central

    Tucker, K; Fadool, DA

    2002-01-01

    The whole-cell configuration of the patch-clamp technique, immunoprecipitation experiments and unilateral naris occlusions were used to investigate whether the voltage-gated potassium channel Kv1.3 was a substrate for neurotrophin-induced tyrosine phosphorylation and subsequent functional modulation of current properties in cultured rat olfactory bulb (OB) neurons. Membrane proteins of the OB included all three Trk receptor kinases, but the truncated form of the receptor, lacking an intact kinase domain, was the predominant form of the protein for TrkA and TrkC, while TrkB was predominantly found as the full-length receptor. Acute (15 min) stimulation of OB neurons with bath application of 50 ng ml−1 brain-derived neurotrophic factor (BDNF), which is a selective ligand for TrkB, caused suppression of the whole-cell outward current and no changes in the kinetics of inactivation or deactivation. Acute stimulation with either nerve growth factor or neurotrophin-3 failed to evoke any changes in Kv1.3 function in the OB neurons. Chronic exposure to BDNF (days) caused an increase in the magnitude of Kv1.3 current and speeding of the inactivation and deactivation of the channel. Acute BDNF-induced activation of TrkB receptors significantly increased tyrosine phosphorylation of Kv1.3 in the OB, as shown using a combined immunoprecipitation and Western blot analysis. With unilateral naris occlusion, the acute BDNF-induced tyrosine phosphorylation of Kv1.3 was increased in neurons lacking odour sensory experience. In summary, the duration of neurotrophin exposure and the sensory-dependent state of a neuron can influence the degree of phosphorylation of a voltage-gated ion channel and its concomitant functional modulation by neurotrophins. PMID:12122142

  15. 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

  16. Ion-dependent gating of kainate receptors.

    PubMed

    Bowie, Derek

    2010-01-01

    Ligand-gated ion channels are an important class of signalling protein that depend on small chemical neurotransmitters such as acetylcholine, l-glutamate, glycine and gamma-aminobutyrate for activation. Although numerous in number, neurotransmitter substances have always been thought to drive the receptor complex into the open state in much the same way and not rely substantially on other factors. However, recent work on kainate-type (KAR) ionotropic glutamate receptors (iGluRs) has identified an exception to this rule. Here, the activation process fails to occur unless external monovalent anions and cations are present. This absolute requirement of ions singles out KARs from all other ligand-gated ion channels, including closely related AMPA- and NMDA-type iGluR family members. The uniqueness of ion-dependent gating has earmarked this feature of KARs as a putative target for the development of selective ligands; a prospect all the more compelling with the recent elucidation of distinct anion and cation binding pockets. Despite these advances, much remains to be resolved. For example, it is still not clear how ion effects on KARs impacts glutamatergic transmission. I conclude by speculating that further analysis of ion-dependent gating may provide clues into how functionally diverse iGluRs families emerged by evolution. Consequently, ion-dependent gating of KARs looks set to continue to be a subject of topical inquiry well into the future.

  17. 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

  18. Channel gating pore: a new therapeutic target.

    PubMed

    Kornilov, Polina; Peretz, Asher; Attali, Bernard

    2013-09-01

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

  19. Upregulation of voltage-gated Na+ channels by long-term activation of the ghrelin-growth hormone secretagogue receptor in clonal GC somatotropes.

    PubMed

    Dominguez, Belisario; Felix, Ricardo; Monjaraz, Eduardo

    2009-05-01

    A central question in adenohypophyseal cell physiology concerns the role of transmembrane ionic fluxes in the initiation of the hormone secretion process. In the current report, we investigated the effects of the growth hormone (GH) secretagogues ghrelin and GH-releasing peptide-6 (GHRP-6) on the regulation of the functional expression of voltage-gated Na(+) channels using the tumoral somatotrope GC cell line as a model. Cells were cultured under control conditions or in presence of the GH secretagogues (GHS) for 96 h, and Na(+) currents (I(Na)) were characterized in whole cell patch-clamp experiments. GHS treatment significantly increased I(Na) density in a dose-dependent manner. The effects of GHRP-6 were accompanied by an augment in conductance without changes in the kinetics and the voltage dependence of the currents, suggesting an increase in the number of channels in the cell membrane. Sustained inhibition of L-type Ca(2+) channel activity decreased I(Na) density and prevented the effects of the GHS, whereas long-term exposure to an L-channel agonist increased I(Na) density and enhanced the actions of GHRP-6, indicating that Ca(2+) entry through these channels plays a role in the regulation of Na(+) channel expression. Likewise, GHRP-6 failed to enhance Na(+) channel expression in the presence of membrane-permeable inhibitors of protein kinases A and C, as well as the Ca(2+)/calmodulin-dependent kinase II. Conversely, treatment with a cAMP analog or a protein kinase C activator enhanced both basal and GHS-induced secretion of GH measured by enzyme-linked immunoassay, suggesting that GHRP-6 acting through the ghrelin receptor and different signaling pathways enhances Na(+) channel membrane expression, which favors hormone release from GC somatotropes.

  20. Stereoselective modulatory actions of oleamide on GABAA receptors and voltage-gated Na+ channels in vitro: a putative endogenous ligand for depressant drug sites in CNS

    PubMed Central

    Verdon, Bernard; Zheng, Jian; Nicholson, Russell A; Ganelli, C Robin; Lees, George

    2000-01-01

    cis-9,10-octadecenoamide (‘oleamide') accumulates in CSF on sleep deprivation. It induces sleep in animals (the trans form is inactive) but its cellular actions are poorly characterized. We have used electrophysiology in cultures from embryonic rat cortex and biochemical studies in mouse nerve preparations to address these issues. Twenty μM cis-oleamide (but not trans) reversibly enhanced GABAA currents and depressed the frequency of spontaneous excitatory and inhibitory synaptic activity in cultured networks. cis-oleamide stereoselectively blocked veratridine-induced (but not K+-induced) depolarisation of mouse synaptoneurosomes (IC50, 13.9 μM). The cis isomer stereoselectively blocked veratridine-induced (but not K+-induced) [3H]-GABA release from mouse synaptosomes (IC50, 4.6 μM). At 20 μM cis-oleamide, but not trans, produced a marked inhibition of Na+ channel-dependent rises in intrasynaptosomal Ca2+. The physiological significance of these observations was examined by isolating Na+ spikes in cultured pyramidal neurones. Sixty-four μM cis-oleamide did not significantly alter the amplitude, rate of rise or duration of unitary action potentials (1 Hz). cis-Oleamide stereoselectively suppressed sustained repetitive firing (SRF) in these cells with an EC50 of 4.1 μM suggesting a frequency- or state-dependent block of voltage-gated Na+ channels. Oleamide is a stereoselective modulator of both postsynaptic GABAA receptors and presynaptic or somatic voltage-gated Na+ channels which are crucial for synaptic inhibition and conduction. The modulatory actions are strikingly similar to those displayed by sedative or anticonvulsant barbiturates and a variety of general anaesthetics. Oleamide may represent an endogenous modulator for drug receptors and an important regulator of arousal. PMID:10694234

  1. 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

  2. The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2.

    PubMed

    Sklodowski, Kamil; Riedelsberger, Janin; Raddatz, Natalia; Riadi, Gonzalo; Caballero, Julio; Chérel, Isabelle; Schulze, Waltraud; Graf, Alexander; Dreyer, Ingo

    2017-03-16

    The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H(+)-ATPase-energized K(+) uptake. Moreover, through reversible post-translational modifications it can also function as an open, K(+)-selective channel, which taps a 'potassium battery', providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

  3. The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2

    PubMed Central

    Sklodowski, Kamil; Riedelsberger, Janin; Raddatz, Natalia; Riadi, Gonzalo; Caballero, Julio; Chérel, Isabelle; Schulze, Waltraud; Graf, Alexander; Dreyer, Ingo

    2017-01-01

    The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a ‘potassium battery’, providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals. PMID:28300158

  4. The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2

    NASA Astrophysics Data System (ADS)

    Sklodowski, Kamil; Riedelsberger, Janin; Raddatz, Natalia; Riadi, Gonzalo; Caballero, Julio; Chérel, Isabelle; Schulze, Waltraud; Graf, Alexander; Dreyer, Ingo

    2017-03-01

    The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a ‘potassium battery’, providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

  5. Hysteresis in voltage-gated channels.

    PubMed

    Villalba-Galea, Carlos A

    2016-09-30

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

  6. The gating of the CFTR channel.

    PubMed

    Moran, Oscar

    2017-01-01

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

  7. Continuous delta opioid receptor activation reduces neuronal voltage gated sodium channel (NaV1.7) levels through activation of protein kinase C in painful diabetic neuropathy

    PubMed Central

    Chattopadhyay, Munmun; Mata, Marina; Fink, David J.

    2012-01-01

    The NaV1.7 tetrodotoxin-sensitive voltage-gated sodium channel isoform plays a critical role in nociception. In rodent models of diabetic neuropathy, increased NaV1.7 in dorsal root ganglion (DRG) neurons correlates with the emergence of pain-related behaviors characteristic of painful diabetic neuropathy (PDN). We examined the effect of transgene-mediated expression of enkephalin on pain-related behaviors and their biochemical correlates in DRG neurons. Transfection of DRG neurons by subcutaneous inoculation of a herpes simplex virus (HSV)-based vector expressing proenkephalin (PE) reversed nocisponsive behavioral responses to heat, cold, and mechanical pressure characteristic of PDN. Vector-mediated enkephalin production in vivo prevented the increase in DRG NaV1.7 observed in PDN, an effect that correlated with inhibition of phosphorylation of p38 MAP kinase and protein kinase C (PKC). Primary DRG neurons in vitro exposed to 45 mM glucose for 18 hrs also demonstrated an increase in NaV1.7 and increased phosphorylation of p38 and PKC; these changes were prevented by transfection in vitro with the enkephalin-expressing vector. The effect of hyperglycemia on NaV1.7 production in vitro was mimicked by exposure to PMA, and blocked by the myristolated PKC inhibitor 20–28 or the p38 inhibitor SB202190; the effect of vector-mediated enkephalin on NaV1.7 levels was prevented by naltrindole. The results of these studies suggest that activation of the presynaptic delta opioid receptor by enkephalin prevents the increase in neuronal NaV1.7 in DRG through inhibition of PKC and p38. These results establish a novel interaction between the delta opioid receptor and voltage gated sodium channels. PMID:18579738

  8. Gα14 subunit-mediated inhibition of voltage-gated Ca2+ and K+ channels via neurokinin-1 receptors in rat celiac-superior mesenteric ganglion neurons.

    PubMed

    Sugino, Shigekazu; Farrag, Mohamed; Ruiz-Velasco, Victor

    2016-03-01

    The mechanisms by which G proteins modulate voltage-gated Ca(2+)channel currents (CaV), particularly CaV2.2 and CaV2.3, are voltage dependent (VD) or voltage independent (VI). VD pathways are typically mediated by Gαi/oand GαSsubfamilies. On the other hand, VI inhibition modulation is coupled to the Gαqsubfamily and signaling pathways downstream of phospholipase C stimulation. In most studies, this latter pathway has been shown to be linked to Gαqand/or Gα11protein subunits. However, there are no studies that have examined whether natively expressed Gα14subunits (Gαqsubfamily member) couple G protein-coupled receptors (GPCR) with CaV2.2 channels. We report that Gα14subunits functionally couple the substance P (SP)/neurokinin-1 (NK-1) receptor pathway to CaV2.2 channels in acutely dissociated rat celiac-superior mesenteric ganglion (CSMG) neurons. Exposure of CSMG neurons to SP blocked the CaV2.2 currents in a predominantly VD manner that was pertussis toxin and cholera toxin resistant, as well as Gαq/11independent. However, silencing Gα14subunits significantly attenuated the SP-mediated Ca(2+)current block. In another set of experiments, exposure of CSMG neurons to SP led to the inhibition of KCNQ K(+)M-currents. The SP-mediated M-current block was significantly reduced in neurons transfected with Gα14small-interference RNA. Finally, overexpression of the GTP-bound Gαq/11binding protein RGS2 did not alter the block of M-currents by SP but significantly abolished the oxotremorine methiodide-mediated M-current inhibition. Taken together, these results provide evidence of a new Gα14-coupled signaling pathway that modulates CaV2.2 and M-currents via SP-stimulated NK-1 receptors in CSMG neurons.

  9. Activation of CRH receptor type 1 expressed on glutamatergic neurons increases excitability of CA1 pyramidal neurons by the modulation of voltage-gated ion channels.

    PubMed

    Kratzer, Stephan; Mattusch, Corinna; Metzger, Michael W; Dedic, Nina; Noll-Hussong, Michael; Kafitz, Karl W; Eder, Matthias; Deussing, Jan M; Holsboer, Florian; Kochs, Eberhard; Rammes, Gerhard

    2013-01-01

    Corticotropin-releasing hormone (CRH) plays an important role in a substantial number of patients with stress-related mental disorders, such as anxiety disorders and depression. CRH has been shown to increase neuronal excitability in the hippocampus, but the underlying mechanisms are poorly understood. The effects of CRH on neuronal excitability were investigated in acute hippocampal brain slices. Population spikes (PS) and field excitatory postsynaptic potentials (fEPSP) were evoked by stimulating Schaffer-collaterals and recorded simultaneously from the somatic and dendritic region of CA1 pyramidal neurons. CRH was found to increase PS amplitudes (mean ± Standard error of the mean; 231.8 ± 31.2% of control; n = 10) while neither affecting fEPSPs (104.3 ± 4.2%; n = 10) nor long-term potentiation (LTP). However, when Schaffer-collaterals were excited via action potentials (APs) generated by stimulation of CA3 pyramidal neurons, CRH increased fEPSP amplitudes (119.8 ± 3.6%; n = 8) and the magnitude of LTP in the CA1 region. Experiments in slices from transgenic mice revealed that the effect on PS amplitude is mediated exclusively by CRH receptor 1 (CRHR1) expressed on glutamatergic neurons. The effects of CRH on PS were dependent on phosphatase-2B, L- and T-type calcium channels and voltage-gated potassium channels but independent on intracellular Ca(2+)-elevation. In patch-clamp experiments, CRH increased the frequency and decay times of APs and decreased currents through A-type and delayed-rectifier potassium channels. These results suggest that CRH does not affect synaptic transmission per se, but modulates voltage-gated ion currents important for the generation of APs and hence elevates by this route overall neuronal activity.

  10. Activation of CRH receptor type 1 expressed on glutamatergic neurons increases excitability of CA1 pyramidal neurons by the modulation of voltage-gated ion channels

    PubMed Central

    Kratzer, Stephan; Mattusch, Corinna; Metzger, Michael W.; Dedic, Nina; Noll-Hussong, Michael; Kafitz, Karl W.; Eder, Matthias; Deussing, Jan M.; Holsboer, Florian; Kochs, Eberhard; Rammes, Gerhard

    2013-01-01

    Corticotropin-releasing hormone (CRH) plays an important role in a substantial number of patients with stress-related mental disorders, such as anxiety disorders and depression. CRH has been shown to increase neuronal excitability in the hippocampus, but the underlying mechanisms are poorly understood. The effects of CRH on neuronal excitability were investigated in acute hippocampal brain slices. Population spikes (PS) and field excitatory postsynaptic potentials (fEPSP) were evoked by stimulating Schaffer-collaterals and recorded simultaneously from the somatic and dendritic region of CA1 pyramidal neurons. CRH was found to increase PS amplitudes (mean ± Standard error of the mean; 231.8 ± 31.2% of control; n = 10) while neither affecting fEPSPs (104.3 ± 4.2%; n = 10) nor long-term potentiation (LTP). However, when Schaffer-collaterals were excited via action potentials (APs) generated by stimulation of CA3 pyramidal neurons, CRH increased fEPSP amplitudes (119.8 ± 3.6%; n = 8) and the magnitude of LTP in the CA1 region. Experiments in slices from transgenic mice revealed that the effect on PS amplitude is mediated exclusively by CRH receptor 1 (CRHR1) expressed on glutamatergic neurons. The effects of CRH on PS were dependent on phosphatase-2B, L- and T-type calcium channels and voltage-gated potassium channels but independent on intracellular Ca2+-elevation. In patch-clamp experiments, CRH increased the frequency and decay times of APs and decreased currents through A-type and delayed-rectifier potassium channels. These results suggest that CRH does not affect synaptic transmission per se, but modulates voltage-gated ion currents important for the generation of APs and hence elevates by this route overall neuronal activity. PMID:23882180

  11. Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels

    PubMed Central

    Moran, Yehu; Kahn, Roy; Cohen, Lior; Gur, Maya; Karbat, Izhar; Gordon, Dalia; Gurevitz, Michael

    2007-01-01

    Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Navs (voltage-gated Na+ channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to receptor site-3. To examine the potency and mode of interaction of Av3 with insect Navs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65±0.46 pmol/100 mg), to compete well with the site-3 toxin LqhαIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (Ki=21.4±7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNav1, but not that of mammalian Navs expressed in Xenopus oocytes. Moreover, like other site-3 toxins, the activity of Av3 was synergically enhanced by ligands of receptor site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other site-3 toxins. These analyses have portrayed a toxin that might interact with receptor site-3 in a different fashion compared with other ligands of this site. This assumption was corroborated by a D1701R mutation in DmNav1, which has been shown to abolish the activity of all other site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of receptor site-3, and raises Av3 as a unique model for design of selective anti-insect compounds. PMID:17492942

  12. Concentration dependence of sodium permeation and sodium ion interactions in the cyclic AMP-gated channels of mammalian olfactory receptor neurons.

    PubMed

    Balasubramanian, S; Lynch, J W; Barry, P H

    1997-09-01

    The dependence of currents through the cyclic nucleotide-gated (CNG) channels of mammalian olfactory receptor neurons (ORNs) on the concentration of NaCl was studied in excised inside-out patches from their dendritic knobs using the patch-clamp technique. With a saturating concentration (100 microM) of adenosine 3',5'-cyclic monophosphate (cAMP), the changes in the reversal potential of macroscopic currents were studied at NaCl concentrations from 25 to 300 mM. In symmetrical NaCl solutions without the addition of divalent cations, the current-voltage relations were almost linear, reversing close to 0 mV. When the external NaCl concentration was maintained at 150 mM and the internal concentrations were varied, the reversal potentials of the cAMP-activated currents closely followed the Na+ equilibrium potential indicating that PCl/PNa approximately 0. However, at low external NaCl concentrations (< or = 100 mM) there was some significant chloride permeability. Our results further indicated that Na+ currents through these channels: (i) did not obey the independence principle; (ii) showed saturation kinetics with K(m)s in the range of 100-150 mM and (iii) displayed a lack of voltage dependence of conductance in asymmetric solutions that suggested that ion-binding sites were situated midway along the channel. Together, these characteristics indicate that the permeation properties of the olfactory CNG channels are significantly different from those of photoreceptor CNG channels.

  13. 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.

  14. Sigma-1 receptor stimulation attenuates calcium influx through activated L-type Voltage Gated Calcium Channels in purified retinal ganglion cells.

    PubMed

    Mueller, Brett H; Park, Yong; Daudt, Donald R; Ma, Hai-Ying; Akopova, Irina; Stankowska, Dorota L; Clark, Abbot F; Yorio, Thomas

    2013-02-01

    Sigma-1 receptors (σ-1rs) exert neuroprotective effects on retinal ganglion cells (RGCs) both in vivo and in vitro. This receptor has unique properties through its actions on several voltage-gated and ligand-gated channels. The purpose of this study was to investigate the role that σ-1rs play in regulating cell calcium dynamics through activated L-type Voltage Gated Calcium Channels (L-type VGCCs) in purified RGCs. RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy1.1 antibody. Calcium imaging was used to measure changes in intracellular calcium after depolarizing the cells with potassium chloride (KCl) in the presence or absence of two σ-1r agonists [(+)-SKF10047 and (+)-Pentazocine], one σ-1r antagonist (BD1047), and one L-type VGCC antagonist (Verapamil). Finally, co-localization studies were completed to assess the proximity of σ-1r with L-type VGCCs in purified RGCs. VGCCs were activated using KCl (20 mM). Pre-treatment with a known L-type VGCC blocker demonstrated a 57% decrease of calcium ion influx through activated VGCCs. Calcium imaging results also demonstrated that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Antagonist treatment using BD1047 demonstrated a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs, implying that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in purified RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in purified RGCs. This is the

  15. 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

  16. Tonic current through GABAA receptors and hyperpolarization-activated cyclic nucleotide-gated channels modulate resonance properties of rat subicular pyramidal neurons.

    PubMed

    Sah, Nirnath; Sikdar, Sujit K

    2014-07-01

    The subiculum, considered to be the output structure of the hippocampus, modulates information flow from the hippocampus to various cortical and sub-cortical areas such as the nucleus accumbens, lateral septal region, thalamus, nucleus gelatinosus, medial nucleus and mammillary nuclei. Tonic inhibitory current plays an important role in neuronal physiology and pathophysiology by modulating the electrophysiological properties of neurons. While the alterations of various electrical properties due to tonic inhibition have been studied in neurons from different regions, its influence on intrinsic subthreshold resonance in pyramidal excitatory neurons expressing hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is not known. Using pharmacological agents, we show the involvement of α5βγ GABAA receptors in the picrotoxin-sensitive tonic current in subicular pyramidal neurons. We further investigated the contribution of tonic conductance in regulating subthreshold electrophysiological properties using current clamp and dynamic clamp experiments. We demonstrate that tonic GABAergic inhibition can actively modulate subthreshold properties, including resonance due to HCN channels, which can potentially alter the response dynamics of subicular pyramidal neurons in an oscillating neuronal network.

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

    PubMed Central

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

    2013-01-01

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

  18. Pregnenolone sulfate activates basic region leucine zipper transcription factors in insulinoma cells: role of voltage-gated Ca2+ channels and transient receptor potential melastatin 3 channels.

    PubMed

    Müller, Isabelle; Rössler, Oliver G; Thiel, Gerald

    2011-12-01

    The neurosteroid pregnenolone sulfate activates a signaling cascade in insulinoma cells involving activation of extracellular signal-regulated protein kinase and enhanced expression of the transcription factor Egr-1. Here, we show that pregnenolone sulfate stimulation leads to a significant elevation of activator protein-1 (AP-1) activity in insulinoma cells. Expression of the basic region leucine zipper (bZIP) transcription factors c-Jun and c-Fos is up-regulated in insulinoma cells and pancreatic β-cells in primary culture after pregnenolone sulfate stimulation. Up-regulation of a chromatin-embedded c-Jun promoter/luciferase reporter gene transcription in pregnenolone sulfate-stimulated insulinoma cells was impaired when the AP-1 binding sites were mutated, indicating that these motifs function as pregnenolone sulfate response elements. In addition, phosphorylation of cAMP response element (CRE)-binding protein is induced and transcription of a CRE-controlled reporter gene is stimulated after pregnenolone sulfate treatment, indicating that the CRE functions as a pregnenolone sulfate response element as well. Pharmacological and genetic experiments revealed that both L-type Ca(2+) channels and transient receptor potential melastatin 3 (TRPM3) channels are essential for connecting pregnenolone sulfate stimulation with enhanced AP-1 activity and bZIP-mediated transcription in insulinoma cells. In contrast, pregnenolone sulfate stimulation did not enhance AP-1 activity or c-Jun and c-Fos expression in pituitary corticotrophs that express functional L-type Ca(2+) channels but only trace amounts of TRPM3. We conclude that expression of L-type Ca(2+) channels is not sufficient to activate bZIP-mediated gene transcription by pregnenolone sulfate. Rather, additional expression of TRPM3 or depolarization of the cells is required to connect pregnenolone sulfate stimulation with enhanced gene transcription.

  19. Evidence for the role of lipid rafts and sphingomyelin in Ca2+-gating of Transient Receptor Potential channels in trigeminal sensory neurons and peripheral nerve terminals.

    PubMed

    Sághy, Éva; Szőke, Éva; Payrits, Maja; Helyes, Zsuzsanna; Börzsei, Rita; Erostyák, János; Jánosi, Tibor Zoltán; Sétáló, György; Szolcsányi, János

    2015-10-01

    Transient Receptor Potential (TRP) cation channels, such as TRP Vanilloid 1 and TRP Ankyrin repeat domain 1 (TRPV1 and TRPA1) are nocisensors playing important role to signal pain. Two "melastatin" TRP receptors, like TRPM8 and TRPM3 are also expressed in a subgroup of primary sensory neurons. These channels serve as thermosensors with unique thermal sensitivity ranges and are activated also by several exogenous and endogenous chemical ligands inducing conformational changes from various allosteric ("multisteric") sites. We analysed the role of plasma membrane microdomains of lipid rafts on isolated trigeminal (TRG) neurons and TRPV1-expressing CHO cell line by measuring agonist-induced Ca2+ transients with ratiometric technique. Stimulation-evoked calcitonin gene related peptide (CGRP) release from sensory nerve endings of the isolated rat trachea by radioimmunoassay was also measured. Lipid rafts were disrupted by cleaving sphingomyelin (SM) with sphingomyelinase (SMase), cholesterol depletion with methyl β-cyclodextrin (MCD) and ganglioside breakdown with myriocin. It has been revealed that intracellular Ca2+ increase responses evoked by the TRPV1 agonist capsaicin, the TRPA1 agonsits allyl isothiocyanate (AITC) and formaldehyde as well as the TRPM8 activator icilin were inhibited after SMase, MCD and myriocin incubation but the response to the TRPM3 agonist pregnenolon sulphate was not altered. Extracellular SMase treatment did not influence the thapsigargin-evoked Ca2+-release from intracellular stores. Besides the cell bodies, SMase also inhibited capsaicin- or AITC-evoked CGRP release from peripheral sensory nerve terminals, this provides the first evidence for the importance of lipid raft integrity in TRPV1 and TRPA1 gating on capsaicin-sensitive nerve terminals. SM metabolites, ceramide and sphingosine, did not influence TRPA1 and TRPV1 activation on TRG neurons, TRPV1-expressing CHO cell line, and nerve terminals. We suggest, that the hydrophobic

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

    PubMed

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

    2013-01-01

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

  2. Immunohistochemical identification of cells expressing ATP-gated cation channels (P2X receptors) in the adult rat thyroid

    PubMed Central

    GLASS, RAINER; BURNSTOCK, GEOFFREY

    2001-01-01

    We carried out immunohistochemistry and western blotting of fresh frozen sections and crude extracts from adult rat thyroids. The histochemical and immunoblotting studies were performed with P2X receptor antibodies from 2 different sources. P2X-immunopositive cells were identified by fluorescence double labelling and confocal microscopy. Results of the western blotting experiments showed double bands of approximately 70 kDa and 140 kDa for all 7 P2X receptor subtypes with both sets of antibodies. Histochemical stains with antibodies from both sources also gave essentially identical results. P2X1, P2X2 and P2X6 receptors were detected exclusively in vascular smooth muscle; P2X5 and P2X7 receptors were also present on vascular smooth muscle. Endothelial cells stained for P2X3, P2X4 and P2X7 receptors. Thyroid follicular cells displayed immunoreactivity for P2X3, P2X4 and P2X5 receptors. No immunostaining for P2X receptors was observed on C-cells. Possible roles for the broad expression of P2X receptor subtypes in the rat thyroid are discussed. PMID:11430696

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

    PubMed Central

    1996-01-01

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

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

    PubMed

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

    2010-08-18

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

  5. Conjoint occurrence of GABAB receptor antibodies in Lambert-Eaton myasthenic syndrome with antibodies to the voltage gated calcium channel.

    PubMed

    Dogan Onugoren, Müjgan; Rauschka, Helmut; Bien, Christian G

    2014-08-15

    Antibodies (abs) to the GABAB receptor have been recently found to be responsible for immune-mediated encephalitis with dominant seizures. They are in approximately 50% of cases associated with small-cell lung cancer (SCLC). GABAB receptors are mainly located in the hippocampus, thalamus and cerebellum in the presynaptic and postsynaptic regions of synapses. The main function of these receptors is to reduce activity states of neurons. In some instances, GABAB receptor abs in these patients were accompanied by other antibodies, among them VGCC abs (Lancaster et al., 2010, Boronat et al., 2011). VGCC abs cause paraneoplastic Lambert Eaton myasthenic syndrome (LEMS) by reduction of presynaptic VGCCs (Titulaer et al., 2011). In the domain of CNS disease, VGCC abs have been found in association with paraneoplastic cerebellar ataxia (Mason et al., 1997) and rarely and at low titres also in other paraneoplastic encephalopathies together with Hu abs (Lennon et al., 1995). It has been a long-standing debate if abs in paraneoplastic conditions associate rather with the neurological syndrome or the tumour. Here, we describe the conjoint occurrence of abs to the GABAB receptor and to the VGCC in a patient with SCLC presenting only symptoms of the peripheral nervous system giving another example of the latter hypothesis.

  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. 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

  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. Introduction to Thematic Minireview Series on Celebrating the Discovery of the Cysteine Loop Ligand-gated Ion Channel Superfamily

    PubMed Central

    Stephenson, F. Anne

    2012-01-01

    The year 2012 marks the 25th anniversary of the discovery of the Cys loop ligand-gated ion channel superfamily of neurotransmitter receptors. This minireview series celebrates this with a series of articles reviewing current information for each of the family members, nicotinic acetylcholine receptors, glycine receptors, GABAA receptors, serotonin-3 (5-HT3) receptors, and glutamate-gated chloride ion channels of proteasome invertebrate phyla. PMID:23038255

  10. Microtransplantation of ligand-gated receptor-channels from fresh or frozen nervous tissue into Xenopus oocytes: a potent tool for expanding functional information.

    PubMed

    Eusebi, F; Palma, E; Amici, M; Miledi, R

    2009-05-01

    Despite huge improvements in neurobiological approaches for investigating the functional properties of neurotransmitter receptors and ion channels, many difficulties are still encountered when focusing on the human brain. Electrophysiological studies aimed at performing direct determinations on human nervous tissue are limited by neurosurgery and also by pathophysiological conditions prevailing before and after the resective operation. The electrophysiological study of receptors and channels becomes difficult also in animal models when the cells are not accessible and/or the experiments last many hours, during which the examined nervous tissue usually becomes unhealthy. To increase the possibility of doing optimal electrophysiological recordings, addressed to investigate the functional properties of receptors and channels, more than two decades ago, foreign mRNAs were injected into Xenopus oocytes to heterologously express the receptors; and about a decade ago cell membranes were injected into the oocytes to directly transplant the native receptors. While the first approach needs complex procedures for mRNA isolation, the membrane preparations are simpler to obtain and the embedded receptors are transplanted in their own membrane, with their own glycosylation and together with any ancillary proteins they may have. Using injections of membranes isolated from fresh nervous tissues several issues have already been addressed and many questions can be answered in the near future. Strikingly, with this approach it has been possible to "resuscitate" receptors and ion channels from tissues kept frozen for many years. This review focuses on recently obtained information and on some new lines of biological research using receptor microtransplantation into oocytes.

  11. Ligand-Gated Ion Channels: Permeation and Activation1

    NASA Astrophysics Data System (ADS)

    Lynch, Joseph W.; Barry, Peter H.

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

  12. Voltage-gated ion channel Kv4.3 is associated with Rap guanine nucleotide exchange factors and regulates angiotensin receptor type 1 signaling to small G-protein Rap.

    PubMed

    Potapova, Irina A; Cohen, Ira S; Doronin, Sergey V

    2007-09-01

    The voltage-gated potassium channel Kv4.3 was coexpressed with its beta-subunit Kv channel-interacting protein 2 and the angiotensin type 1 receptor in HEK-293 cells. Proteomic analysis of proteins coimmunoprecipitated with Kv4.3 revealed that Kv4.3 is associated with Rap guanine nucleotide exchange factors MR-GEF and EPAC-1. Previously, we demonstrated that Kv4.3 interacts with the angiotensin type 1 receptor in HE293 cells and cardiac myocytes. On the basis of this, we investigated the angiotensin type 1 receptor signaling to small G-proteins Ras and Rap-1 in the presence and absence of the Kv4.3-Kv channel-interacting protein 2 macromolecular complex. Ras activation was not significantly affected by coexpression of Kv4.3 and Kv channel-interacting protein 2. Ras exhibited a rapid activation-inactivation pattern with maximum activity at 2.5 min after addition of angiotensin II. In contrast, activation of Rap-1 was affected dramatically by coexpression of Kv4.3 and Kv channel-interacting protein 2 with the angiotensin type 1 receptor. In the absence of Kv4.3 and Kv channel-interacting protein 2, stimulation of the angiotensin type 1 receptor resulted in steady activation of Rap-1 that reached a plateau 25 min after addition of angiotensin II. In the presence of Kv4.3 and Kv channel-interacting protein 2, Rap-1 reaches a maximum activity 2.5 min after addition of angiotensin II and then deactivates rapidly, demonstrating a pattern of activation similar to that of Ras. Our findings show that Kv4.3 regulates angiotensin type 1 receptor signaling to the small G-protein Rap-1.

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

    PubMed

    Huang, Junting; Zamponi, Gerald W

    2016-10-18

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

  14. 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.

  15. 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

  16. mRNAs coding for neurotransmitter receptors and voltage-gated sodium channels in the adult rabbit visual cortex after monocular deafferentiation

    PubMed Central

    Nguyen, Quoc-Thang; Matute, Carlos; Miledi, Ricardo

    1998-01-01

    It has been postulated that, in the adult visual cortex, visual inputs modulate levels of mRNAs coding for neurotransmitter receptors in an activity-dependent manner. To investigate this possibility, we performed a monocular enucleation in adult rabbits and, 15 days later, collected their left and right visual cortices. Levels of mRNAs coding for voltage-activated sodium channels, and for receptors for kainate/α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-d-aspartate (NMDA), γ-aminobutyric acid (GABA), and glycine were semiquantitatively estimated in the visual cortices ipsilateral and contralateral to the lesion by the Xenopus oocyte/voltage-clamp expression system. This technique also allowed us to study some of the pharmacological and physiological properties of the channels and receptors expressed in the oocytes. In cells injected with mRNA from left or right cortices of monocularly enucleated and control animals, the amplitudes of currents elicited by kainate or AMPA, which reflect the abundance of mRNAs coding for kainate and AMPA receptors, were similar. There was no difference in the sensitivity to kainate and in the voltage dependence of the kainate response. Responses mediated by NMDA, GABA, and glycine were unaffected by monocular enucleation. Sodium channel peak currents, activation, steady-state inactivation, and sensitivity to tetrodotoxin also remained unchanged after the enucleation. Our data show that mRNAs for major neurotransmitter receptors and ion channels in the adult rabbit visual cortex are not obviously modified by monocular deafferentiation. Thus, our results do not support the idea of a widespread dynamic modulation of mRNAs coding for receptors and ion channels by visual activity in the rabbit visual system. PMID:9501250

  17. 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.

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

    PubMed Central

    1994-01-01

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

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

    PubMed

    Eastwood, Amy L; Goodman, Miriam B

    2012-10-01

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

  20. Allosteric Voltage Gating of Potassium Channels I

    PubMed Central

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

    1999-01-01

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

  1. 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

  2. Simultaneous Optical Recording in Multiple Cells by Digital Holographic Microscopy of Chloride Current Associated to Activation of the Ligand-Gated Chloride Channel GABAA Receptor

    PubMed Central

    Jourdain, Pascal; Boss, Daniel; Rappaz, Benjamin; Moratal, Corinne; Hernandez, Maria-Clemencia; Depeursinge, Christian

    2012-01-01

    Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABAA mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABAA receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABAA receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM. PMID:23236427

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

    PubMed

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

    2017-03-06

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

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

    PubMed

    Venkataraman, Gaurav; Srikumar, Deepa; Holmgren, Miguel

    2014-06-09

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

  5. Conserved Gating Elements in TRPC4 and TRPC5 Channels*

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2002-01-01

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

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

    PubMed Central

    Tóth, Balázs; Iordanov, Iordan; Csanády, László

    2014-01-01

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

  8. Structure-Function Map of the Receptor Site for β-Scorpion Toxins in Domain II of Voltage-gated Sodium Channels*

    PubMed Central

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

    2011-01-01

    Voltage-gated sodium (Nav) channels are the molecular targets of β-scorpion toxins, which shift the voltage dependence of activation to more negative membrane potentials by a voltage sensor-trapping mechanism. Molecular determinants of β-scorpion toxin (CssIV) binding and action on rat brain sodium channels are located in the S1-S2 (IIS1-S2) and S3-S4 (IIS3-S4) extracellular linkers of the voltage-sensing module in domain II. In IIS1-S2, mutations of two amino acid residues (Glu779 and Pro782) significantly altered the toxin effect by reducing binding affinity. In IIS3-S4, six positions surrounding the key binding determinant, Gly845, define a hot spot of high-impact residues. Two of these substitutions (A841N and L846A) reduced voltage sensor trapping. The other three substitutions (N842R, V843A, and E844N) increased voltage sensor trapping. These bidirectional effects suggest that the IIS3-S4 loop plays a primary role in determining both toxin affinity and efficacy. A high resolution molecular model constructed with the Rosetta-Membrane modeling system reveals interactions of amino acid residues in sodium channels that are crucial for toxin action with residues in CssIV that are required for its effects. In this model, the wedge-shaped CssIV inserts between the IIS1-S2 and IIS3-S4 loops of the voltage sensor, placing key amino acid residues in position to interact with binding partners in these extracellular loops. These results provide new molecular insights into the voltage sensor-trapping model of toxin action and further define the molecular requirements for the development of antagonists that can prevent or reverse toxicity of scorpion toxins. PMID:21795675

  9. 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

  10. Preferential use of unobstructed lateral portals as the access route to the pore of human ATP-gated ion channels (P2X receptors).

    PubMed

    Samways, Damien S K; Khakh, Baljit S; Dutertre, Sébastien; Egan, Terrance M

    2011-08-16

    P2X receptors are trimeric cation channels with widespread roles in health and disease. The recent crystal structure of a P2X4 receptor provides a 3D view of their topology and architecture. A key unresolved issue is how ions gain access to the pore, because the structure reveals two different pathways within the extracellular domain. One of these is the central pathway spanning the entire length of the extracellular domain and covering a distance of ≈70 Å. The second consists of three lateral portals, adjacent to the membrane and connected to the transmembrane pore by short tunnels. Here, we demonstrate the preferential use of the lateral portals. Owing to their favorable diameters and equivalent spacing, the lateral portals split the task of ion supply threefold and minimize an ion's diffusive path before it succumbs to transmembrane electrochemical gradients.

  11. 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.

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

    PubMed

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

    2013-01-01

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

  13. Structural determinants of skeletal muscle ryanodine receptor gating.

    PubMed

    Ramachandran, Srinivas; Chakraborty, Asima; Xu, Le; Mei, Yingwu; Samsó, Montserrat; Dokholyan, Nikolay V; Meissner, Gerhard

    2013-03-01

    Ryanodine receptor type 1 (RyR1) releases Ca(2+) from intracellular stores upon nerve impulse to trigger skeletal muscle contraction. Effector binding at the cytoplasmic domain tightly controls gating of the pore domain of RyR1 to release Ca(2+). However, the molecular mechanism that links effector binding to channel gating is unknown due to lack of structural data. Here, we used a combination of computational and electrophysiological methods and cryo-EM densities to generate structural models of the open and closed states of RyR1. Using our structural models, we identified an interface between the pore-lining helix (Tyr-4912-Glu-4948) and a linker helix (Val-4830-Val-4841) that lies parallel to the cytoplasmic membrane leaflet. To test the hypothesis that this interface controls RyR1 gating, we designed mutations in the linker helix to stabilize either the open (V4830W and T4840W) or closed (H4832W and G4834W) state and validated them using single channel experiments. To further confirm this interface, we designed mutations in the pore-lining helix to stabilize the closed state (Q4947N, Q4947T, and Q4947S), which we also validated using single channel experiments. The channel conductance and selectivity of the mutations that we designed in the linker and pore-lining helices were indistinguishable from those of WT RyR1, demonstrating our ability to modulate RyR1 gating without affecting ion permeation. Our integrated computational and experimental approach significantly advances the understanding of the structure and function of an unusually large ion channel.

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

    PubMed

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

    2005-01-01

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

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

    PubMed

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

    2015-01-01

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

  16. Coupling between Voltage Sensors and Activation Gate in Voltage-gated K+ Channels

    PubMed Central

    Lu, Zhe; Klem, Angela M.; Ramu, Yajamana

    2002-01-01

    Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1–S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5–S6, equivalent to M1–M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4–S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues. PMID:12407078

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

    PubMed

    Pankratov, Yuriy; Lalo, Ulyana

    2014-09-15

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

  18. Kinetic Contributions to Gating by Interactions Unique to N-methyl-d-aspartate (NMDA) Receptors*

    PubMed Central

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

    2015-01-01

    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

  19. 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

  20. 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

  1. Chloride dependence of hyperpolarization-activated chloride channel gates

    PubMed Central

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

    1999-01-01

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

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

    PubMed

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

    2008-03-20

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

  3. 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

  4. 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

  5. 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

  6. 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

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

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

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

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

    PubMed Central

    Kariev, Alisher M.; Green, Michael E.

    2012-01-01

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

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

    PubMed

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

    2012-05-01

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

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

    PubMed

    Ryu, Sujung; Yellen, Gary

    2012-11-01

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

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

    PubMed

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

    1997-01-16

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

  12. 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

  13. Surfaces and boundaries in the mechanosensitive channel gating

    NASA Astrophysics Data System (ADS)

    Sukharev, Sergei

    2009-03-01

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

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

    PubMed

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

    2013-07-01

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

  15. 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.

  16. 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.

  17. 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

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

    PubMed

    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.

  19. On the Evolution of Voltage Gated Ion Channels

    NASA Astrophysics Data System (ADS)

    Brenner, Michael

    2006-03-01

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

  20. 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.

  1. Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone.

    PubMed

    Buckingham, Steven D; Higashino, Yoshiaki; Sattelle, David B

    2009-11-01

    The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D(f)) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D(f) motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.

  2. Potassium channels and their evolving gates.

    PubMed

    Jan, L Y; Jan, Y N

    1994-09-08

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

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

    PubMed Central

    Elinder, Fredrik; Liin, Sara I.

    2017-01-01

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

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

    PubMed

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

    1996-05-01

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

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

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

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

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

    PubMed

    Yellen, Gary

    2002-09-05

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

  7. The mechano-gated K(2P) channel TREK-1.

    PubMed

    Dedman, Alexandra; Sharif-Naeini, Reza; Folgering, Joost H A; Duprat, Fabrice; Patel, Amanda; Honoré, Eric

    2009-03-01

    The versatility of neuronal electrical activity is largely conditioned by the expression of different structural and functional classes of K+ channels. More than 80 genes encoding the main K+ channel alpha subunits have been identified in the human genome. Alternative splicing, heteromultimeric assembly, post-translational modification and interaction with auxiliary regulatory subunits further increase the molecular and functional diversity of K+ channels. Mammalian two-pore domain K+ channels (K(2P)) make up one class of K+ channels along with the inward rectifiers and the voltage- and/or calcium-dependent K+ channels. Each K(2P) channel subunit is made up of four transmembrane segments and two pore-forming (P) domains, which are arranged in tandem and function as either homo- or heterodimeric channels. This novel structural arrangement is associated with unusual gating properties including "background" or "leak" K+ channel activity, in which the channels show constitutive activity at rest. In this review article, we will focus on the lipid-sensitive mechano-gated K(2P) channel TREK-1 and will emphasize on the polymodal function of this "unconventional" K+ channel.

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

    PubMed

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

    2011-06-01

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

  9. 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

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

    PubMed

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

    2016-01-01

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

  11. A Gate Hinge Controls the Epithelial Calcium Channel TRPV5

    PubMed Central

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

    2017-01-01

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

  12. Spatial Distribution of Calcium-Gated Chloride Channels in Olfactory Cilia

    PubMed Central

    French, Donald A.; Badamdorj, Dorjsuren; Kleene, Steven J.

    2010-01-01

    Background In vertebrate olfactory receptor neurons, sensory cilia transduce odor stimuli into changes in neuronal membrane potential. The voltage changes are primarily caused by the sequential openings of two types of channel: a cyclic-nucleotide-gated (CNG) cationic channel and a calcium-gated chloride channel. In frog, the cilia are 25 to 200 µm in length, so the spatial distributions of the channels may be an important determinant of odor sensitivity. Principal Findings To determine the spatial distribution of the chloride channels, we recorded from single cilia as calcium was allowed to diffuse down the length of the cilium and activate the channels. A computational model of this experiment allowed an estimate of the spatial distribution of the chloride channels. On average, the channels were concentrated in a narrow band centered at a distance of 29% of the ciliary length, measured from the base of the cilium. This matches the location of the CNG channels determined previously. This non-uniform distribution of transduction proteins is consistent with similar findings in other cilia. Conclusions On average, the two types of olfactory transduction channel are concentrated in the same region of the cilium. This may contribute to the efficient detection of weak stimuli. PMID:21209888

  13. Molecular diversity of GABA-gated chloride channels in the rat anterior pituitary.

    PubMed

    Boué-Grabot, E; Dufy, B; Garret, M

    1995-12-15

    mRNA expression of GABA-gated Cl(-)-channels in rat antepituitary was evaluated by using an reverse-transcribed (RT)-polymerase chain reaction (RT-PCR) method with degenerate and specific oligonucleotides. The main result of our findings is that the antepituitary expresses mRNAs encoding alpha 4 and rho 1 GABA receptor subunits. These two subunits are believed to be, respectively, constituents of benzodiazepine-insensitive GABAA and GABAC receptors in the CNS. This molecular analysis is consistent with the pharmacological diversity of GABA receptors in pituitary cells.

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  16. 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

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

    PubMed

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

    1999-09-01

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

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

    PubMed

    Kraus-Friedmann, N

    2000-03-01

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

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

    PubMed

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

    2014-11-01

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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

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

    PubMed

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

    2008-12-30

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

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

    PubMed

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

    2010-10-15

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

  8. 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

  9. Mechanisms of Activation of Voltage-Gated Potassium Channels

    PubMed Central

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

    2014-01-01

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

  10. 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

  11. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

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

  13. The Pharmacology of Cyclic Nucleotide-Gated Channels: Emerging from the Darkness

    PubMed Central

    Brown, R. Lane; Strassmaier, Timothy; Brady, James D.; Karpen, Jeffrey W.

    2008-01-01

    Cyclic nucleotide-gated (CNG) ion channels play a central role in vision and olfaction, generating the electrical responses to light in photoreceptors and to odorants in olfactory receptors. These channels have been detected in many other tissues where their functions are largely unclear. The use of gene knockouts and other methods have yielded some information, but there is a pressing need for potent and specific pharmacological agents directed at CNG channels. To date there has been very little systematic effort in this direction - most of what can be termed CNG channel pharmacology arose from testing reagents known to target protein kinases or other ion channels, or by accident when researchers were investigating other intracellular pathways that may regulate the activity of CNG channels. Predictably, these studies have not produced selective agents. However, taking advantage of emerging structural information and the increasing knowledge of the biophysical properties of these channels, some promising compounds and strategies have begun to emerge. In this review we discuss progress on two fronts, cyclic nucleotide analogs as both activators and competitive inhibitors, and inhibitors that target the pore or gating machinery of the channel. We also discuss the potential of these compounds for treating certain forms of retinal degeneration. PMID:17073662

  14. Ryanodine receptors as leak channels.

    PubMed

    Guerrero-Hernández, Agustín; Ávila, Guillermo; Rueda, Angélica

    2014-09-15

    Ryanodine receptors are Ca(2+) release channels of internal stores. This review focuses on those situations and conditions that transform RyRs from a finely regulated ion channel to an unregulated Ca(2+) leak channel and the pathological consequences of this alteration. In skeletal muscle, mutations in either CaV1.1 channel or RyR1 results in a leaky behavior of the latter. In heart cells, RyR2 functions normally as a Ca(2+) leak channel during diastole within certain limits, the enhancement of this activity leads to arrhythmogenic situations that are tackled with different pharmacological strategies. In smooth muscle, RyRs are involved more in reducing excitability than in stimulating contraction so the leak activity of RyRs in the form of Ca(2+) sparks, locally activates Ca(2+)-dependent potassium channels to reduce excitability. In neurons the enhanced activity of RyRs is associated with the development of different neurodegenerative disorders such as Alzheimer and Huntington diseases. It appears then that the activity of RyRs as leak channels can have both physiological and pathological consequences depending on the cell type and the metabolic condition.

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

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

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

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

    PubMed Central

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

    2008-01-01

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

  17. 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.

  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. Voltage-dependent gating of NR1/2B NMDA receptors

    PubMed Central

    Clarke, Richard J; Johnson, Jon W

    2008-01-01

    Ligand-gated ion channels are activated by agonist binding, but may also be modulated by membrane voltage. N-Methyl-d-aspartate receptors (NMDARs) exhibit especially strong voltage dependence due to channel block by external Mg2+ (Mgo2+). Here we demonstrate that activity of NMDARs composed of NR1 and NR2B subunits (NR1/2B receptors) is enhanced by depolarization even in 0 Mgo2+, causing slow current relaxations in response to rapid voltage changes. We present a kinetic model of receptor activation that incorporates voltage-dependent gating-associated NR2B subunit conformational changes. The model accurately reproduces current relaxations during depolarizations and subsequent repolarizations in 0 Mgo2+. Model simulations in physiological Mgo2+ concentrations show that voltage-dependent receptor gating also underlies the slow component of Mgo2+ unblock, a phenomenon that previously was shown to influence Mgo2+ unblock kinetics during dendritic spikes. We propose that voltage-dependent gating of NR1/2B receptors confers enhanced voltage and time dependence on NMDAR-mediated signalling. PMID:18936081

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

    PubMed

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

    2012-01-20

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

  1. 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

  2. Design of a Gated Molecular Proton Channel

    SciTech Connect

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

    2011-01-17

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

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

    PubMed

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

    2009-09-16

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

  4. Stimulation-dependent gating of TRPM3 channel in planar lipid bilayers.

    PubMed

    Uchida, Kunitoshi; Demirkhanyan, Lusine; Asuthkar, Swapna; Cohen, Alejandro; Tominaga, Makoto; Zakharian, Eleonora

    2016-03-01

    The transient receptor potential melastatin (TRPM)-3 channel is critical for various physiologic processes. In somatosensory neurons, TRPM3 has been implicated in temperature perception and inflammatory hyperalgesia, whereas in pancreatic β-cells the channel has been linked to glucose-induced insulin release. As a typical representative of the TRP family, TRPM3 is highly polymodal. In cells, it is activated by heat and chemical agonists, including pregnenolone sulfate (PS) and nifedipine (Nif). To define the nuances of TRPM3 channel activity and its modulators, we succeeded in incorporating the TRPM3 protein into planar lipid bilayers. We found that phosphatidylinositol-4,5-bisphosphate (PIP2) or clotrimazole is necessary for channel opening by PS. Unlike PS, the presence of Nif alone sufficed to induce TRPM3 activity and demonstrated distinct gating behavior. We also performed an extensive thermodynamic analysis of TRPM3 activation and found that TRPM3 exhibited slight temperature sensitivity in the bilayers. In the absence of other agonists TRPM3 channels remained closed upon heat-induced stimulation, but opened in the presence of PIP2, although with only a low open-probability profile. Together, our results elucidate the details peculiar to TRPM3 channel function in an isolated system. We confirmed its direct gating by PS and PIP2, but found a lack of the strong intrinsic temperature sensitivity common to other thermosensitive TRP channels.

  5. Voltage change-induced gating transitions of the rabbit skeletal muscle Ca2+ release channel

    PubMed Central

    Zahradníková, A; Mészáros, L G

    1998-01-01

    We used the planar lipid bilayer method to study single ryanodine receptor Ca2+ release channels (RyRCs) from fast skeletal muscle of the rabbit. We found that changes in membrane voltage directly induced gating transitions of the RyRC: (i) in the steady state, even at activating Ca2+ concentrations (20 μm), at a constant membrane potential the channels resided in a low open probability (Po) state (inactivated-, I-mode), and (ii) upon abrupt changes of voltage, the apparent inactivation of the RyRCs was relieved, resulting in a rapid and transient increase in Po. The magnitude of the Po increase was a function of both the duration and the amplitude of the applied prepulse, but was independent of the channel activity during the prepulse. The voltage-induced Po increase probably involved major conformational changes of the channel, as it resulted in substantial alterations in the gating pattern of the channels: the voltage change-induced increase in Po was accompanied by the rapid appearance of two types of channel activity (high (H) and low (L) open probability modes). The response of the RyRC to voltage changes raises the interesting possibility that the activation of RyRC in situ might involve electrical events, i.e. a possible dipole-dipole coupling between the release channel and the voltage sensor. PMID:9547378

  6. Cardiac Mechano-Gated Ion Channels and Arrhythmias

    PubMed Central

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

    2015-01-01

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

  7. 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

  8. Comparison of gating dynamics of different IP3R channels with immune algorithm searching for channel parameter distributions

    NASA Astrophysics Data System (ADS)

    Cai, Xiuhong; Li, Xiang; Qi, Hong; Wei, Fang; Chen, Jianyong; Shuai, Jianwei

    2016-10-01

    The gating properties of the inositol 1, 4, 5-trisphosphate (IP3) receptor (IP3R) are determined by the binding and unbinding capability of Ca2+ ions and IP3 messengers. With the patch clamp experiments, the stationary properties have been discussed for Xenopus oocyte type-1 IP3R (Oo-IP3R1), type-3 IP3R (Oo-IP3R3) and Spodoptera frugiperda IP3R (Sf-IP3R). In this paper, in order to provide insights about the relation between the observed gating characteristics and the gating parameters in different IP3Rs, we apply the immune algorithm to fit the parameters of a modified DeYoung-Keizer model. By comparing the fitting parameter distributions of three IP3Rs, we suggest that the three types of IP3Rs have the similar open sensitivity in responding to IP3. The Oo-IP3R3 channel is easy to open in responding to low Ca2+ concentration, while Sf-IP3R channel is easily inhibited in responding to high Ca2+ concentration. We also show that the IP3 binding rate is not a sensitive parameter for stationary gating dynamics for three IP3Rs, but the inhibitory Ca2+ binding/unbinding rates are sensitive parameters for gating dynamics for both Oo-IP3R1 and Oo-IP3R3 channels. Such differences may be important in generating the spatially and temporally complex Ca2+ oscillations in cells. Our study also demonstrates that the immune algorithm can be applied for model parameter searching in biological systems.

  9. Cytoprotection of kidney epithelial cells by compounds that target amino acid gated chloride channels.

    PubMed

    Venkatachalam, M A; Weinberg, J M; Patel, Y; Saikumar, P; Dong, Z

    1996-02-01

    Glycine, strychnine and certain chloride channel blockers were reported to protect cells against lethal cell injury. These effects have been attributed to interactions with membrane proteins related to CNS glycine gated chloride channel receptors. We have investigated the pharmacology of these actions. Madin-Darby canine kidney (MDCK) epithelial cells were depleted of adenosine triphosphate (ATP) by incubation in glucose free medium containing a mitochondrial uncoupler. Medium Ca2+ was adjusted to 100 nM in the presence of an ionophore such that intracellular Ca2+ did not increase, and Ca(2+)-related injury mechanisms were inhibited. This permitted more sensitive quantitation of protection against cell injury attributable to glycine or other agents whose actions might be related to those of the amino acid. Two classes of compounds showed cytoprotective activity in this system: (1) ligands at chloride channel receptors, such as glycine, strychnine and avermectin B1a; (2) chloride channel blockers, including cyanotriphenylboron and niflumic acid, both of which are known to bind to channel domains of CNS glycine receptors. Morphological and functional studies showed that the compounds preserved plasma membrane integrity, but permitted cell swelling. Substitution of medium chloride by gluconate, or chloride salts by sucrose, did not substantially modify lethal damage or its prevention by glycine or other drugs. The compounds did not modify ATP declines. At least for some compounds, cytoprotection appeared to be specific to structural features on the molecules. These observations are consistent with the hypothesis that a plasma membrane protein related to glycine-gated chloride channel receptors plays a significant role in cell injury, but indicate that the mechanisms of injury and protection by compounds active in this system are not related to chloride fluxes.

  10. 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

  11. 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.

  12. A single amino acid gates the KcsA channel.

    PubMed

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

    2014-08-08

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

  13. 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.

  14. Voltage-gated Potassium Channels as Therapeutic Drug Targets

    PubMed Central

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

    2009-01-01

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

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

    PubMed

    Thompson, Jill; Begenisich, Ted

    2012-03-01

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

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

    PubMed Central

    Thompson, Jill

    2012-01-01

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

  17. Stimulation-dependent gating of TRPM3 channel in planar lipid bilayers

    PubMed Central

    Uchida, Kunitoshi; Demirkhanyan, Lusine; Asuthkar, Swapna; Cohen, Alejandro; Tominaga, Makoto; Zakharian, Eleonora

    2016-01-01

    The transient receptor potential melastatin (TRPM)-3 channel is critical for various physiologic processes. In somatosensory neurons, TRPM3 has been implicated in temperature perception and inflammatory hyperalgesia, whereas in pancreatic β-cells the channel has been linked to glucose-induced insulin release. As a typical representative of the TRP family, TRPM3 is highly polymodal. In cells, it is activated by heat and chemical agonists, including pregnenolone sulfate (PS) and nifedipine (Nif). To define the nuances of TRPM3 channel activity and its modulators, we succeeded in incorporating the TRPM3 protein into planar lipid bilayers. We found that phosphatidylinositol-4,5-bisphosphate (PIP2) or clotrimazole is necessary for channel opening by PS. Unlike PS, the presence of Nif alone sufficed to induce TRPM3 activity and demonstrated distinct gating behavior. We also performed an extensive thermodynamic analysis of TRPM3 activation and found that TRPM3 exhibited slight temperature sensitivity in the bilayers. In the absence of other agonists TRPM3 channels remained closed upon heat-induced stimulation, but opened in the presence of PIP2, although with only a low open-probability profile. Together, our results elucidate the details peculiar to TRPM3 channel function in an isolated system. We confirmed its direct gating by PS and PIP2, but found a lack of the strong intrinsic temperature sensitivity common to other thermosensitive TRP channels.—Kunitoshi, U., Demirkhanyan, L., Asuthkar, S., Cohen, A., Tominaga, M., Zakharian, E. Stimulation-dependent gating of TRPM3 channel in planar lipid bilayers. PMID:26655382

  18. Voltage- and cold-dependent gating of single TRPM8 ion channels

    PubMed Central

    Skryma, Roman; Bidaux, Gabriel; Magleby, Karl L.; Scholfield, C. Norman; McGeown, J. Graham; Prevarskaya, Natalia

    2011-01-01

    Transient receptor potential (TRP) channels play critical roles in cell signaling by coupling various environmental factors to changes in membrane potential that modulate calcium influx. TRP channels are typically activated in a polymodal manner, thus integrating multiple stimuli. Although much progress has been made, the underlying mechanisms of TRP channel activation are largely unknown. The TRPM8 cation channel has been extensively investigated as a major neuronal cold sensor but is also activated by voltage, calcium store depletion, and some lipids as well as by compounds that produce cooling sensations, such as menthol or icilin. Several models of TRPM8 activation have been proposed to explain the interaction between these diverse stimuli. However, a kinetic scheme is not yet available that can describe the detailed single-channel kinetics to gain further insight into the underlying gating mechanism. To work toward this goal, we investigated voltage-dependent single-channel gating in cell-attached patches at two different temperatures (20 and 30°C) using HEK293 cells stably expressing TRPM8. Both membrane depolarization and cooling increased channel open probability (Po) mainly by decreasing the duration of closed intervals, with a smaller increase in the duration of open intervals. Maximum likelihood analysis of dwell times at both temperatures indicated gating in a minimum of five closed and two open states, and global fitting over a wide range of voltages identified a seven-state model that described the voltage dependence of Po, the single-channel kinetics, and the response of whole-cell currents to voltage ramps and steps. The major action of depolarization and cooling was to accelerate forward transitions between the same two sets of adjacent closed states. The seven-state model provides a general mechanism to account for TRPM8 activation by membrane depolarization at two temperatures and can serve as a starting point for further investigations of

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

    PubMed

    Panyi, Gyorgy; Deutsch, Carol

    2006-11-01

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

  20. Emerging concepts for G protein-gated inwardly rectifying potassium (GIRK) channels in health and disease

    PubMed Central

    Lüscher, Christian; Slesinger, Paul A.

    2010-01-01

    G protein-gated inwardly rectifying potassium (GIRK) channels hyperpolarize neurons in response to the activation of many G-protein coupled receptors and thus control the excitability of neurons through GIRK-mediated self-inhibition, slow synaptic potentials and volume transmission. GIRK channel function and trafficking are highly dependent on their subunit composition. Pharmacological investigations of GIRK channels and studies in animal models suggest that GIRK activity has an important role in physiological responses, including pain perception and memory modulation. Moreover, abnormal GIRK function has been implicated in altering neuronal excitability and cell death that may be important in the pathophysiology of human diseases such as epilepsy, Down’s syndrome, Parkinson’s disease and drug addiction. GIRK channels may therefore prove to be a valuable new therapeutic target for treating these health problems. PMID:20389305

  1. Structural insights into Ca2+-activated long-range allosteric channel gating of RyR1

    PubMed Central

    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

    2016-01-01

    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 Ca2+-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 Ca2+ 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

  2. Intramembrane aromatic interactions influence the lipid sensitivities of pentameric ligand-gated ion channels.

    PubMed

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

    2015-01-23

    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.

  3. Studies of multimodal gating of the sodium channel.

    PubMed

    Keynes, Richard D

    2002-01-01

    Chandler and Meves found that in squid axons perfused with NaF a small flow of Na+ ions persisted in the inactivated state, and that the Na+ channel therefore has more than one open state. Studies by Correa and Bezanilla on single patches in squid axons showed that such steady currents arose from reopening of the channel at a relatively low frequency. Currents with comparable properties are generated in mammalian brain cells and elsewhere. The existence of a third mode of gating was established by Patlak and Ortiz when they showed that in frog muscle fibres there were occasionally quite large bursts of late openings. Again, similar behaviour has been observed in other types of muscle and in brain cells. It is suggested that the voltage gating of all ionic channels involves a screw-helical mechanism, operating in steps each transferring unit charge. For segment S4 in domain IV of Na+ channels, three charges have to be transferred to reach the initial open state, and a fourth for fast inactivation to take place. The single late openings in the inactivated steady state may be explained by the transfer of a fifth charge in IVS4, while the larger bursts of reopening involve a modulation of the mechanism of fast inactivation.

  4. 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.

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

    PubMed

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-07-02

    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.

  6. 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

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

    PubMed Central

    Buraei, Zafir; Yang, Jian

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

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

    PubMed Central

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

    2016-01-01

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

  10. Intersubunit Physical Couplings Fostered By The Left Flipper Domain Facilitate Channel Opening Of P2X4 Receptors.

    PubMed

    Wang, Jin; Sun, Liang-Fei; Cui, Wen-Wen; Zhao, Wen-Shan; Ma, Xue-Fei; Li, Bin; Liu, Yan; Yang, Yang; Hu, You-Min; Huang, Li-Dong; Cheng, Xiao-Yang; Li, Lingyong; Lu, Xiang-Yang; Tian, Yun; Yu, Ye

    2017-03-16

    P2X receptors are ATP-gated trimeric channels with important roles in diverse pathophysiological functions. A detailed understanding of the mechanism underlying the gating process of these receptors is thus fundamentally important and may open new therapeutic avenues. The left flipper (LF) domain of P2X receptors is a flexible loop structure and its coordinated motions together with the dorsal fin (DF) domain are crucial for the channel gating of the P2X receptors. However, the mechanism underlying the crucial role of the LF domain in the channel gating remains obscure. Here, we propose that the ATP-induced allosteric changes of the LF domain enable it to foster intersubunit physical couplings among the DF and two lower body domains, which is pivotal for the channel gating of P2X4 receptors. Metadynamics analysis indicated that these newly established intersubunit couplings correlate well with the ATP-bound open state of the receptors. Moreover, weakening or strengthening these physical interactions with engineered intersubunit metal bridges remarkably decreased or increased the open probability of the receptors, respectively. Further disulfide crosslinking and covalent modification confirmed that the intersubunit physical couplings among the DF and two lower body domains fostered by the LF domain at the open state act as an integrated structural element that is stringently required for the channel gating of P2X4 receptors. Our observations provide new mechanistic insights into P2X receptor activation and will stimulate development of new allosteric modulators of P2X receptors.

  11. 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

  12. Pore conformations and gating mechanism of a Cys-loop receptor.

    PubMed

    Paas, Yoav; Gibor, Gilad; Grailhe, Regis; Savatier-Duclert, Nathalie; Dufresne, Virginie; Sunesen, Morten; de Carvalho, Lia Prado; Changeux, Jean-Pierre; Attali, Bernard

    2005-11-01

    Neurons regulate the propagation of chemoelectric signals throughout the nervous system by opening and closing ion channels, a process known as gating. Here, histidine-based metal-binding sites were engineered along the intrinsic pore of a chimeric Cys-loop receptor to probe state-dependent Zn(2+)-channel interactions. Patterns of Zn(2+) ion binding within the pore reveal that, in the closed state, the five pore-lining segments adopt an oblique orientation relative to the axis of ion conduction and constrict into a physical gate at their intracellular end. The interactions of Zn(2+) with the open state indicate that the five pore-lining segments should rigidly tilt to enable the movement of their intracellular ends away from the axis of ion conduction, so as to open the constriction (i.e., the gate). Alignment of the functional results with the 3D structure of an acetylcholine receptor allowed us to generate structural models accounting for the closed and open pore conformations and for a gating mechanism of a Cys-loop receptor.

  13. Mechanism of gating of T-type calcium channels

    PubMed Central

    1990-01-01

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

  14. Two-photon scanning photochemical microscopy: mapping ligand-gated ion channel distributions.

    PubMed Central

    Denk, W

    1994-01-01

    The locations and densities of ionotropic membrane receptors, which are responsible for receiving synaptic transmission throughout the nervous system, are of prime importance in understanding the function of neural circuits. It is shown that the highly localized liberation of "caged" neurotransmitters by two-photon absorption-mediated photoactivation can be used in conjunction with recording the induced whole-cell current to determine the distribution of ligand-gated ion channels. The technique is potentially sensitive enough to detect individual channels with diffraction-limited spatial resolution. Images of the distribution of nicotinic acetylcholine receptors on cultured BC3H1 cells were obtained using a photoactivatable precursor of the nicotinic agonist carbamoylcholine. Images PMID:7517555

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

    PubMed

    Kruger, Larisa C; Isom, Lori L

    2016-06-01

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

  16. Structure and gating of CLC channels and exchangers

    PubMed Central

    Accardi, Alessio

    2015-01-01

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

  17. 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

  18. Pannexin-1 as a potentiator of ligand-gated receptor signaling.

    PubMed

    Isakson, Brant E; Thompson, Roger J

    2014-01-01

    Pannexins are a class of plasma membrane spanning proteins that presumably form a hexameric, non-selective ion channel. Although similar in secondary structure to the connexins, pannexins notably do not form endogenous gap junctions and act as bona fide ion channels. The pannexins have been primarily studied as ATP-release channels, but the overall diversity of their functions is still being elucidated. There is an intriguing theme with pannexins that has begun to develop. In this review we analyze several recent reports that converge on the idea that pannexin channels (namely Panx1) can potentiate ligand-gated receptor signaling. Although the literature remains sparse, this emerging concept appears consistent between both ionotropic and metabotropic receptors of several ligand families.

  19. 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

  20. Bidirectional regulation of dendritic voltage-gated potassium channels by the fragile X mental retardation protein.

    PubMed

    Lee, Hye Young; Ge, Woo-Ping; Huang, Wendy; He, Ye; Wang, Gordon X; Rowson-Baldwin, Ashley; Smith, Stephen J; Jan, Yuh Nung; Jan, Lily Yeh

    2011-11-17

    How transmitter receptors modulate neuronal signaling by regulating voltage-gated ion channel expression remains an open question. Here we report dendritic localization of mRNA of Kv4.2 voltage-gated potassium channel, which regulates synaptic plasticity, and its local translational regulation by fragile X mental retardation protein (FMRP) linked to fragile X syndrome (FXS), the most common heritable mental retardation. FMRP suppression of Kv4.2 is revealed by elevation of Kv4.2 in neurons from fmr1 knockout (KO) mice and in neurons expressing Kv4.2-3'UTR that binds FMRP. Moreover, treating hippocampal slices from fmr1 KO mice with Kv4 channel blocker restores long-term potentiation induced by moderate stimuli. Surprisingly, recovery of Kv4.2 after N-methyl-D-aspartate receptor (NMDAR)-induced degradation also requires FMRP, likely due to NMDAR-induced FMRP dephosphorylation, which turns off FMRP suppression of Kv4.2. Our study of FMRP regulation of Kv4.2 deepens our knowledge of NMDAR signaling and reveals a FMRP target of potential relevance to FXS.

  1. 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

  2. Unravelling calcium-release channel gating: clues from a 'hot' disease.

    PubMed Central

    McCarthy, T V; Mackrill, J J

    2004-01-01

    Ryanodine receptors (RyRs) are a family of intracellular channels that mediate Ca2+ release from the endoplasmic and sarcoplasmic reticulum. More than 50 distinct point mutations in one member of this family, RyR1, cause malignant hyperthermia, a potentially lethal pharmacogenetic disorder of skeletal muscle. These mutations are not randomly distributed throughout the primary structure of RyR1, but are grouped in three discrete clusters. In this issue of the Biochemical Journal, Kobayashi et al. present evidence that interdomain interactions between two of these mutation-enriched regions play a key role in the gating mechanism of RyR1. PMID:15154833

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

    PubMed

    González-Ramírez, Ricardo; Felix, Ricardo

    2017-03-31

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

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

    PubMed

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

    2016-09-01

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

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

    PubMed Central

    Keynes, R D; Elinder, F

    1999-01-01

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

  6. Decavanadate modulates gating of TRPM4 cation channels.

    PubMed

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

    2004-11-01

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

  7. A Critical Gating Switch at a Modulatory Site in Neuronal Kir3 Channels

    PubMed Central

    Adney, Scott K.; Ha, Junghoon; Meng, Xuan-Yu; Kawano, Takeharu

    2015-01-01

    Inwardly rectifying potassium channels enforce tight control of resting membrane potential in excitable cells. The Kir3.2 channel, a member of the Kir3 subfamily of G-protein-activated potassium channels (GIRKs), plays several roles in the nervous system, including key responsibility in the GABAB pathway of inhibition, in pain perception pathways via opioid receptors, and is also involved in alcoholism. PKC phosphorylation acts on the channel to reduce activity, yet the mechanism is incompletely understood. Using the heterologous Xenopus oocyte system combined with molecular dynamics simulations, we show that PKC modulation of channel activity is dependent on Ser-196 in Kir3.2 such that, when this site is phosphorylated, the channel is less sensitive to PKC inhibition. This reduced inhibition is dependent on an interaction between phospho-Ser (SEP)-196 and Arg-201, reducing Arg-201 interaction with the sodium-binding site Asp-228. Neutralization of either SEP-196 or Arg-201 leads to a channel with reduced activity and increased sensitivity to PKC inhibition. This study clarifies the role of Ser-196 as an allosteric modulator of PKC inhibition and suggests that the SEP-196/Arg-201 interaction is critical for maintaining maximal channel activity. SIGNIFICANCE STATEMENT The inwardly rectifying potassium 3.2 (Kir3.2) channel is found principally in neurons that regulate diverse brain functions, including pain perception, alcoholism, and substance addiction. Activation or inhibition of this channel leads to changes in neuronal firing and chemical message transmission. The Kir3.2 channel is subject to regulation by intracellular signals including sodium, G-proteins, ethanol, the phospholipid phosphatidylinositol bis-phosphate, and phosphorylation by protein kinases. Here, we take advantage of the recently published structure of Kir3.2 to provide an in-depth molecular view of how phosphorylation of a specific residue previously thought to be the target of PKC promotes

  8. The First Extracellular Linker Is Important for Several Aspects of the Gating Mechanism of Human TRPA1 Channel

    PubMed Central

    Marsakova, Lenka; Barvik, Ivan; Zima, Vlastimil; Zimova, Lucie; Vlachova, Viktorie

    2017-01-01

    Transient receptor potential ankyrin 1 (TRPA1) is an excitatory ion channel involved in pain, inflammation and itching. This channel gates in response to many irritant and proalgesic agents, and can be modulated by calcium and depolarizing voltage. While the closed-state structure of TRPA1 has been recently resolved, also having its open state is essential for understanding how this channel works. Here we use molecular dynamics simulations combined with electrophysiological measurements and systematic mutagenesis to predict and explore the conformational changes coupled to the expansion of the presumptive channel's lower gate. We show that, upon opening, the upper part of the sensor module approaches the pore domain of an adjacent subunit and the conformational dynamics of the first extracellular flexible loop may govern the voltage-dependence of multimodal gating, thereby serving to stabilize the open state of the channel. These results are generally important in understanding the structure and function of TRPA1 and offer new insights into the gating mechanism of TRPA1 and related channels. PMID:28197074

  9. 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

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

    PubMed

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

    2016-11-01

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

  11. Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel.

    PubMed

    Hilf, Ricarda J C; Dutzler, Raimund

    2009-01-01

    The X-ray structure of a pentameric ligand-gated ion channel from Erwinia chrysanthemi (ELIC) has recently provided structural insight into this family of ion channels at high resolution. The structure shows a homo-pentameric protein with a barrel-stave architecture that defines an ion-conduction pore located on the fivefold axis of symmetry. In this structure, the wide aqueous vestibule that is encircled by the extracellular ligand-binding domains of the five subunits narrows to a discontinuous pore that spans the lipid bilayer. The pore is constricted by bulky hydrophobic residues towards the extracellular side, which probably serve as barriers that prevent the diffusion of ions. This interrupted pore architecture in ELIC thus depicts a non-conducting conformation of a pentameric ligand-gated ion channel, the thermodynamically stable state in the absence of bound ligand. As ligand binding promotes pore opening in these ion channels and the specific ligand for ELIC has not yet been identified, we have turned our attention towards a homologous protein from the cyanobacterium Gloebacter violaceus (GLIC). GLIC was shown to form proton-gated channels that are activated by a pH decrease on the extracellular side and that do not desensitize after activation. Both prokaryotic proteins, ELIC and GLIC form ion channels that are selective for cations over anions with poor discrimination among monovalent cations, characteristics that resemble the conduction properties of the cation-selective branch of the family that includes acetylcholine and serotonin receptors. Here we present the X-ray structure of GLIC at 3.1 A resolution. The structure reveals a conformation of the channel that is distinct from ELIC and that probably resembles the open state. In combination, both structures suggest a novel gating mechanism for pentameric ligand-gated ion channels where channel opening proceeds by a change in the tilt of the pore-forming helices.

  12. Structural basis for potentiation by alcohols and anaesthetics in a ligand-gated ion channel

    PubMed Central

    Sauguet, Ludovic; Howard, Rebecca J.; Malherbe, Laurie; Lee, Ui S.; Corringer, Pierre-Jean; Harris, R. Adron; Delarue, Marc

    2014-01-01

    Ethanol alters nerve signalling by interacting with proteins in the central nervous system, particularly pentameric ligand-gated ion channels. A recent series of mutagenesis experiments on Gloeobacter violaceus ligand-gated ion channel, a prokaryotic member of this family, identified a single-site variant that is potentiated by pharmacologically relevant concentrations of ethanol. Here we determine crystal structures of the ethanol-sensitized variant in the absence and presence of ethanol and related modulators, which bind in a transmembrane cavity between channel subunits and may stabilize the open form of the channel. Structural and mutagenesis studies defined overlapping mechanisms of potentiation by alcohols and anaesthetics via the inter-subunit cavity. Furthermore, homology modelling show this cavity to be conserved in human ethanol-sensitive glycine and GABA(A) receptors, and to involve residues previously shown to influence alcohol and anaesthetic action on these proteins. These results suggest a common structural basis for ethanol potentiation of an important class of targets for neurological actions of ethanol. PMID:23591864

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

    PubMed

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

    2014-01-07

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

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

    PubMed Central

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

    1998-01-01

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

  15. An mRNA encoding a putative GABA-gated chloride channel is expressed in the human cardiac conduction system.

    PubMed

    Garret, M; Bascles, L; Boue-Grabot, E; Sartor, P; Charron, G; Bloch, B; Margolskee, R F

    1997-04-01

    GABA-gated chloride channels are the main inhibitory neurotransmitter receptors in the CNS. Conserved domains among members of previously described GABAA receptor subunits were used to design degenerate sense and antisense oligonucleotides. A PCR product from this amplification was used to isolate a full-length cDNA. The predicted protein has many of the features shared by other members of the ligand-gated ion channel family. This channel subunit has significant amino acid identity (25-40%) with members of GABAA and GABAC receptor subunits and thus may represent a new subfamily of the GABA receptor channel. Although we cannot rule out that this clone encodes a receptor for an unidentified ligand, it was termed GABA chi. This gene is mainly expressed in placenta and in heart; however, placenta appears to express only an unspliced mRNA. In situ hybridization reveals that the GABA chi subunit mRNA is present in the electrical conduction system of the human heart. Our results suggest that novel GABA receptors expressed outside of the CNS may regulate cardiac function.

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

    PubMed

    Carr, Michael J

    2013-10-01

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

  17. Contributions of Conserved Residues at the Gating Interface of Glycine Receptors*

    PubMed Central

    Pless, Stephan A.; Leung, Ada W. Y.; Galpin, Jason D.; Ahern, Christopher A.

    2011-01-01

    Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in α1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 α1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 → Gln. PMID:21835920

  18. Domain-based identification and analysis of glutamate receptor ion channels and their relatives in prokaryotes.

    PubMed

    Ger, Mao-Feng; Rendon, Gloria; Tilson, Jeffrey L; Jakobsson, Eric

    2010-10-06

    Voltage-gated and ligand-gated ion channels are used in eukaryotic organisms for the purpose of electrochemical signaling. There are prokaryotic homologues to major eukaryotic channels of these sorts, including voltage-gated sodium, potassium, and calcium channels, Ach-receptor and glutamate-receptor channels. The prokaryotic homologues have been less well characterized functionally than their eukaryotic counterparts. In this study we identify likely prokaryotic functional counterparts of eukaryotic glutamate receptor channels by comprehensive analysis of the prokaryotic sequences in the context of known functional domains present in the eukaryotic members of this family. In particular, we searched the nonredundant protein database for all proteins containing the following motif: the two sections of the extracellular glutamate binding domain flanking two transmembrane helices. We discovered 100 prokaryotic sequences containing this motif, with a wide variety of functional annotations. Two groups within this family have the same topology as eukaryotic glutamate receptor channels. Group 1 has a potassium-like selectivity filter. Group 2 is most closely related to eukaryotic glutamate receptor channels. We present analysis of the functional domain architecture for the group of 100, a putative phylogenetic tree, comparison of the protein phylogeny with the corresponding species phylogeny, consideration of the distribution of these proteins among classes of prokaryotes, and orthologous relationships between prokaryotic and human glutamate receptor channels. We introduce a construct called the Evolutionary Domain Network, which represents a putative pathway of domain rearrangements underlying the domain composition of present channels. We believe that scientists interested in ion channels in general, and ligand-gated ion channels in particular, will be interested in this work. The work should also be of interest to bioinformatics researchers who are interested in the use

  19. Methamphetamine acutely inhibits voltage-gated calcium channels but chronically up-regulates L-type channels.

    PubMed

    Andres, Marilou A; Cooke, Ian M; Bellinger, Frederick P; Berry, Marla J; Zaporteza, Maribel M; Rueli, Rachel H; Barayuga, Stephanie M; Chang, Linda

    2015-07-01

    In neurons, calcium (Ca(2+) ) channels regulate a wide variety of functions ranging from synaptic transmission to gene expression. They also induce neuroplastic changes that alter gene expression following psychostimulant administration. Ca(2+) channel blockers have been considered as potential therapeutic agents for the treatment of methamphetamine (METH) dependence because of their ability to reduce drug craving among METH users. Here, we studied the effects of METH exposure on voltage-gated Ca(2+) channels using SH-SY5Y cells as a model of dopaminergic neurons. We found that METH has different short- and long-term effects. A short-term effect involves immediate (< 5 min) direct inhibition of Ca(2+) ion movements through Ca(2+) channels. Longer exposure to METH (20 min or 48 h) selectively up-regulates the expression of only the CACNA1C gene, thus increasing the number of L-type Ca(2+) channels. This up-regulation of CACNA1C is associated with the expression of the cAMP-responsive element-binding protein (CREB), a known regulator of CACNA1C gene expression, and the MYC gene, which encodes a transcription factor that putatively binds to a site proximal to the CACNA1C gene transcription initiation site. The short-term inhibition of Ca(2+) ion movement and later, the up-regulation of Ca(2+) channel gene expression together suggest the operation of cAMP-responsive element-binding protein- and C-MYC-mediated mechanisms to compensate for Ca(2+) channel inhibition by METH. Increased Ca(2+) current density and subsequent increased intracellular Ca(2+) may contribute to the neurodegeneration accompanying chronic METH abuse. Methamphetamine (METH) exposure has both short- and long-term effects. Acutely, methamphetamine directly inhibits voltage-gated calcium channels. Chronically, neurons compensate by up-regulating the L-type Ca(2+) channel gene, CACNA1C. This compensatory mechanism is mediated by transcription factors C-MYC and CREB, in which CREB is linked to the

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

    PubMed

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

    2015-09-23

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

  1. 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.

  2. 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.

  3. 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.

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

    PubMed Central

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

    2016-01-01

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

  5. Emerging models of glutamate receptor ion channel structure and function.

    PubMed

    Mayer, Mark L

    2011-10-12

    Excitatory synaptic transmission in the brain is mediated by ligand-gated ion channels (iGluRs) activated by glutamate. Distinct from other neurotransmitter receptors, the extracellular domains of iGluRs are loosely packed assemblies with two clearly distinct layers, each of which has both local and global 2-fold axes of symmetry. By contrast, the iGluR transmembrane segments have 4-fold symmetry and share a conserved pore loop architecture found in tetrameric voltage-gated ion channels. The striking layered architecture of iGluRs revealed by the 3.6 Å resolution structure of an AMPA receptor homotetramer likely arose from gene fusion events that occurred early in evolution. Although this modular design has greatly facilitated biophysical and structural studies on individual iGluR domains, and suggested conserved mechanisms for iGluR gating, recent work is beginning to reveal unanticipated diversity in the structure, allosteric regulation, and assembly of iGluR subtypes.

  6. Multisite Binding of a General Anesthetic to the Prokaryotic Pentameric Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC)*

    PubMed Central

    Spurny, Radovan; Billen, Bert; Howard, Rebecca J.; Brams, Marijke; Debaveye, Sarah; Price, Kerry L.; Weston, David A.; Strelkov, Sergei V.; Tytgat, Jan; Bertrand, Sonia; Bertrand, Daniel; Lummis, Sarah C. R.; Ulens, Chris

    2013-01-01

    Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABAA/C receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7–β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels. PMID:23364792

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2014-04-02

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    1996-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. 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.

  13. A model of the closed form of the nicotinic acetylcholine receptor m2 channel pore.

    PubMed

    Kim, Sanguk; Chamberlain, Aaron K; Bowie, James U

    2004-08-01

    The nicotinic acetylcholine receptor is a neurotransmitter-gated ion channel in the postsynaptic membrane. It is composed of five homologous subunits, each of which contributes one transmembrane helix--the M2 helix--to create the channel pore. The M2 helix from the delta subunit is capable of forming a channel by itself. Although a model of the receptor was recently proposed based on a low-resolution, cryo-electron microscopy density map, we found that the model does not explain much of the other available experimental data. Here we propose a new model of the M2 channel derived solely from helix packing and symmetry constraints. This model agrees well with experimental results from solid-state NMR, chemical reactivity, and mutagenesis experiments. The model depicts the channel pore, the channel gate, and the residues responsible for cation specificity.

  14. Pre-synaptic GABA receptors inhibit glutamate release through GIRK channels in rat cerebral cortex.

    PubMed

    Ladera, Carolina; del Carmen Godino, María; José Cabañero, María; Torres, Magdalena; Watanabe, Masahiko; Luján, Rafael; Sánchez-Prieto, José

    2008-12-01

    Neuronal G protein-gated inwardly rectifying potassium (GIRK) channels mediate the slow inhibitory effects of many neurotransmitters post-synaptically. However, no evidence exists that supports that GIRK channels play any role in the inhibition of glutamate release by GABA(B) receptors. In this study, we show for the first time that GABA(B) receptors operate through two mechanisms in nerve terminals from the cerebral cortex. As shown previously, GABA(B) receptors reduces glutamate release and the Ca(2+) influx mediated by N-type Ca(2+) channels in a mode insensitive to the GIRK channel blocker tertiapin-Q and consistent with direct inhibition of this voltage-gated Ca(2+) channel. However, by means of weak stimulation protocols, we reveal that GABA(B) receptors also reduce glutamate release mediated by P/Q-type Ca(2+) channels, and that these responses are reversed by the GIRK channel blocker tertiapin-Q. Consistent with the functional interaction between GABA(B) receptors and GIRK channels at nerve terminals we demonstrate by immunogold electron immunohistochemistry that pre-synaptic boutons of asymmetric synapses co-express GABA(B) receptors and GIRK channels, thus suggesting that the functional interaction of these two proteins, found at the post-synaptic level, also occurs at glutamatergic nerve terminals.

  15. Shellfish toxins targeting voltage-gated sodium channels.

    PubMed

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

    2013-11-28

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

  16. Voltage-gated sodium channels and metastatic disease.

    PubMed

    Brackenbury, William J

    2012-01-01

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

  17. 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

  18. 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

  19. Nicotinic Acetylcholine Receptors at the Single-Channel Level.

    PubMed

    Bouzat, Cecilia; Sine, Steven M

    2017-03-05

    Over the past four decades, the patch clamp technique and nicotinic acetylcholine (nACh) receptors have established an enduring partnership. Like all good partnerships, each partner has proven significant in its own right, while their union has spurred innumerable advances in life science research. A member and prototype of the superfamily of pentameric ligand-gated ion channels, the nACh receptor is a chemo-electric transducer, binding nerve-released ACh and rapidly opening its channel to cation flow to elicit cellular excitation. A subject of a Nobel Prize in Physiology or Medicine, the patch clamp technique provides unprecedented resolution of currents through single ion channels in their native cellular environments. Here, focusing on muscle and α7 nACh receptors, we describe the extraordinary contribution of the patch clamp technique toward understanding how they activate in response to neurotransmitter, how subtle structural and mechanistic differences among nACh receptor subtypes translate into significant physiological differences, and how nACh receptors are being exploited as therapeutic drug targets.

  20. DIDS modifies the conductance, gating, and inactivation mechanisms of the cardiac ryanodine receptor.

    PubMed Central

    Hill, Adam Parker; Sitsapesan, Rebecca

    2002-01-01

    The effects of the covalent modifier of amino groups, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on the single-channel properties of purified sheep cardiac ryanodine receptors (RyR) incorporated into planar phospholipid bilayers were investigated. DIDS increased single-channel conductance and open probability (P(o)) and induced unique modifications to the voltage-dependence of gating. The effects of DIDS on conduction and gating were irreversible within the time scale of the experiments, and both effects were dependent on the permeant ion. DIDS induced a greater increase in conductance with Ca(2+) (20%) compared with K(+) (8%) as the permeant ion. After modification by DIDS, all channels could be rapidly inactivated in a voltage-dependent manner. The open probability of the DIDS-modified channel decreased with increasing positive or negative transmembrane potentials; however, inactivation was only observed at negative potentials. Our results demonstrate that inactivation of RyR channels is dependent on the ligand activating the channel, and this will have consequences for the control and termination of sarcoplasmic reticulum Ca(2+) release in cardiac cells. PMID:12023226

  1. Contribution of valine 7' of TMD2 to gating of neuronal alpha3 receptor subtypes.

    PubMed

    Nieves-Cintrón, Madeline; Caballero-Rivera, Daniel; Navedo, Manuel F; Lasalde-Dominicci, José A

    2006-12-01

    The second transmembrane domain (TMD2) of the Cys-loop family of ligand-gated ion channels forms the channel pore. The functional role of the amino acid residues contributing to the channel pore in neuronal nicotinic alpha3 receptors is not well understood. We characterized the contribution of TMD2 position V7' to channel gating in neuronal nicotinic alpha3 receptors. Site-directed mutagenesis was used to substitute position alpha3 (V7') with four different amino acids (A, F, S, or Y) and coexpressed each mutant subunit with wild-type (WT) beta2 or beta4 subunits in Xenopus oocytes. Whole-cell voltage clamp experiments show that substitution for an alanine, serine, or phenylalanine decreased by 2.3-6.2-fold the ACh-EC(50) for alpha3beta2 and alpha3beta4 receptor subtypes. Interestingly, mutation V7'Y did not produce a significant change in ACh-EC(50) when coexpressed with the beta2 subunit but showed a significant approximately two-fold increase with beta4. Similar responses were obtained with nicotine as the agonist. The antagonist sensitivity of the mutant channels was assessed by using dihydro-beta-erythroidine (DHbetaE) and methyllycaconitine (MLA). The apparent potency of DHbetaE as an antagonist increased by approximately 3.7- and 11-fold for the alpha3beta2 V7'S and V7'F mutants, respectively, whereas no evident changes in antagonist potency were observed for the V7'A and V7'Y mutants. The V7'S and V7'F mutations increase MLA antagonist potency for the alpha3beta4 receptor by approximately 6.2- and approximately 9.3-fold, respectively. The V7'A mutation selectively increases the MLA antagonist potency for the alpha3beta4 receptor by approximately 18.7-fold. These results indicate that position V7' contributes to channel gating kinetics and pharmacology of the neuronal nicotinic alpha3 receptors.

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

    PubMed Central

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

    2014-01-01

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

  3. 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.

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

    PubMed Central

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

    2010-01-01

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

  5. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  7. A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels.

    PubMed

    Zhang, Roger S; Wright, Jordan D; Pless, Stephan A; Nunez, John-Jose; Kim, Robin Y; Li, Jenny B W; Yang, Runying; Ahern, Christopher A; Kurata, Harley T

    2015-06-19

    ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.

  8. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel

    PubMed Central

    Nagel, Georg; Szellas, Tanjef; Huhn, Wolfram; Kateriya, Suneel; Adeishvili, Nona; Berthold, Peter; Ollig, Doris; Hegemann, Peter; Bamberg, Ernst

    2003-01-01

    Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii. ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii, were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H+ and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracellular H+. ChR2 is expressed mainly in C. reinhardtii under low-light conditions, suggesting involvement in photoreception in dark-adapted cells. The predicted seven-transmembrane α helices of ChR2 are characteristic for G protein-coupled receptors but reflect a different motif for a cation-selective ion channel. Finally, we demonstrate that ChR2 may be used to depolarize small or large cells, simply by illumination. PMID:14615590

  9. 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.

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

    PubMed

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

    2016-03-04

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

  11. 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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2007-02-16

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

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

    PubMed

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

    2003-01-01

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

  15. 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

  16. Unambiguous observation of blocked states reveals altered, blocker-induced, cardiac ryanodine receptor gating

    PubMed Central

    Mukherjee, Saptarshi; Thomas, N. Lowri; Williams, Alan J.

    2016-01-01

    The flow of ions through membrane channels is precisely regulated by gates. The architecture and function of these elements have been studied extensively, shedding light on the mechanisms underlying gating. Recent investigations have focused on ion occupancy of the channel’s selectivity filter and its ability to alter gating, with most studies involving prokaryotic K+ channels. Some studies used large quaternary ammonium blocker molecules to examine the effects of altered ionic flux on gating. However, the absence of blocking events that are visibly distinct from closing events in K+ channels makes unambiguous interpretation of data from single channel recordings difficult. In this study, the large K+ conductance of the RyR2 channel permits direct observation of blocking events as distinct subconductance states and for the first time demonstrates the differential effects of blocker molecules on channel gating. This experimental platform provides valuable insights into mechanisms of blocker-induced modulation of ion channel gating. PMID:27703263

  17. Selective alteration of sodium channel gating by Australian funnel-web spider toxins.

    PubMed

    Nicholson, G M; Little, M J; Tyler, M; Narahashi, T

    1996-01-01

    The actions of potent mammalian neurotoxins isolated from the venom of two Australian funnel-web spiders were investigated using both electrophysiological and neurochemical techniques. Whole-cell patch clamp recording of sodium currents in rat dorsal root ganglion neurons revealed that versutoxin (VTX), isolated from the venom of Hadronyche versuta, produced a concentration-dependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation and a reduction in peak TTX-S sodium current. In contrast, VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. VTX also shifted the voltage dependence of sodium channel activation in the hyperpolarizing direction and increased the rate of recovery from inactivation. Ion flux studies performed in rat brain synaptosomes also revealed that robustoxin (RTX), from the venom of Atrax robustus, and VTX both produced a partial activation of 22Na+ flux and an inhibition of batrachotoxin-activated 22Na+ flux. This inhibition of flux through batrachotoxin-activated channels was not due to an interaction with neurotoxin receptor site 1 since [3H]saxitoxin binding was unaffected. In addition, the partial activation of 22Na+ flux was not enhanced in the presence of alpha-scorpion toxin and further experiments suggest that VTX also enhances [3H]batrachotoxin binding. These selective actions of funnel-web spider toxins on sodium channel function are comparable to those of alpha-scorpion and sea anemone toxins which bind to neurotoxin receptor site 3 on the channel to slow channel inactivation profoundly. Also, these modifications of sodium channel gating and kinetics are consistent with actions of the spider toxins to produce repetitive firing of action potentials.

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

    PubMed

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

    2009-01-01

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

  19. Signaling by purinergic receptors and channels in the pituitary gland

    PubMed Central

    Stojilkovic, Stanko S.; He, Mu-Lan; Koshimizu, Taka-aki; Balik, Ales; Zemkova, Hana

    2009-01-01

    Adenosine 5′-triphosphate is frequently released by cells and acts as an agonist for G protein-coupled P2Y receptors and ligand-gated P2X cationic channels in numerous tissues. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5′-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors. In the pituitary gland, adenosine 5′-triphosphate is released from the endings of magnocellular hypothalamic neurons and by anterior pituitary cells through pathway(s) that are still not well characterized. This gland also expresses several members of each family of purinergic receptors. P2X and adenosine receptors are co-expressed in the somata and nerve terminals of vasopressin-releasing neurons as well as in some secretory pituitary cells. P2X receptors stimulate electrical activity and modulate InsP3-dependent calcium release from intracellular stores, whereas adenosine receptors terminate electrical activity. Calcium-mobilizing P2Y receptors are predominantly expressed in non-secretory cells of the anterior and posterior pituitary. PMID:19467293

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

    PubMed

    Chen, Xixi; Johnston, Daniel

    2006-12-01

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

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

    PubMed

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

    2003-06-01

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

  2. Two-pore channels (TPCs): Novel voltage-gated ion channels with pleiotropic functions.

    PubMed

    Feijóo-Bandín, Sandra; García-Vence, María; García-Rúa, Vanessa; Roselló-Lletí, Esther; Portolés, Manuel; Rivera, Miguel; González-Juanatey, José Ramón; Lago, Francisca

    2017-01-02

    Two-pore channels (TPC1-3) comprise a subfamily of the eukaryotic voltage-gated ion channels (VGICs) superfamily that are mainly expressed in acidic stores in plants and animals. TPCS are widespread across the animal kingdom, with primates, mice and rats lacking TPC3, and mainly act as Ca(+) and Na(+) channels, although it was also suggested that they could be permeable to other ions. Nowadays, TPCs have been related to the development of different diseases, including Parkinson´s disease, obesity or myocardial ischemia. Due to this, their study has raised the interest of the scientific community to try to understand their mechanism of action in order to be able to develop an efficient drug that could regulate TPCs activity. In this review, we will provide an updated view regarding TPCs structure, function and activation, as well as their role in different pathophysiological processes.

  3. 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

  4. 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'.

  5. Modal gating transitions in cardiac ryanodine receptors during increases of Ca2+ concentration produced by photolysis of caged Ca2+.

    PubMed

    Zahradníková, A; Dura, M; Györke, S

    1999-08-01

    Channel adaptation is a basic property of the sarcoplasmic reticulum Ca2+-release channels/ryanodine receptors (RyRs). It allows channel activity to decay during sustained increases in the concentration of activating Ca2+. Despite the potential physiological importance of this self-confining process, its molecular mechanism is not well understood. To define the mechanism of adaptation we studied the dynamics of cardiac Ca2+-release channel (RyR) gating using the planar lipid bilayer technique in combination with photolysis of caged Ca2+ (DM-nitrophen). Channels activated by rapid and sustained increases in Ca2+ concentration (from 0.1 to 0.5 micromol/l) displayed three distinct gating modes, manifested as current records with frequent and long openings (H-mode), with rare and short openings (L-mode), and with no openings (I-mode). H-mode channel activity occurred primarily at early times while L- and I-modes predominated at late times after the rapid Ca2+ concentration increase. The decrease in probability of H-mode, mirrored by an increase in the probability of the I-mode, proceeded with a time constant similar to that observed for spontaneous decay in channel activity (i.e., adaptation) in ensemble average records. These results indicate that RyR adaptation transpires by a shift of channel gating from a high open probability mode to low open probability and inactivated modes of the channel.

  6. Adult forebrain NMDA receptors gate social motivation and social memory.

    PubMed

    Jacobs, Stephanie; Tsien, Joe Z

    2017-02-01

    Motivation to engage in social interaction is critical to ensure normal social behaviors, whereas dysregulation in social motivation can contribute to psychiatric diseases such as schizophrenia, autism, social anxiety disorders and post-traumatic stress disorder (PTSD). While dopamine is well known to regulate motivation, its downstream targets are poorly understood. Given the fact that the dopamine 1 (D1) receptors are often physically coupled with the NMDA receptors, we hypothesize that the NMDA receptor activity in the adult forebrain principal neurons are crucial not only for learning and memory, but also for the proper gating of social motivation. Here, we tested this hypothesis by examining sociability and social memory in inducible forebrain-specific NR1 knockout mice. These mice are ideal for exploring the role of the NR1 subunit in social behavior because the NR1 subunit can be selectively knocked out after the critical developmental period, in which NR1 is required for normal development. We found that the inducible deletion of the NMDA receptors prior to behavioral assays impaired, not only object and social recognition memory tests, but also resulted in profound deficits in social motivation. Mice with ablated NR1 subunits in the forebrain demonstrated significant decreases in sociability compared to their wild type counterparts. These results suggest that in addition to its crucial role in learning and memory, the NMDA receptors in the adult forebrain principal neurons gate social motivation, independent of neuronal development.

  7. 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.

  8. Single acetylcholine receptor channel currents recorded at high hydrostatic pressures.

    PubMed Central

    Heinemann, S H; Stühmer, W; Conti, F

    1987-01-01

    A technique for performing patch-clamp experiments under high hydrostatic (oil) pressure is described. The method allows the transfer of whole cell or membrane patches in a recording configuration into a pressure vessel, where pressure can be increased up to 60 MPa (approximately equal to 600 bar). We have studied in this way the pressure dependence of single acetylcholine receptor channels in excised "outside-out" membrane patches from cultured rat muscle cells. In the range of 0.1 to 60 MPa the open channel conductance in 140 mM NaCl solutions did not vary by more than 2%, which implies that the translocation of sodium ions through the channel pore does not involve steps with significant activation volumes. At high acetylcholine concentrations (20 microM) bursts of single-channel activity allowed measurements of the mean open and mean closed times of the channel. Pressurization to 40 MPa increased both mean open and mean closed times giving apparent activation volumes of about 59 and 139 A3, respectively. This implies a net volume increase of 80 A3, associated with the transition from the agonist-free state to the open state of the channel, which may be partially associated with the agonist-binding step. All the observed pressure effects were reversible. The activation volumes for the gating of acetylcholine receptor channels are comparable to those of sodium and potassium channels in the squid giant axon, suggesting that there is some basic common mechanism in the operation of ion-channel proteins. Images PMID:2437577

  9. 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).

  10. The Styryl Dye FM1-43 Suppresses Odorant Responses in a Subset of Olfactory Neurons by Blocking Cyclic Nucleotide-gated (CNG) Channels*

    PubMed Central

    Breunig, Esther; Kludt, Eugen; Czesnik, Dirk; Schild, Detlev

    2011-01-01

    Many olfactory receptor neurons use a cAMP-dependent transduction mechanism to transduce odorants into depolarizations. This signaling cascade is characterized by a sequence of two currents: a cation current through cyclic nucleotide-gated channels followed by a chloride current through calcium-activated chloride channels. To date, it is not possible to interfere with these generator channels under physiological conditions with potent and specific blockers. In this study we identified the styryl dye FM1-43 as a potent blocker of native olfactory cyclic nucleotide-gated channels. Furthermore, we characterized this substance to stain olfactory receptor neurons that are endowed with cAMP-dependent transduction. This allows optical differentiation and pharmacological interference with olfactory receptor neurons at the level of the signal transduction. PMID:21646359

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-05-06

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

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

    PubMed Central

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

    2016-01-01

    The ryanodine receptors (RyRs) are intracellular calcium channels responsible for rapid release of Ca2+ 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

  14. Receptors and Channels Targeted by Synthetic Cannabinoid Receptor Agonists and Antagonists

    PubMed Central

    Pertwee, R.G.

    2010-01-01

    It is widely accepted that non-endogenous compounds that target CB1 and/or CB2 receptors possess therapeutic potential for the clinical management of an ever growing number of disorders. Just a few of these disorders are already treated with Δ9-tetrahydrocannabinol or nabilone, both CB1/CB2 receptor agonists, and there is now considerable interest in expanding the clinical applications of such agonists and also in exploiting CB2-selective agonists, peripherally restricted CB1/CB2 receptor agonists and CB1/CB2 antagonists and inverse agonists as medicines. Already, numerous cannabinoid receptor ligands have been developed and their interactions with CB1 and CB2 receptors well characterized. This review describes what is currently known about the ability of such compounds to bind to, activate, inhibit or block non-CB1, non-CB2 G protein-coupled receptors such as GPR55, transmitter gated channels, ion channels and nuclear receptors in an orthosteric or allosteric manner. It begins with a brief description of how each of these ligands interacts with CB1 and/or CB2 receptors. PMID:20166927

  15. Functional Coupling of Ca2+ Channels and Ryanodine Receptors in Cardiac Myocytes

    NASA Astrophysics Data System (ADS)

    Sham, James S. K.; Cleemann, Lars; Morad, Martin

    1995-01-01

    In skeletal muscle, dihydropyridine receptors are functionally coupled to ryanodine receptors of the sarcoplasmic reticulum in triadic or diadic junctional complexes. In cardiac muscle direct physical or functional couplings have not been demonstrated. We have tested the hypothesis of functional coupling of L-type Ca2+ channels and ryanodine receptors in rat cardiac myocytes by comparing the efficacies of Ca2+ in triggering Ca2+ release when the ion enters the cell via the Ca2+ channels or the Na^+/Ca2+ exchanger. Ca2+ transported through the Ca2+ channels was 20-160 times more effective than Ca2+ influx via the Na^+/Ca2+ exchanger in gating Ca2+ release from the sarcoplasmic reticulum, suggesting privileged communication between Ca2+ channels and ryanodine receptors. In support of this hypothesis we found that Ca2+ channels were inactivated by Ca2+ release from the sarcoplasmic reticulum, even though the myoplasmic Ca2+ concentrations were buffered with 10 mM EGTA. The data thus suggest privileged cross signaling between the dihydropyridine and ryanodine receptors such that Ca2+ flux through either the Ca2+ channel or the ryanodine receptor alters the gating kinetics of the other channel.

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

    PubMed

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

    2017-03-21

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

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

    PubMed Central

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

    2015-01-01

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

  18. 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

  19. X-ray structures define human P2X3 receptor gating cycle and antagonist action

    NASA Astrophysics Data System (ADS)

    Mansoor, Steven E.; Lü, Wei; Oosterheert, Wout; Shekhar, Mrinal; Tajkhorshid, Emad; Gouaux, Eric

    2016-10-01

    P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human P2X receptors. The mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structures of the pore-forming transmembrane domains of these receptors remain unclear. Here we report X-ray crystal structures of the human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/closed-pore/desensitized and antagonist-bound/closed states. The open state structure harbours an intracellular motif we term the ‘cytoplasmic cap’, which stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. The competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements that underlie P2X receptor gating and provide a foundation for the development of new pharmacological agents.

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

    SciTech Connect

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

    2010-11-12

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

  1. Voltage-gated sodium channel blockers as immunomodulators.

    PubMed

    Roselli, Francesco; Livrea, Paolo; Jirillo, Emilio

    2006-01-01

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

  2. 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

  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.

  4. Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

    PubMed Central

    Degani-Katzav, Nurit; Gortler, Revital; Gorodetzki, Lilach; Paas, Yoav

    2016-01-01

    The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and β subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds. By electrophysiological characterization of these mutants, we found heteromeric assemblies with two equivalent Glu-binding sites at β/α intersubunit interfaces, where the GluClβ and GluClα subunits, respectively, contribute the “principal” and “complementary” components of the putative Glu-binding pockets. We identified a mutation in the IVM-binding site (far away from the Glu-binding sites), which significantly increased the sensitivity of the heteromeric mutant receptor to both Glu and IVM, and improved the receptor subunits’ cooperativity. We further characterized this heteromeric GluClR mutant as a receptor having a third Glu-binding site at an α/α intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three α and two β subunits arranged in a counterclockwise β-α-β-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces. PMID:26792524

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

    PubMed Central

    Morton, Russell A.; Valenzuela, C. Fernando

    2015-01-01

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2017-01-10

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

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

    PubMed

    Dougherty, Kevin; Covarrubias, Manuel

    2006-12-01

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

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

    PubMed

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

    2008-06-01

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

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

    PubMed

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

    2015-11-17

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

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

    PubMed Central

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

    2015-01-01

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

  12. 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.

  13. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2013-04-15

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

  16. Axonal voltage-gated ion channels as pharmacological targets for pain.

    PubMed

    Moldovan, Mihai; Alvarez, Susana; Romer Rosberg, Mette; Krarup, Christian

    2013-05-15

    Upon peripheral nerve injury (caused by trauma or disease process) axons of the dorsal root ganglion (DRG) somatosensory neurons have the ability to sprout and regrow/remyelinate to reinnervate distant target tissue or form a tangled scar mass called a neuroma. This regenerative response can become maladaptive leading to a persistent and debilitating pain state referred to as chronic pain corresponding to the clinical description of neuropathic/chronic inflammatory pain. There is little agreement to what causes peripheral chronic pain other than hyperactivity of the nociceptive DRG neurons which ultimately depends on the function of voltage-gated ion channels. This review focuses on the pharmacological modulators of voltage-gated ion channels known to be present on axonal membrane which represents by far the largest surface of DRG neurons. Blockers of voltage-gated Na(+) channels, openers of voltage-gated K(+) channels and blockers of hyperpolarization-activated cyclic nucleotide-gated channels that were found to reduce neuronal activity were also found to be effective in neuropathic and inflammatory pain states. The isoforms of these channels present on nociceptive axons have limited specificity. The rationale for considering axonal voltage-gated ion channels as targets for pain treatment comes from the accumulating evidence that chronic pain states are associated with a dysregulation of these channels that could alter their specificity and make them more susceptible to pharmacological modulation. This drives the need for further development of subtype-specific voltage-gated ion channels modulators, as well as clinically available neurophysiological techniques for monitoring axonal ion channel function in peripheral nerves.

  17. X-ray structure of a prokaryotic pentameric ligand-gated ion channel.

    PubMed

    Hilf, Ricarda J C; Dutzler, Raimund

    2008-03-20

    Pentameric ligand-gated ion channels (pLGICs) are key players in the early events of electrical signal transduction at chemical synapses. The family codes for a structurally conserved scaffold of channel proteins that open in response to the binding of neurotransmitter molecules. All proteins share a pentameric organization of identical or related subunits that consist of an extracellular ligand-binding domain followed by a transmembrane channel domain. The nicotinic acetylcholine receptor (nAChR) is the most thoroughly studied member of the pLGIC family (for recent reviews see refs 1-3). Two sources of structural information provided an architectural framework for the family. The structure of the soluble acetylcholine-binding protein (AChBP) defined the organization of the extracellular domain and revealed the chemical basis of ligand interaction. Electron microscopy studies of the nAChR from Torpedo electric ray have yielded a picture of the full-length protein and have recently led to the interpretation of an electron density map at 4.0 A resolution. Despite the wealth of experimental information, high-resolution structures of any family member have so far not been available. Until recently, the pLGICs were believed to be only expressed in multicellular eukaryotic organisms. The abundance of prokaryotic genome sequences, however, allowed the identification of several homologous proteins in bacterial sources. Here we present the X-ray structure of a prokaryotic pLGIC from the bacterium Erwinia chrysanthemi (ELIC) at 3.3 A resolution. Our study reveals the first structure of a pLGIC at high resolution and provides an important model system for the investigation of the general mechanisms of ion permeation and gating within the family.

  18. G-protein-gated inwardly rectifying potassium channels modulate respiratory depression by opioids

    PubMed Central

    Montandon, Gaspard; Ren, Jun; Victoria, Nicole C.; Liu, Hattie; Wickman, Kevin; Greer, John J.; Horner, Richard L.

    2015-01-01

    Background Drugs acting on μ-opioid receptors (MOR) are widely used as analgesics, but present side-effects including life-threatening respiratory depression. MOR are G-protein-coupled receptors inhibiting neuronal activity through calcium channels, adenylyl cyclase, and/or G-protein–gated inwardly-rectifying potassium (GIRK) channels. The pathways underlying MOR-dependent inhibition of rhythmic breathing are unknown. Methods Using a combination of genetic, pharmacological and physiological tools in rodents in vivo, we aimed to identify the role of GIRK channels in MOR-mediated inhibition of respiratory circuits. Results GIRK channels were expressed in the ventrolateral medulla, a neuronal population regulating rhythmic breathing, and GIRK channel activation with flupirtine reduced respiratory rate in rats (percentage of baseline rate in mean±SD: 79.4±7.4%, n=7), wild-type mice (82.6±3.8%, n=3), but not in mice lacking the GIRK2 subunit, an integral subunit of neuronal GIRK channels (GIRK2−/−, 101.0±1.9%, n=3). Application of the MOR agonist DAMGO to the ventrolateral medulla depressed respiratory rate, an effect partially reversed by the GIRK channel blocker Tertiapin Q (baseline: 42.1±7.4 breath/min, DAMGO: 26.1±13.4 breath/min, TertiapinQ+DAMGO: 33.9±9.8 breath/min, n=4). Importantly, DAMGO applied to the ventrolateral medulla failed to reduce rhythmic breathing in GIRK2−/− mice (percentage of baseline rate: 103.2±12.1%, n=4), whereas it considerably reduced rate in wild-type mice (62.5±17.7% of baseline, n=4). Respiratory rate depression by systemic injection of the opioid analgesic fentanyl was markedly reduced in GIRK2−/− (percentage of baseline: 12.8±15.8%, n=5) compared to wild-type mice (72.9±27.3%). Conclusion Overall, these results identify that GIRK channels contribute to respiratory inhibition by MOR, an essential step toward understanding respiratory depression by opioids. PMID:26675532

  19. Differential CaMKII regulation by voltage-gated calcium channels in the striatum.

    PubMed

    Pasek, Johanna G; Wang, Xiaohan; Colbran, Roger J

    2015-09-01

    Calcium signaling regulates synaptic plasticity and many other functions in striatal medium spiny neurons to modulate basal ganglia function. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a major calcium-dependent signaling protein that couples calcium entry to diverse cellular changes. CaMKII activation results in autophosphorylation at Thr286 and sustained calcium-independent CaMKII activity after calcium signals dissipate. However, little is known about the mechanisms regulating striatal CaMKII. To address this, mouse brain slices were treated with pharmacological modulators of calcium channels and punches of dorsal striatum were immunoblotted for CaMKII Thr286 autophosphorylation as an index of CaMKII activation. KCl depolarization increased levels of CaMKII autophosphorylation ~2-fold; this increase was blocked by an LTCC antagonist and was mimicked by treatment with pharmacological LTCC activators. The chelation of extracellular calcium robustly decreased basal CaMKII autophosphorylation within 5min and increased levels of total CaMKII in cytosolic fractions, in addition to decreasing the phosphorylation of CaMKII sites in the GluN2B subunit of NMDA receptors and the GluA1 subunit of AMPA receptors. We also found that the maintenance of basal levels of CaMKII autophosphorylation requires low-voltage gated T-type calcium channels, but not LTCCs or R-type calcium channels. Our findings indicate that CaMKII activity is dynamically regulated by multiple calcium channels in the striatum thus coupling calcium entry to key downstream substrates.

  20. X-ray structures define human P2X3 receptor gating cycle and antagonist action

    PubMed Central

    Mansoor, Steven E.; Lü, Wei; Oosterheert, Wout; Shekhar, Mrinal; Tajkhorshid, Emad; Gouaux, Eric

    2016-01-01

    Summary P2X receptors are trimeric, non-selective cation channels activated by ATP that play important roles in cardiovascular, neuronal and immune systems. Despite their central function in human physiology and as potential targets of therapeutic agents, there are no structures of human P2X receptors. Mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structure of the pore-forming transmembrane domains remain unclear. We report x-ray crystal structures of human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/desensitized and antagonist-bound closed states. The open state structure harbors an intracellular motif we term the “cytoplasmic cap”, that stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. Competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements underpinning P2X receptor gating and provide a foundation for development of new pharmacologic agents. PMID:27626375

  1. 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.

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

    PubMed

    Bähring, Robert; Covarrubias, Manuel

    2011-02-01

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

  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.

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

    PubMed Central

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

    2016-01-01

    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 Ca2+ 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

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

    PubMed Central

    1992-01-01

    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

  6. Phylogenetic shadowing of a histamine-gated chloride channel involved in insect vision.

    PubMed

    Iovchev, Mladen; Boutanaev, Alexander; Ivanov, Ivaylo; Wolstenholme, Adrian; Nurminsky, Dmitry; Semenov, Eugene

    2006-01-01

    A recently identified gene, hclA (synonym: ort), codes for an ionotrophic histamine receptor subunit in Drosophila melanogaster, and known hclA mutations lead to defects in the visual system, neurologic disorders and changed responsiveness to neurotoxins. To investigate whether this novel class of receptors is common across the Insecta, we analysed the genomes of 15 other insect species (Diptera, Hymenoptera, Coleoptera, Lepidoptera) and revealed orthologs of hclA in all of them. The predicted receptor domain of HCLA is extensively conserved (86-100% of identity) among the 16 proteins. Minor changes in the amino acid sequence that includes the putative transmembrane domains (TMs) 1-3 were found in non-drosophilid species only. Substantial amino acid variability was observed in the signal polypeptides, the intracellular loop domains and in TM4, in good accordance with known data on sequence variations in ligand-gated ion channels. Pairwise comparisons revealed three consensus sequences for N-glycosylation, conserved in HCLAs of all species studied, as well as a drosophilid-specific putative phosphorylation site. Real-time PCR analysis demonstrated that hclA-mRNA is abundant in heads of adult Drosophila. However, species- and sex-specific variations of the hclA expression levels were also observed.

  7. 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

    2015-09-05

    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.

  8. 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.

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

    PubMed

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

    2013-06-07

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

  10. Subunit-specific mechanisms and proton sensitivity of NMDA receptor channel block.

    PubMed

    Dravid, Shashank M; Erreger, Kevin; Yuan, Hongjie; Nicholson, Katherine; Le, Phuong; Lyuboslavsky, Polina; Almonte, Antoine; Murray, Ernest; Mosely, Cara; Barber, Jeremy; French, Adam; Balster, Robert; Murray, Thomas F; Traynelis, Stephen F

    2007-05-15

    We have compared the potencies of structurally distinct channel blockers at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptors. The IC50 values varied with stereochemistry and subunit composition, suggesting that it may be possible to design subunit-selective channel blockers. For dizocilpine (MK-801), the differential potency of MK-801 stereoisomers determined at recombinant NMDA receptors was confirmed at native receptors in vitro and in vivo. Since the proton sensor is tightly linked both structurally and functionally to channel gating, we examined whether blocking molecules that interact in the channel pore with the gating machinery can differentially sense protonation of the receptor. Blockers capable of remaining trapped in the pore during agonist unbinding showed the strongest dependence on extracellular pH, appearing more potent at acidic pH values that promote channel closure. Determination of pK(a) values for channel blockers suggests that the ionization of ketamine but not of other blockers can influence its pH-dependent potency. Kinetic modelling and single channel studies suggest that the pH-dependent block of NR1/NR2A by (-)MK-801 but not (+)MK-801 reflects an increase in the MK-801 association rate even though protons reduce channel open probability and thus MK-801 access to its binding site. Allosteric modulators that alter pH sensitivity alter the potency of MK-801, supporting the interpretation that the pH sensitivity of MK-801 binding reflects the changes at the proton sensor rather than a secondary effect of pH. These data suggest a tight coupling between the proton sensor and the ion channel gate as well as unique subunit-specific mechanisms of channel block.

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

    PubMed

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

    2011-10-05

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

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

    PubMed

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

    2011-07-21

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

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

    PubMed

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

    2013-10-23

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-10-01

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

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

    PubMed

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

    2013-02-01

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

  16. 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

  17. Clinical Features of Neuromuscular Disorders in Patients with N-Type Voltage-Gated Calcium Channel Antibodies

    PubMed Central

    Totzeck, Andreas; Mummel, Petra; Kastrup, Oliver; Hagenacker, Tim

    2016-01-01

    Neuromuscular junction disorders affect the pre- or postsynaptic nerve to muscle transmission due to autoimmune antibodies. Members of the group like myasthenia gravis and Lambert-Eaton syndrome have pathophysiologically distinct characteristics. However, in practice, distinction may be difficult. We present a series of three patients with a myasthenic syndrome, dropped-head syndrome, bulbar and respiratory muscle weakness and positive testing for anti-N-type voltage-gated calcium channel antibodies. In two cases anti-acetylcholin receptor antibodies were elevated, anti-P/Q-type voltage-gated calcium channel antibodies were negative. All patients initially responded to pyridostigmine with a non-response in the course of the disease. While one patient recovered well after treatment with intravenous immunoglobulins, 3,4-diaminopyridine, steroids and later on immunosuppression with mycophenolate mofetil, a second died after restriction of treatment due to unfavorable cancer diagnosis, the third patient declined treatment. Although new antibodies causing neuromuscular disorders were discovered, clinical distinction has not yet been made. Our patients showed features of pre- and postsynaptic myasthenic syndrome as well as severe dropped-head syndrome and bulbar and axial muscle weakness, but only anti-N-type voltage-gated calcium channel antibodies were positive. When administered, one patient benefited from 3,4-diaminopyridine. We suggest that this overlap-syndrome should be considered especially in patients with assumed seronegative myasthenia gravis and lack of improvement under standard therapy. PMID:28078065

  18. Tunable Calcium Current through TRPV1 Receptor Channels*S⃞

    PubMed Central

    Samways, Damien S. K.; Khakh, Baljit S.; Egan, Terrance M.

    2008-01-01

    TRPV1 receptors are polymodal cation channels that open in response to diverse stimuli including noxious heat, capsaicin, and protons. Because Ca2+ is vital for TRPV1 signaling, we sought to precisely measure its contribution to TRPV1 responses and discovered that the Ca2+ current was tuned by the mode of activation. Using patch clamp photometry, we found that the fraction of the total current carried by Ca2+ (called the Pf%) was significantly smaller for TRPV1 currents evoked by protons than for those evoked by capsaicin. Using site-directed mutagenesis, we discovered that the smaller Pf% was due to protonation of three acidic amino acids (Asp646, Glu648, and Glu651) that are located in the mouth of the pore. Thus, in keeping with recent reports of time-dependent changes in the ionic permeability of some ligand-gated ion channels, we now show for the first time that the physiologically important Ca2+ current of the TRPV1 receptor is also dynamic and depends on the mode of activation. This current is significantly smaller when the receptor is activated by a change in pH, owing to atomic scale interactions of H+ and Ca2+ with the fixed negative charge of side chains in the pore. PMID:18775990

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

    PubMed Central

    Wang, Andrew; Zocchi, Giovanni

    2011-01-01

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

  20. Promiscuous gating modifiers target the voltage sensor of K(v)7.2, TRPV1, and H(v)1 cation channels.

    PubMed

    Kornilov, Polina; Peretz, Asher; Lee, Yoonji; Son, Karam; Lee, Jin Hee; Refaeli, Bosmat; Roz, Netta; Rehavi, Moshe; Choi, Sun; Attali, Bernard

    2014-06-01

    Some of the fascinating features of voltage-sensing domains (VSDs) in voltage-gated cation channels (VGCCs) are their modular nature and adaptability. Here we examined the VSD sensitivity of different VGCCs to 2 structurally related nontoxin gating modifiers, NH17 and NH29, which stabilize K(v)7.2 potassium channels in the closed and open states, respectively. The effects of NH17 and NH29 were examined in Chinese hamster ovary cells transfected with transient receptor potential vanilloid 1 (TRPV1) or K(v)7.2 channels, as well as in dorsal root ganglia neurons, using the whole-cell patch-clamp technique. NH17 and NH29 exert opposite effects on TRPV1 channels, operating, respectively, as an activator and a blocker of TRPV1 currents (EC50 and IC50 values ranging from 4 to 40 μM). Combined mutagenesis, electrophysiology, structural homology modeling, molecular docking, and molecular dynamics simulation indicate that both compounds target the VSDs of TRPV1 channels, which, like vanilloids, are involved in π-π stacking, H-bonding, and hydrophobic interactions. Reflecting their promiscuity, the drugs also affect the lone VSD proton channel mVSOP. Thus, the same gating modifier can promiscuously interact with different VGCCs, and subtle differences at the VSD-ligand interface will dictate whether the gating modifier stabilizes channels in either the closed or the open state.

  1. 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

  2. STIM1 activates CRAC channels through rotation of the pore helix to open a hydrophobic gate

    PubMed Central

    Yamashita, Megumi; Yeung, Priscilla S.-W.; Ing, Christopher E.; McNally, Beth A.; Pomès, Régis; Prakriya, Murali

    2017-01-01

    Store-operated Ca2+ release-activated Ca2+ (CRAC) channels constitute a major pathway for Ca2+ influx and mediate many essential signalling functions in animal cells, yet how they open remains elusive. Here, we investigate the gating mechanism of the human CRAC channel Orai1 by its activator, stromal interacting molecule 1 (STIM1). We find that two rings of pore-lining residues, V102 and F99, work together to form a hydrophobic gate. Mutations of these residues to polar amino acids produce channels with leaky gates that conduct ions in the resting state. STIM1-mediated channel activation occurs through rotation of the pore helix, which displaces the F99 residues away from the pore axis to increase pore hydration, allowing ions to flow through the V102-F99 hydrophobic band. Pore helix rotation by STIM1 also explains the dynamic coupling between CRAC channel gating and ion selectivity. This hydrophobic gating mechanism has implications for CRAC channel function, pharmacology and disease-causing mutations. PMID:28220789

  3. 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.

  4. STIM1 activates CRAC channels through rotation of the pore helix to open a hydrophobic gate

    NASA Astrophysics Data System (ADS)

    Yamashita, Megumi; Yeung, Priscilla S.-W.; Ing, Christopher E.; McNally, Beth A.; Pomès, Régis; Prakriya, Murali

    2017-02-01

    Store-operated Ca2+ release-activated Ca2+ (CRAC) channels constitute a major pathway for Ca2+ influx and mediate many essential signalling functions in animal cells, yet how they open remains elusive. Here, we investigate the gating mechanism of the human CRAC channel Orai1 by its activator, stromal interacting molecule 1 (STIM1). We find that two rings of pore-lining residues, V102 and F99, work together to form a hydrophobic gate. Mutations of these residues to polar amino acids produce channels with leaky gates that conduct ions in the resting state. STIM1-mediated channel activation occurs through rotation of the pore helix, which displaces the F99 residues away from the pore axis to increase pore hydration, allowing ions to flow through the V102-F99 hydrophobic band. Pore helix rotation by STIM1 also explains the dynamic coupling between CRAC channel gating and ion selectivity. This hydrophobic gating mechanism has implications for CRAC channel function, pharmacology and disease-causing mutations.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Denning, Elizabeth J.; Woolf, Thomas B.

    2009-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2013-03-01

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

  9. 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

  10. The pharmacology of voltage-gated sodium channels in sensory neurones.

    PubMed

    Docherty, Reginald J; Farmer, Clare E

    2009-01-01

    Voltage-gated sodium channels (VGSCs) are vital for the normal functioning of most excitable cells. At least nine distinct functional subtypes of VGSCs are recognized, corresponding to nine genes for their pore-forming alpha-subunits. These have different developmental expression patterns, different tissue distributions in the adult and are differentially regulated at the cellular level by receptor-coupled cell signalling systems. Unsurprisingly, VGSC blockers are found to be useful as drugs in diverse clinical applications where excessive excitability of tissue leads to pathological dysfunction, e.g. epilepsy or cardiac tachyarrhythmias. The effects of most clinically useful VGSC blockers are use-dependent, i.e. their efficacy depends on channel activity. In addition, many natural toxins have been discovered that interact with VGSCs in complex ways and they have been used as experimental probes to study the structure and function of the channels and to better understand how drugs interact with the channels. Here we have attempted to summarize the properties of VGSCs in sensory neurones, discuss how they are regulated by cell signalling systems and we have considered briefly current concepts of their physiological function. We discuss in detail how drugs and toxins interact with archetypal VGSCs and where possible consider how they act on VGSCs in peripheral sensory neurones. Increasingly, drugs that block VGSCs are being used as systemic analgesic agents in chronic pain syndromes, but the full potential for VGSC blockers in this indication is yet to be realized and other applications in sensory dysfunction are also possible. Drugs targeting VGSC subtypes in sensory neurones are likely to provide novel systemic analgesics that are tissue-specific and perhaps even disease-specific, providing much-needed novel therapeutic approaches for the relief of chronic pain.

  11. 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.

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

    PubMed

    Tan, Nhi; Lansman, Jeffry B

    2014-08-01

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

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

    PubMed Central

    Tan, Nhi; Lansman, Jeffry B

    2014-01-01

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

  14. Agonists block currents through acetylcholine receptor channels.

    PubMed Central

    Sine, S M; Steinbach, J H

    1984-01-01

    We have examined the effects of high concentrations of cholinergic agonists on currents through single acetylcholine receptor (AChR) channels on clonal BC3H1 cells. We find that raised concentrations of acetylcholine (ACh; above 300 microM) or carbamylcholine (Carb; above 1,000 microM) produce a voltage- and concentration-dependent reduction in the mean single-channel current. Raised concentrations of suberyldicholine (Sub; above 3 microM) produce a voltage- and concentration-dependent increase in the number of brief duration low-conductance interruptions of open-channel currents. These observations can be quantitatively described by a model in which agonist molecules enter and transiently occlude the ion-channel of the AChR. PMID:6478036

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

    PubMed Central

    1996-01-01

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

  16. 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

  17. 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

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

    PubMed

    Nayak, Tapan K; Sikdar, S K

    2007-10-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1986-03-01

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

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

    PubMed

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

    2014-09-02

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

  1. Pore architecture and ion sites in acid-sensing ion channels and P2X receptors.

    PubMed

    Gonzales, Eric B; Kawate, Toshimitsu; Gouaux, Eric

    2009-07-30

    Acid-sensing ion channels are proton-activated, sodium-selective channels composed of three subunits, and are members of the superfamily of epithelial sodium channels, mechanosensitive and FMRF-amide peptide-gated ion channels. These ubiquitous eukaryotic ion channels have essential roles in biological activities as diverse as sodium homeostasis, taste and pain. Despite their crucial roles in biology and their unusual trimeric subunit stoichiometry, there is little knowledge of the structural and chemical principles underlying their ion channel architecture and ion-binding sites. Here we present the structure of a functional acid-sensing ion channel in a desensitized state at 3 A resolution, the location and composition of the approximately 8 A 'thick' desensitization gate, and the trigonal antiprism coordination of caesium ions bound in the extracellular vestibule. Comparison of the acid-sensing ion channel structure with the ATP-gated P2X(4) receptor reveals similarity in pore architecture and aqueous vestibules, suggesting that there are unanticipated yet common structural and mechanistic principles.

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

    PubMed Central

    2016-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  4. Coordinated modulation of cellular signaling through ligand-gated ion channels in Hydra vulgaris (Cnidaria, Hydrozoa).

    PubMed

    Pierobon, Paola

    2012-01-01

    Cnidarians lack well developed organs, but they have evolved the molecular and cellular components needed to assemble a nervous system. The apparent 'simplicity' of the cnidarian nervous net does not occur at the cellular level, but rather in the organisation of conducting systems. Cnidarian neurons are in fact electrically excitable, show the typical extended morphology and are connected by chemical synapses or gap junctions. They have been regarded as peptidergic, given the wealth of neuropeptides generally distributed along neurites and in cell bodies, supporting the hypothesis of a modulatory role in neurotransmission. However, the presence of clear-cored, as well as dense-cored synaptic vesicles in cnidarian neurons suggests both fast and slow synaptic transmission mechanisms. In fact, biochemical and functional evidence indicates that classical neurotransmitters and their metabolic partners are present in cnidarian tissues, where they are involved in coordinating motility and behavior. We have identified and characterized in Hydra tissues receptors to the inhibitory and excitatory amino acid neurotransmitters, GABA, glycine and NMDA, that are similar to mammalian ionotropic receptors in terms of their biochemical and pharmacological properties. These receptors appear to regulate pacemaker activities and their physiological correlates; in the live animal, they also affect feeding behavior, namely the duration and termination of the response elicited by reduced glutathione, with opposite actions of GABA and glycine or NMDA, respectively. These results suggest that modulation of cellular signaling through ligand-gated-ion channels is an ancient characteristic in the animal kingdom, and that the pharmacological properties of these receptors have been highly conserved during evolution.

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

    NASA Astrophysics Data System (ADS)

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

    2010-11-01

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

  6. Regulation of voltage-gated potassium channels by PI(4,5)P2.

    PubMed

    Kruse, Martin; Hammond, Gerald R V; Hille, Bertil

    2012-08-01

    Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) regulates activities of numerous ion channels including inwardly rectifying potassium (K(ir)) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (K(V)) channels might be regulated by PI(4,5)P(2). Wide expression of K(V) channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of K(V) channels by PI(4,5)P(2), we have coexpressed several of them in tsA-201 cells with a G protein-coupled receptor (M(1)R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P(2) with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P(2) at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited K(V)7.1, K(V)7.2/7.3, and K(ir)2.1 channel current by 90-95%. Activation of M(1)R inhibited K(V)7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P(2) regulation of activity of K(V)1.1/K(V)β1.1, K(V)1.3, K(V)1.4, and K(V)1.5/K(V)β1.3, K(V)2.1, K(V)3.4, K(V)4.2, K(V)4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for K(V)1.1/K(V)β1.1 and K(V)3.4, resulting in up-regulation of current density upon activation of M(1)R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P(2) at the plasma membrane by enzymes does not seem to influence activity of most tested K(V) channels, whereas it does strongly inhibit members of the K(V)7 and K(ir) families.

  7. Lipid agonism: The PIP2 paradigm of ligand-gated ion channels.

    PubMed

    Hansen, Scott B

    2015-05-01

    The past decade, membrane signaling lipids emerged as major regulators of ion channel function. However, the molecular nature of lipid binding to ion channels remained poorly described due to a lack of structural information and assays to quantify and measure lipid binding in a membrane. How does a lipid-ligand bind to a membrane protein in the plasma membrane, and what does it mean for a lipid to activate or regulate an ion channel? How does lipid binding compare to activation by soluble neurotransmitter? And how does the cell control lipid agonism? This review focuses on lipids and their interactions with membrane proteins, in particular, ion channels. I discuss the intersection of membrane lipid biology and ion channel biophysics. A picture emerges of membrane lipids as bona fide agonists of ligand-gated ion channels. These freely diffusing signals reside in the plasma membrane, bind to the transmembrane domain of protein, and cause a conformational change that allosterically gates an ion channel. The system employs a catalog of diverse signaling lipids ultimately controlled by lipid enzymes and raft localization. I draw upon pharmacology, recent protein structure, and electrophysiological data to understand lipid regulation and define inward rectifying potassium channels (Kir) as a new class of PIP2 lipid-gated ion channels.

  8. 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.

  9. 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

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

    PubMed Central

    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

    2016-01-01

    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 Ca2+ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca2+ channels in pollen tube tips are core components that regulate Ca2+ gradients by mediating and regulating external Ca2+ influx. Therefore, Ca2+ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ channel for pollen tube guidance in Arabidopsis. PMID:26929345

  11. 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.

  12. 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.

  13. The α2δ subunits of voltage-gated calcium channels.

    PubMed

    Dolphin, Annette C

    2013-07-01

    Voltage-gated calcium channels consist of the main pore-forming α1 subunit, together, except in the case of the T-type channels, with β and α2δ and sometimes γ subunits, which are collectively termed auxiliary or accessory subunits. This review will concentrate on the properties and role of the α2δ subunits of these channels. These proteins are largely extracellular, membrane-associated proteins which influence the trafficking, localization, and biophysical properties of the channels. This article is part of a Special Issue entitled: Calcium channels.

  14. 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

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

    PubMed Central

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

    2015-01-01

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

  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.

  17. Domain and interdomain energetics underlying gating in Shaker-type Kv channels.

    PubMed

    Peyser, Alexander; Gillespie, Dirk; Roth, Roland; Nonner, Wolfgang

    2014-10-21

    To understand gating events with a time-base many orders-of-magnitude slower than that of atomic motion in voltage-gated ion channels such as the Shaker-type KV channels, a multiscale physical model is constructed from the experimentally well-characterized voltage-sensor (VS) domains coupled to a hydrophobic gate. The four VS domains are described by a continuum electrostatic model under voltage-clamp conditions, the control of ion flow by the gate domain is described by a vapor-lock mechanism, and the simple coupling principle is informed by known experimental results and trial-and-error. The configurational energy computed for each element is used to produce a total Hamiltonian that is a function of applied voltage, VS positions, and gate radius. We compute statistical-mechanical expectation values of macroscopic laboratory observables. This approach stands in contrast with molecular-dynamic models which are challenged by increasing scale, and kinetic models which assume a probability distribution rather than derive it from the underlying physics. This generic model predicts well the Shaker charge/voltage and conductance/voltage relations; the tight constraints underlying these results allow us to quantitatively assess the underlying physical mechanisms. The total electrical work picked up by the VS domains is an order-of-magnitude larger than the work required to actuate the gate itself, suggesting an energetic basis for the evolutionary flexibility of the voltage-gating mechanism. The cooperative slide-and-interlock behavior of the VS domains described by the VS-gate coupling relation leads to the experimentally observed bistable gating. This engineering approach should prove useful in the investigation of various elements underlying gating characteristics and degraded behavior due to mutation.

  18. Charybdotoxin blocks voltage-gated K+ channels in human and murine T lymphocytes

    PubMed Central

    1989-01-01

    A variety of scorpion venoms and purified toxins were tested for effects on ion channels in human T lymphocytes, a human T leukemia cell line (Jurkat), and murine thymocytes, using the whole-cell patch-clamp method. Nanomolar concentrations of charbdotoxin (CTX), a purified peptide component of Leiurus quinquestriatus venom known to block Ca2+- activated K+ channels from muscle, blocked "type n" voltage-gated K+ channels in human T lymphoid cells. The Na+ channels occasionally expressed in these cells were unaffected by the toxin. From the time course of development and removal of K+ channel block we determined the rates of CTX binding and unbinding. CTX blocks K+ channels in Jurkat cells with a Kd value between 0.5 and 1.5 nM. Of the three types of voltage-gated K+ channels present in murine thymocytes, types n and n' are blocked by CTX at nanomolar concentrations. The third variety of K+ channels, "type l," is unaffected by CTX. Noxiustoxin (NTX), a purified toxin from Centruroides noxius known to block Ca2+-activated K+ channels, also blocked type n K+ channels with a high degree of potency (Kd = 0.2 nM). In addition, several types of crude scorpion venoms from the genera Androctonus, Buthus, Centruroides, and Pandinus blocked type n channels. We conclude that CTX and NTX are not specific for Ca2+ activated K+ channels and that purified scorpion toxins will provide useful probes of voltage-gated K+ channels in T lymphocytes. The existence of high-affinity sites for scorpion toxin binding may help to classify structurally related K+ channels and provide a useful tool for their biochemical purification. PMID:2475579

  19. Molecular interactions involved in proton-dependent gating in KcsA potassium channels

    PubMed Central

    Posson, David J.; Thompson, Ameer N.; McCoy, Jason G.

    2013-01-01

    The bacterial potassium channel KcsA is gated open by the binding of protons to amino acids on the intracellular side of the channel. We have identified, via channel mutagenesis and x-ray crystallography, two pH-sensing amino acids and a set of nearby residues involved in molecular interactions that influence gating. We found that the minimal mutation of one histidine (H25) and one glutamate (E118) near the cytoplasmic gate completely abolished pH-dependent gating. Mutation of nearby residues either alone or in pairs altered the channel’s response to pH. In addition, mutations of certain pairs of residues dramatically increased the energy barriers between the closed and open states. We proposed a Monod–Wyman–Changeux model for proton binding and pH-dependent gating in KcsA, where H25 is a “strong” sensor displaying a large shift in pKa between closed and open states, and E118 is a “weak” pH sensor. Modifying model parameters that are involved in either the intrinsic gating equilibrium or the pKa values of the pH-sensing residues was sufficient to capture the effects of all mutations. PMID:24218397

  20. Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains

    NASA Astrophysics Data System (ADS)

    Lörinczi, Éva; Gómez-Posada, Juan Camilo; de La Peña, Pilar; Tomczak, Adam P.; Fernández-Trillo, Jorge; Leipscher, Ulrike; Stühmer, Walter; Barros, Francisco; Pardo, Luis A.

    2015-03-01

    Voltage-gated channels open paths for ion permeation upon changes in membrane potential, but how voltage changes are coupled to gating is not entirely understood. Two modules can be recognized in voltage-gated potassium channels, one responsible for voltage sensing (transmembrane segments S1 to S4), the other for permeation (S5 and S6). It is generally assumed that the conversion of a conformational change in the voltage sensor into channel gating occurs through the intracellular S4-S5 linker that provides physical continuity between the two regions. Using the pathophysiologically relevant KCNH family, we show that truncated proteins interrupted at, or lacking the S4-S5 linker produce voltage-gated channels in a heterologous model that recapitulate both the voltage-sensing and permeation properties of the complete protein. These observations indicate that voltage sensing by the S4 segment is transduced to the channel gate in the absence of physical continuity between the modules.

  1. 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

  2. Regulation of ATP-Gated P2X Channels: From Redox Signaling to Interactions with Other Proteins

    PubMed Central

    Leiva-Salcedo, Elías; Rokic, Milos B.; Coddou, Claudio

    2014-01-01

    Abstract Significance: The family of purinergic P2X receptors (P2XRs) is a part of ligand-gated superfamily of channels activated by extracellular adenosine-5′-triphosphate. P2XRs are present in virtually all mammalian tissues as well as in tissues of other vertebrate and nonvertebrate species and mediate a large variety of functions, including fast transmission at central synapses, contraction of smooth muscle cells, platelet aggregation, and macrophage activation to proliferation and cell death. Recent Advances: The recent solving of crystal structure of the zebrafish P2X4.1R is a major advance in the understanding of structural correlates of channel activation and regulation. Combined with growing information obtained in the post-structure era and the reinterpretation of previous work within the context of the tridimensional structure, these data provide a better understanding of how the channel operates at the molecular levels. Critical Issues: This review focuses on the relationship between redox signaling and P2XR function. We also discuss other allosteric modulation of P2XR gating in the physiological/pathophysiological context. This includes the summary of extracellular actions of trace metals, which can be released to the synaptic cleft, pH decrease that happens during ischemia and inflammation, and calcium, an extracellular and intracellular messenger. Future Directions: Our evolving understanding of activation and regulation of P2XRs is helpful in clarifying the mechanism by which these channels trigger and modulate cellular functions. Further research is required to identify the signaling pathways contributing to the regulation of the receptor activity and to develop novel and receptor-specific allosteric modulators, which could be used in vivo with therapeutic potential. Antioxid. Redox Signal. 21, 953–970. PMID:23944253

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

    PubMed

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

    2015-08-07

    Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization. We describe anion channel rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision.

  4. An update on transcriptional and post-translational regulation of brain voltage-gated sodium channels.

    PubMed

    Onwuli, Donatus O; Beltran-Alvarez, Pedro

    2016-03-01

    Voltage-gated sodium channels are essential proteins in brain physiology, as they generate the sodium currents that initiate neuronal action potentials. Voltage-gated sodium channels expression, localisation and function are regulated by a range of transcriptional and post-translational mechanisms. Here, we review our understanding of regulation of brain voltage-gated sodium channels, in particular SCN1A (NaV1.1), SCN2A (NaV1.2), SCN3A (NaV1.3) and SCN8A (NaV1.6), by transcription factors, by alternative splicing, and by post-translational modifications. Our focus is strongly centred on recent research lines, and newly generated knowledge.

  5. Evidence for a role of GABA- and glutamate-gated chloride channels in olfactory memory.

    PubMed

    Boumghar, Katia; Couret-Fauvel, Thomas; Garcia, Mikael; Armengaud, Catherine

    2012-11-01

    In the honeybee, we investigated the role of transmissions mediated by GABA-gated chloride channels and glutamate-gated chloride channels (GluCls) of the mushroom bodies (MBs) on olfactory learning using a single-trial olfactory conditioning paradigm. The GABAergic antagonist picrotoxin (PTX) or the GluCl antagonist L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) was injected alone or in combination into the α-lobes of MBs. PTX impaired early long-term olfactory memory when injected before conditioning or before testing. L-trans-PDC alone induced no significant effect on learning and memory but induced a less specific response to the conditioned odor. When injected before PTX, L-trans-PDC was able to modulate PTX effects. These results emphasize the role of MB GABA-gated chloride channels in consolidation processes and strongly support that GluCls are involved in the perception of the conditioned stimulus.

  6. Ciguatoxins: Cyclic Polyether Modulators of Voltage-gated Iion Channel Function

    PubMed Central

    Nicholson, Graham M.; Lewis, Richard J.

    2006-01-01

    Ciguatoxins are cyclic polyether toxins, derived from marine dinoflagellates, which are responsible for the symptoms of ciguatera poisoning. Ingestion of tropical and subtropical fin fish contaminated by ciguatoxins results in an illness characterised by neurological, cardiovascular and gastrointestinal disorders. The pharmacology of ciguatoxins is characterised by their ability to cause persistent activation of voltage-gated sodium channels, to increase neuronal excitability and neurotransmitter release, to impair synaptic vesicle recycling, and to cause cell swelling. It is these effects, in combination with an action to block voltage-gated potassium channels at high doses, which are believed to underlie the complex of symptoms associated with ciguatera. This review examines the sources, structures and pharmacology of ciguatoxins. In particular, attention is placed on their cellular modes of actions to modulate voltage-gated ion channels and other Na+-dependent mechanisms in numerous cell types and to current approaches for detection and treatment of ciguatera.

  7. 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

  8. Optical control of trimeric P2X receptors and acid-sensing ion channels.

    PubMed

    Browne, Liam E; Nunes, João P M; Sim, Joan A; Chudasama, Vijay; Bragg, Laricia; Caddick, Stephen; North, R Alan

    2014-01-07

    P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

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

    PubMed Central

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

    2014-01-01

    GORK is the only outward-rectifying Kv-like K+ channel expressed in guard cells. Its activity is tightly regulated to facilitate K+ efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward-rectifying K+ channel KAT1. Whereas the population of KAT1 is subject to regulated traffic to and from the plasma membrane, nothing is known about GORK, its distribution and traffic in vivo. We have used transformations with fluorescently-tagged GORK to explore its characteristics in tobacco epidermis and Arabidopsis guard cells. These studies showed that GORK assembles in puncta that reversibly dissociated as a function of the external K+ concentration. Puncta dissociation parallelled the gating dependence of GORK, the speed of response consistent with the rapidity of channel gating response to changes in the external ionic conditions. Dissociation was also suppressed by the K+ channel blocker Ba2+. By contrast, confocal and protein biochemical analysis failed to uncover substantial exo- and endocytotic traffic of the channel. Gating of GORK is displaced to more positive voltages with external K+, a characteristic that ensures the channel facilitates only K+ efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K+ above 1 mm. We suggest that GORK clustering in puncta is related to its gating and conductance, and reflects associated conformational changes and (de)stabilisation of the channel protein, possibly as a platform for transmission and coordination of channel gating in response to external K+. PMID:24517091

  10. Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating.

    PubMed

    Perozo, Eduardo; Kloda, Anna; Cortes, D Marien; Martinac, Boris

    2002-09-01

    In mechanosensitive (MS) channels, gating is initiated by changes in intra-bilayer pressure profiles originating from bilayer deformation. Here we evaluated two physical mechanisms as triggers of MS channel gating: the energetic cost of protein-bilayer hydrophobic mismatches and the geometric consequences of bilayer intrinsic curvature. Structural changes in the Escherichia coli large MS channel (MscL) were studied under nominally zero transbilayer pressures using both patch clamp and EPR spectroscopic approaches. Changes in membrane intrinsic curvature induced by the external addition of lysophosphatidylcholine (LPC) generated massive spectroscopic changes in the narrow constriction that forms the channel 'gate', trapping the channel in the fully open state. Hydrophobic mismatch alone was unable to open the channel, but decreasing bilayer thickness lowered MscL activation energy, stabilizing a structurally distinct closed channel intermediate. We propose that the mechanism of mechanotransduction in MS channels is defined by both local and global asymmetries in the transbilayer pressure profile at the lipid-protein interface.

  11. Energetic role of the paddle motif in voltage gating of Shaker K(+) channels.

    PubMed

    Xu, Yanping; Ramu, Yajamana; Shin, Hyeon-Gyu; Yamakaze, Jayden; Lu, Zhe

    2013-05-01

    Voltage-gated ion channels underlie rapid electric signaling in excitable cells. Electrophysiological studies have established that the N-terminal half of the fourth transmembrane segment ((NT)S4) of these channels is the primary voltage sensor, whereas crystallographic studies have shown that (NT)S4 is not located within a proteinaceous pore. Rather, (NT)S4 and the C-terminal half of S3 ((CT)S3 or S3b) form a helix-turn-helix motif, termed the voltage-sensor paddle. This unexpected structural finding raises two fundamental questions: does the paddle motif also exist in voltage-gated channels in a biological membrane, and, if so, what is its function in voltage gating? Here, we provide evidence that the paddle motif exists in the open state of Drosophila Shaker voltage-gated K(+) channels expressed in Xenopus oocytes and that (CT)S3 acts as an extracellular hydrophobic 'stabilizer' for (NT)S4, thus biasing the gating chemical equilibrium toward the open state.

  12. Transient Receptor Potential Channels in neuropathic pain.

    PubMed

    Basso, Lilian; Altier, Christophe

    2016-10-27

    Neuropathic pain caused by disease or dysfunction of the nervous system is one of the most difficult pain conditions to treat. Symptoms include a hypersensitivity to mechanical and thermal stimuli, processed by specialized nociceptors that constitute the first line of defence of the somatosensory system. The detection of these stimuli depends on the TRP ion channel family, which activates upon damaging pressure, extreme temperature, or toxic endogenous and exogenous chemicals. This review will summarize the current knowledge of the contribution of TRP channels, particularly the thermosensitive TRP, including TRPV1, TRPA1 and TRPM8 channels that play a central role in the sensitization of nociceptive transduction. We will discuss the pharmacology of these receptors and their relative success in preclinical and clinical studies.

  13. Single-Particle Cryo-EM of the Ryanodine Receptor Channel in an Aqueous Environment

    PubMed Central

    Baker, Mariah R.; Fan, Guizhen

    2015-01-01

    Ryanodine receptors (RyRs) are tetrameric ligand-gated Ca2+ release channels that are responsible for the increase of cytosolic Ca2+ concentration leading to muscle contraction. Our current understanding of RyR channel gating and regulation is greatly limited due to the lack of a high-resolution structure of the channel protein. The enormous size and unwieldy shape of Ca2+ release channels make X-ray or NMR methods difficult to apply for high-resolution structural analysis of the full-length functional channel. Single-particle electron cryo-microscopy (cryo-EM) is one of the only effective techniques for the study of such a large integral membrane protein and its molecular interactions. Despite recent developments in cryo-EM technologies and break-through single-particle cryo-EM studies of ion channels, cryospecimen preparation, particularly the presence of detergent in the buffer, remains the main impediment to obtaining atomic-resolution structures of ion channels and a multitude of other integral membrane protein complexes. In this review we will discuss properties of several detergents that have been successfully utilized in cryo-EM studies of ion channels and the emergence of the detergent alternative amphipol to stabilize ion channels for structure-function characterization. Future structural studies of challenging specimen like ion channels are likely to be facilitated by cryo-EM amenable detergents or alternative surfactants. PMID:26913144

  14. Single-particle cryo-EM of the ryanodine receptor channel in an aqueous environment

    PubMed Central

    Baker, Mariah R.; Fan, Guizhen; Serysheva, Irina I.

    2015-01-01

    Ryanodine receptors (RyRs) are tetrameric ligand-gated Ca2+ release channels that are responsible for the increase of cytosolic Ca2+ concentration leading to muscle contraction. Our current understanding of RyR channel gating and regulation is greatly limited due to the lack of a high-resolution structure of the channel protein. The enormous size and unwieldy shape of Ca2+ release channels make X-ray or NMR methods difficult to apply for high-resolution structural analysis of the full-length functional channel. Single-particle electron cryo-microscopy (cryo-EM) is one of the only effective techniques for the study of such a large integral membrane protein and its molecular interactions. Despite recent developments in cryo-EM technologies and break-through single-particle cryo-EM studies of ion channels, cryospecimen preparation, particularly the presence of detergent in the buffer, remains the main impediment to obtaining atomic-resolution structures of ion channels and a multitude of other integral membrane protein complexes. In this review we will discuss properties of several detergents that have been successfully utilized in cryo-EM studies of ion channels and the emergence of the detergent alternative amphipol to stabilize ion channels for structure-function characterization. Future structural studies of challenging specimen like ion channels are likely to be facilitated by cryo-EM amenable detergents or alternative surfactants. PMID:25844145

  15. 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.

  16. Linker-gating ring complex as passive spring and Ca(2+)-dependent machine for a voltage- and Ca(2+)-activated potassium channel.

    PubMed

    Niu, Xiaowei; Qian, Xiang; Magleby, Karl L

    2004-06-10

    Ion channels are proteins that control the flux of ions across cell membranes by opening and closing (gating) their pores. It has been proposed that channels gated by internal agonists have an intracellular gating ring that extracts free energy from agonist binding to open the gates using linkers that directly connect the gating ring to the gates. Here we find for a voltage- and Ca(2+)-activated K+ (BK) channel that shortening the linkers increases channel activity and lengthening the linkers decreases channel activity, both in the presence and absence of intracellular Ca2+. These observations are consistent with a mechanical model in which the linker-gating ring complex forms a passive spring that applies force to the gates in the absence of Ca2+ to modulate the voltage-dependent gating. Adding Ca2+ then changes the force to further activate the channel. Both the passive and Ca(2+)-induced forces contribute to the gating of the channel.

  17. Arresting a Transient Receptor Potential (TRP) Channel

    PubMed Central

    Shukla, Arun K.; Kim, Jihee; Ahn, Seungkirl; Xiao, Kunhong; Shenoy, Sudha K.; Liedtke, Wolfgang; Lefkowitz, Robert J.

    2010-01-01

    β-Arrestins, originally discovered to desensitize activated G protein-coupled receptors, (aka seven-transmembrane receptors, 7TMRs) also mediate 7TMR internalization and G protein-independent signaling via these receptors. More recently, several regulatory roles of β-arrestins for atypical 7TMRs and non-7TM receptors have emerged. Here, we uncover an entirely novel regulatory role of β-arrestins in cross-talk between the angiotensin receptor (AT1aR) and a member of the transient receptor potential (TRP) ion channel family, TRPV4. AT1aR and TRPV4 form a constitutive complex in the plasma membrane, and angiotensin stimulation leads to recruitment of β-arrestin 1 to this complex. Surprisingly, angiotensin stimulation results in ubiquitination of TRPV4, a process that requires β-arrestin 1, and subsequently to internalization and functional down-regulation of TRPV4. β-Arrestin 1 interacts with, and acts as an adaptor for AIP4, an E3 ubiquitin ligase responsible for TRPV4 ubiquitination. Thus, our data provide the first evidence of a functional link between β-arrestins and TRPV4 and uncovers an entirely novel mechanism to maintain appropriate intracellular Ca2+ concentration to avoid excessive Ca2+ signaling. PMID:20650893

  18. Trapping of ivermectin by a pentameric ligand-gated ion channel upon open-to-closed isomerization

    PubMed Central

    Degani-Katzav, Nurit; Klein, Moshe; Har-Even, Moran; Gortler, Revital; Tobi, Ruthi; Paas, Yoav

    2017-01-01

    Ivermectin (IVM) is a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. By activating invertebrate pentameric glutamate-gated chloride channels (GluCl receptors; GluClRs), IVM induces sustained chloride influx and long-lasting membrane hyperpolarization that inhibit neural excitation in nematodes. Although IVM activates the C. elegans heteromeric GluClα/β receptor, it cannot activate a homomeric receptor composed of the C. elegans GluClβ subunits. To understand this incapability, we generated a homopentameric α7-GluClβ chimeric receptor that consists of an extracellular ligand-binding domain of an α7 nicotinic acetylcholine receptor known to be potentiated by IVM, and a chloride-selective channel domain assembled from GluClβ subunits. Application of IVM prior to acetylcholine inhibited the responses of the chimeric α7-GluClβR. Adding IVM to activated α7-GluClβRs, considerably accelerated the decline of ACh-elicited currents and stabilized the receptors in a non-conducting state. Determination of IVM association and dissociation rate constants and recovery experiments suggest that, following initial IVM binding to open α7-GluClβRs, the drug induces a conformational change and locks the ion channel in a closed state for a long duration. We further found that IVM also inhibits the activation by glutamate of a homomeric receptor assembled from the C. elegans full-length GluClβ subunits. PMID:28218274

  19. Trapping of ivermectin by a pentameric ligand-gated ion channel upon open-to-closed isomerization.

    PubMed

    Degani-Katzav, Nurit; Klein, Moshe; Har-Even, Moran; Gortler, Revital; Tobi, Ruthi; Paas, Yoav

    2017-02-20

    Ivermectin (IVM) is a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. By activating invertebrate pentameric glutamate-gated chloride channels (GluCl receptors; GluClRs), IVM induces sustained chloride influx and long-lasting membrane hyperpolarization that inhibit neural excitation in nematodes. Although IVM activates the C. elegans heteromeric GluClα/β receptor, it cannot activate a homomeric receptor composed of the C. elegans GluClβ subunits. To understand this incapability, we generated a homopentameric α7-GluClβ chimeric receptor that consists of an extracellular ligand-binding domain of an α7 nicotinic acetylcholine receptor known to be potentiated by IVM, and a chloride-selective channel domain assembled from GluClβ subunits. Application of IVM prior to acetylcholine inhibited the responses of the chimeric α7-GluClβR. Adding IVM to activated α7-GluClβRs, considerably accelerated the decline of ACh-elicited currents and stabilized the receptors in a non-conducting state. Determination of IVM association and dissociation rate constants and recovery experiments suggest that, following initial IVM binding to open α7-GluClβRs, the drug induces a conformational change and locks the ion channel in a closed state for a long duration. We further found that IVM also inhibits the activation by glutamate of a homomeric receptor assembled from the C. elegans full-length GluClβ subunits.

  20. 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

  1. Gate-controlled topological conducting channels in bilayer graphene

    NASA Astrophysics Data System (ADS)

    Li, Jing; Wang, Ke; McFaul, Kenton J.; Zern, Zachary; Ren, Yafei; Watanabe, Kenji; Taniguchi, Takashi; Qiao, Zhenhua; Zhu, Jun

    2016-12-01

    The existence of inequivalent valleys K and K‧ in the momentum space of 2D hexagonal lattices provides a new electronic degree of freedom, the manipulation of which can potentially lead to new types of electronics, analogous to the role played by electron spin. In materials with broken inversion symmetry, such as an electrically gated bilayer graphene (BLG), the momentum-space Berry curvature Ω carries opposite sign in the K and K‧ valleys. A sign reversal of Ω along an internal boundary of the sheet gives rise to counterpropagating 1D conducting modes encoded with opposite-valley indices. These metallic states are topologically protected against backscattering in the absence of valley-mixing scattering, and thus can carry current ballistically. In BLG, the reversal of Ω can occur at the domain wall of AB- and BA-stacked domains, or at the line junction of two oppositely gated regions. The latter approach can provide a scalable platform to implement valleytronic operations, such as valves and waveguides, but it is technically challenging to realize. Here, we fabricate a dual-split-gate structure in BLG and present evidence of the predicted metallic states in electrical transport. The metallic states possess a mean free path (MFP) of up to a few hundred nanometres in the absence of a magnetic field. The application of a perpendicular magnetic field suppresses the backscattering significantly and enables a junction 400 nm in length to exhibit conductance close to the ballistic limit of 4e2/h at 8 T. Our experiment paves the way to the realization of gate-controlled ballistic valley transport and the development of valleytronic applications in atomically thin materials.

  2. PIP2 regulation of KCNQ channels: biophysical and molecular mechanisms for lipid modulation of voltage-dependent gating.

    PubMed

    Zaydman, Mark A; Cui, Jianmin

    2014-01-01

    Voltage-gated potassium (Kv) channels contain voltage-sensing (VSD) and pore-gate (PGD) structural domains. During voltage-dependent gating, conformational changes in the two domains are coupled giving rise to voltage-dependent opening of the channel. In addition to membrane voltage, KCNQ (Kv7) channel opening requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Recent studies suggest that PIP2 serves as a cofactor to mediate VSD-PGD coupling in KCNQ1 channels. In this review, we put these findings in the context of the current understanding of voltage-dependent gating, lipid modulation of Kv channel activation, and PIP2-regulation of KCNQ channels. We suggest that lipid-mediated coupling of functional domains is a common mechanism among KCNQ channels that may be applicable to other Kv channels and membrane proteins.

  3. PIP2 regulation of KCNQ channels: biophysical and molecular mechanisms for lipid modulation of voltage-dependent gating

    PubMed Central

    Zaydman, Mark A.; Cui, Jianmin

    2014-01-01

    Voltage-gated potassium (Kv) channels contain voltage-sensing (VSD) and pore-gate (PGD) structural domains. During voltage-dependent gating, conformational changes in the two domains are coupled giving rise to voltage-dependent opening of the channel. In addition to membrane voltage, KCNQ (Kv7) channel opening requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Recent studies suggest that PIP2 serves as a cofactor to mediate VSD-PGD coupling in KCNQ1 channels. In this review, we put these findings in the context of the current understanding of voltage-dependent gating, lipid modulation of Kv channel activation, and PIP2-regulation of KCNQ channels. We suggest that lipid-mediated coupling of functional domains is a common mechanism among KCNQ channels that may be applicable to other Kv channels and membrane proteins. PMID:24904429

  4. Regulation of AMPA receptor gating and pharmacology by TARP auxiliary subunits.

    PubMed

    Milstein, Aaron D; Nicoll, Roger A

    2008-07-01

    Presynaptic glutamate release elicits brief waves of membrane depolarization in neurons by activating AMPA receptors. Depending on its precise size and shape, current through AMPA receptors gates downstream processes like NMDA receptor activation and action potential generation. Over a decade of research on AMPA receptor structure and function has identified binding sites on AMPA receptors for agonists, antagonists and allosteric modulators as well as key residues underlying differences in the gating behavior of various AMPA receptor subtypes. However, the recent discovery that AMPA receptors are accompanied in the synaptic membrane by a family of auxiliary subunits known as transmembrane AMPA receptor regulatory proteins (TARPs) has revealed that the kinetics and pharmacology of neuronal AMPA receptors differ in many respects from those predicted by classical studies of AMPA receptors in heterologous systems. Here, we summarize recent work and discuss remaining questions concerning the structure and function of native TARP-AMPA receptor complexes.

  5. Coupled gating of skeletal muscle ryanodine receptors is modulated by Ca2+, Mg2+, and ATP

    PubMed Central

    Porta, Maura; Diaz-Sylvester, Paula L.; Neumann, Jake T.; Escobar, Ariel L.; Fleischer, Sidney

    2012-01-01

    Coupled gating (synchronous openings and closures) of groups of skeletal muscle ryanodine receptors (RyR1), which mimics RyR1-mediated Ca2+ release underlying Ca2+ sparks, was first described by Marx et al. (Marx SO, Ondrias K, Marks AR. Science 281: 818–821, 1998). The nature of the RyR1-RyR1 interactions for coupled gating still needs to be characterized. Consequently, we defined planar lipid bilayer conditions where ∼25% of multichannel reconstitutions contain mixtures of coupled and independently gating RyR1. In ∼10% of the cases, all RyRs (2–10 channels; most frequently 3–4) gated in coupled fashion, allowing for quantification. Our results indicated that coupling required cytosolic solutions containing ATP/Mg2+ and high (50 mM) luminal Ca2+ (Calum) or Sr2+ solutions. Bursts of coupled activity (events) started and ended abruptly, with all channels activating/deactivating within ∼300 μs. Coupled RyR1 were heterogeneous, where highly active RyR1 (“drivers”) seemed open during the entire coupled event (Po = 1), while other RyR1s (“followers”) displayed abundant flickering and smaller amplitude. Drivers mean open time increased with cytosolic Ca2+ (Cacyt) or caffeine, whereas followers flicker frequency was Cacyt independent and more sensitive to inhibition by cytosolic Mg2+. Coupled events were insensitive to varying lumen-to-cytosol Ca2+ fluxes from ∼1 to 8 pA, which does not corroborate coupling of neighboring RyR1 by local Ca2+-induced Ca2+ release. However, coupling requires specific Calum sites, as it was lost when Calum was replaced by luminal Ba2+ or Mg2+. In summary, coupled events reveal complex interactions among heterogeneous RyR1, differentially modulated by cytosolic ATP/Mg2+, Cacyt, and Calum, which under cell-like ionic conditions may parallel synchronous RyR1 gating during Ca2+ sparks. PMID:22785120

  6. Surface dynamics of voltage-gated ion channels

    PubMed Central

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

    2016-01-01

    ABSTRACT Neurons encode information in fast changes of the membrane potential, and thus electrical membrane properties are critically important for the integration and processing of synaptic inputs by a neuron. These electrical properties are largely determined by ion channels embedded in the membrane. The distribution of most ion channels in the membrane is not spatially uniform: they undergo activity-driven changes in the range of minutes to days. Even in the range of milliseconds, the composition and topology of ion channels are not static but engage in highly dynamic processes including stochastic or activity-dependent transient association of the pore-forming and auxiliary subunits, lateral diffusion, as well as clustering of different channels. In this review we briefly discuss the potential impact of mobile sodium, calcium and potassium ion channels and the functional significance of this for individual neurons and neuronal networks. PMID:26891382

  7. Calmodulin and Ca(2+) control of voltage gated Na(+) channels.

    PubMed

    Gabelli, Sandra B; Yoder, Jesse B; Tomaselli, Gordon F; Amzel, L Mario

    2016-01-01

    The structures of the cytosolic portion of voltage activated sodium channels (CTNav) in complexes with calmodulin and other effectors in the presence and the absence of calcium provide information about the mechanisms by which these effectors regulate channel activity. The most studied of these complexes, those of Nav1.2 and Nav1.5, show details of the conformations and the specific contacts that are involved in channel regulation. Another voltage activated sodium channel, Nav1.4, shows significant calcium dependent inactivation, while its homolog Nav1.5 does not. The available structures shed light on the possible localization of the elements responsible for this effect. Mutations in the genes of these 3 Nav channels are associated with several disease conditions: Nav1.2, neurological conditions; Nav1.4, syndromes involving skeletal muscle; and Nav1.5, cardiac arrhythmias. Many of these disease-specific mutations are located at the interfaces involving CTNav and its effectors.

  8. Physical basis of apparent pore-dilation of ATP-activated P2X receptor channels

    PubMed Central

    Li, Mufeng; Toombes, Gilman E S; Silberberg, Shai D; Swartz, Kenton J

    2016-01-01

    The selectivity of ion channels is fundamental for their roles in electrical and chemical signaling, and ion homeostasis. Although most ion channels exhibit stable ion selectivity, the prevailing view for purinergic P2X receptor channels, transient receptor potential V1 (TRPV1) channels and acid sensing ion channels (ASICs) is that their ion conduction pores dilate upon prolonged activation. We investigated this mechanism in P2X receptors and found that the hallmark shift in equilibrium potential observed with prolonged channel activation does not result from pore dilation, but from time-dependent alterations in the concentration of intracellular ions. We derived a physical model to calculate ion concentration changes during patch-clamp recordings, which validates our experimental findings and provides a quantitative guideline for effectively controlling ion concentration. Our results have fundamental implications for understanding ion permeation and gating in P2X receptor channels, and more broadly for using patch-clamp techniques to study ion channels and neuronal excitability. PMID:26389841

  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.

  10. Voltage-gated sodium channel modulation by scorpion α-toxins

    PubMed Central

    Bosmans, Frank; Tytgat, Jan

    2007-01-01

    Voltage-gated Na+ channels are integral membrane proteins that function as a gateway for a selective permeation of sodium ions across biological membranes. In this way, they are crucial players for the generation of action potentials in excitable cells. Voltage-gated Na+ channels are encoded by at least nine genes in mammals. The different isoforms have remarkably similar functional properties, but small changes in function and pharmacology are biologically well-defined, as underscored by mutations that cause several diseases and by modulation of a myriad of compounds respectively. This review will stress on the modulation of voltage-gated Na+ channels by scorpion alpha-toxins. Nature has designed these two classes of molecules as if they were predestined to each other: an inevitable ‘encounter’ between a voltage-gated Na+ channel isoform and an alpha-toxin from scorpion venom indeed results in a dramatically changed Na+ current phenotype with clear-cut consequences on electrical excitability and sometimes life or death. This fascinating aspect justifies an overview on scorpion venoms, their alpha-toxins and the Na+ channel targets they are built for, as well as on the molecular determinants that govern the selectivity and affinity of this ‘inseparable duo’. PMID:17087986

  11. A new electro-optical approach for conductance measurement: an assay for the study of drugs acting on ligand-gated ion channels

    PubMed Central

    Menegon, A.; Pitassi, S.; Mazzocchi, N.; Redaelli, L.; Rizzetto, R.; Rolland, J. F.; Poli, C.; Imberti, M.; Lanati, A.; Grohovaz, F.

    2017-01-01

    Ligand gated ion channels are involved in many pathophysiological processes and represent a relevant, although challenging, target for drug discovery. We propose an innovative electro-optical approach to their analysis able to derive membrane conductance values from the local membrane potential changes imposed by test current pulses and measured by fast voltage-sensitive fluorescent dyes. We exploited the potential of this proprietary method by developing a drug testing system called “ionChannel Optical High-content Microscope” (ionChannelΩ). This automated platform was validated by testing the responses of reference drugs on cells expressing different ligand-gated ion channels. Furthermore, a double-blind comparison with FLIPR and automated patch-clamp was performed on molecules designed to act as antagonists of the P2RX7 receptor. ionChannelΩ proved highly reliable in all tests, resulting faster and more cost-effective than electrophysiological techniques. Overall, ionChannelΩ is amenable to the study of ligand gated ion channels that are receiving less attention due to limitations in current assays. PMID:28322303

  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. How and why are calcium currents curtailed in the skeletal muscle voltage‐gated calcium channels?

    PubMed Central

    Tuluc, Petronel

    2017-01-01

    Abstract Voltage‐gated calcium channels represent the sole mechanism converting electrical signals of excitable cells into cellular functions such as contraction, secretion and gene regulation. Specific voltage‐sensing domains detect changes in membrane potential and control channel gating. Calcium ions entering through the channel function as second messengers regulating cell functions, with the exception of skeletal muscle, where CaV1.1 essentially does not function as a channel but activates calcium release from intracellular stores. It has long been known that calcium currents are dispensable for skeletal muscle contraction. However, the questions as to how and why the channel function of CaV1.1 is curtailed remained obscure until the recent discovery of a developmental CaV1.1 splice variant with normal channel functions. This discovery provided new means to study the molecular mechanisms regulating the channel gating and led to the understanding that in skeletal muscle, calcium currents need to be restricted to allow proper regulation of fibre type specification and to prevent mitochondrial damage. PMID:27896815

  14. 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.

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

    PubMed Central

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

    2005-01-01

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

  16. Recessed source concept in nanoscale vertical surrounding gate (VSG) MOSFETs for controlling short-channel effects

    NASA Astrophysics Data System (ADS)

    Subrahmanyam, B.; Jagadesh Kumar, M.

    2009-02-01

    In the recent past, vertical surrounding gate (VSG) MOSFETs have gained importance since defining their nanoscale channel length no longer depends on lithographic limitations and since they can lead to high packing densities. However, as the channel lengths decrease below 100 nm, VSG MOSFETs too suffer from short-channel effects due to the coupling between the drain and source side charges. In this paper, we demonstrate that using a recessed source, the short-channel effects in nanoscale VSG MOSFETs can be effectively controlled.

  17. The cys-loop ligand-gated ion channel gene superfamily of the parasitoid wasp, Nasonia vitripennis.

    PubMed

    Jones, A K; Bera, A N; Lees, K; Sattelle, D B

    2010-03-01

    Members of the cys-loop ligand-gated ion channel (cysLGIC) superfamily mediate chemical neurotransmission and are studied extensively as potential targets of drugs used to treat neurological disorders, such as Alzheimer's disease. Insect cys-loop LGICs also have central roles in the nervous system and are targets of highly successful insecticides. Here, we describe the cysLGIC superfamily of the parasitoid wasp, Nasonia vitripennis, which is emerging as a highly useful model organism and is deployed as a biological control of insect pests. The wasp superfamily consists of 26 genes, which is the largest insect cysLGIC superfamily characterized, whereas Drosophila melanogaster, Apis mellifera and Tribolium castaneum have 23, 21 and 24, respectively. As with Apis, Drosophila and Tribolium, Nasonia possesses ion channels predicted to be gated by acetylcholine, gamma-amino butyric acid, glutamate and histamine, as well as orthologues of the Drosophila pH-sensitive chloride channel (pHCl), CG8916 and CG12344. Similar to other insects, wasp cysLGIC diversity is broadened by alternative splicing and RNA A-to-I editing, which may also serve to generate species-specific receptor isoforms. These findings on N. vitripennis enhance our understanding of cysLGIC functional genomics and provide a useful basis for the study of their function in the wasp model, as well as for the development of improved insecticides that spare a major beneficial insect species.

  18. A reduced mechanical model for cAMP-modulated gating in HCN channels

    PubMed Central

    Weißgraeber, Stephanie; Saponaro, Andrea; Thiel, Gerhard; Hamacher, Kay

    2017-01-01

    We developed an in silico mechanical model to analyze the process of cAMP-induced conformational modulations in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which conduct cations across the membrane of mammalian heart and brain cells. The structural analysis reveals a quaternary twist in the cytosolic parts of the four subunits in the channel tetramer. This motion augments the intrinsic dynamics of the very same protein structure. The pronounced differences between the cAMP bound and unbound form include a mutual interaction between the C-linker of the cyclic nucleotide binding domain (CNBD) and the linker between the S4 and S5 transmembrane domain of the channel. This allows a mechanistic annotation of the twisting motion in relation to the allosteric modulation of voltage-dependent gating of this channel by cAMP. PMID:28074902

  19. A reduced mechanical model for cAMP-modulated gating in HCN channels

    NASA Astrophysics Data System (ADS)

    Weißgraeber, Stephanie; Saponaro, Andrea; Thiel, Gerhard; Hamacher, Kay

    2017-01-01

    We developed an in silico mechanical model to analyze the process of cAMP-induced conformational modulations in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which conduct cations across the membrane of mammalian heart and brain cells. The structural analysis reveals a quaternary twist in the cytosolic parts of the four subunits in the channel tetramer. This motion augments the intrinsic dynamics of the very same protein structure. The pronounced differences between the cAMP bound and unbound form include a mutual interaction between the C-linker of the cyclic nucleotide binding domain (CNBD) and the linker between the S4 and S5 transmembrane domain of the channel. This allows a mechanistic annotation of the twisting motion in relation to the allosteric modulation of voltage-dependent gating of this channel by cAMP.

  20. The STIM1-binding site nexus remotely controls Orai1 channel gating

    PubMed Central

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

    2016-01-01

    The ubiquitously expressed Orai Ca2+ channels are gated through a unique process of intermembrane coupling with the Ca2+-sensing STIM proteins. Despite the significance of Orai1-mediated Ca2+ signals, how gating of Orai1 is triggered by STIM1 remains unknown. A widely held gating model invokes STIM1 binding directly to Orai1 pore-forming helix. Here we report that an Orai1 C-terminal STIM1-binding site, situated far from the N-terminal pore helix, alone provides the trigger that is necessary and sufficient for channel gating. We identify a critical ‘nexus' within Orai1 connecting the peripheral C-terminal STIM1-binding site to the Orai1 core helices. Mutation of the nexus transforms Orai1 into a persistently open state exactly mimicking the action of STIM1. We suggest that the Orai1 nexus transduces the STIM1-binding signal through a conformational change in the inner core helices, and that STIM1 remotely gates the Orai1 channel without the necessity for direct STIM1 contact with the pore-forming helix. PMID:27929067

  1. 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

  2. Ethanol potentiation of GABAergic transmission in cultured spinal cord neurons involves gamma-aminobutyric acidA-gated chloride channels

    SciTech Connect

    Mehta, A.K.; Ticku, M.K.

    1988-08-01

    The interaction of ethanol with gamma-aminobutyric acid (GABA)-mediated 36-Cl-influx and its modulation by various drugs was investigated in C57 mice spinal cord cultured neurons. Ethanol (5-100 mM) potentiated the effect of GABA on /sup 36/Cl-influx; whereas at concentrations greater than or equal to 50 mM ethanol activated Cl- channels directly. The effect of ethanol was specific for GABAA receptor-gated Cl- channels, as ethanol did not potentiate glycine-induced /sup 36/Cl-influx in the same neurons. Both the enhancing and direct effects of ethanol on /sup 36/Cl-influx were blocked by GABA antagonists like bicuculline, picrotoxinin and inverse agonists of the benzodiazepine site like the imidazodiazepine R015-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo (1,5 alpha), (1,4)benzodiazepine-3-carboxylate) and N-methyl-beta-carboline-3-carboxamide (FG-7142). Ethanol potentiating effect of GABA-induced /sup 36/Cl-influx was also reversed by methyl-6,7-dimethyl-4-ethyl-beta-carboline-3-carboxylate. The effects of the inverse agonists were blocked by the benzodiazepine receptor antagonist R015-1788. Both R015-4513 and FG-7142 reversed direct and GABA potentiating effects of ethanol effect at concentrations lower than those that exhibit inverse agonistic activity in the /sup 36/Cl-influx assay in cultured neurons. These results suggest that ethanol facilitation of GABAAergic transmission involves GABA receptor-gated Cl- channels and that this interaction may be responsible for some of the pharmacological effects of ethanol.

  3. 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.

  4. Evolution of voltage-gated ion channels at the emergence of Metazoa.

    PubMed

    Moran, Yehu; Barzilai, Maya Gur; Liebeskind, Benjamin J; Zakon, Harold H

    2015-02-15

    Voltage-gated ion channels are large transmembrane proteins that enable the passage of ions through their pore across the cell membrane. These channels belong to one superfamily and carry pivotal roles such as the propagation of neuronal and muscular action potentials and the promotion of neurotransmitter secretion in synapses. In this review, we describe in detail the current state of knowledge regarding the evolution of these channels with a special emphasis on the metazoan lineage. We highlight the contribution of the genomic revolution to the understanding of ion channel evolution and for revealing that these channels appeared long before the appearance of the first animal. We also explain how the elucidation of channel selectivity properties and function in non-bilaterian animals such as cnidarians (sea anemones, corals, jellyfish and hydroids) can contribute to the study of channel evolution. Finally, we point to open questions and future directions in this field of research.

  5. Anti-voltage-gated potassium channel Kv1.4 antibodies in myasthenia gravis.

    PubMed

    Romi, Fredrik; Suzuki, Shigeaki; Suzuki, Norihiro; Petzold, Axel; Plant, Gordon T; Gilhus, Nils Erik

    2012-07-01

    Myasthenia gravis (MG) is an autoimmune disease characterized by skeletal muscle weakness mainly caused by acetylcholine receptor antibodies. MG can be divided into generalized and ocular, and into early-onset (<50 years of age) and late-onset (≥50 years of age). Anti-Kv1.4 antibodies targeting α-subunits (Kv1.4) of the voltage-gated potassium K(+) channel occurs frequently among patients with severe MG, accounting for 18% of a Japanese MG population. The aim of this study was to characterize the clinical features and serological associations of anti-Kv1.4 antibodies in a Caucasian MG population with mild and localized MG. Serum samples from 129 Caucasian MG patients with mainly ocular symptoms were tested for the presence of anti-Kv1.4 antibodies and compared to clinical and serological parameters. There were 22 (17%) anti-Kv1.4 antibody-positive patients, most of them women with late-onset MG, and all of them with mild MG. This contrasts to the Japanese anti-Kv1.4 antibody-positive patients who suffered from severe MG with bulbar symptoms, myasthenic crisis, thymoma, myocarditis and prolonged QT time on electrocardiography, despite equal anti-Kv1.4 antibody occurrence in both populations. No other clinical or serological parameters influenced anti-Kv1.4 antibody occurrence.

  6. Cyclic nucleotide gated channels and related signaling components in plant innate immunity.

    PubMed

    Ma, Wei; Smigel, Andries; Verma, Rajeev; Berkowitz, Gerald A

    2009-04-01

    Although plants lack the mobile sentry cells present in animal innate immune systems, plants have developed complex innate immune reactions triggering basal resistance and the hypersensitive response (HR). Cytosolic Ca(2+) elevation is considered to be an important early event in this pathogen response signal transduction cascade. Plasma membrane (PM)-localized cyclic nucleotide gated channels (CNGCs) contribute to the cytosolic Ca(2+) rise upon pathogen perception. Recent work suggests that some PM-localized leucine-rich-repeat receptor-like kinases (LRR-RLKs) may be involved in the perception of pathogen associated molecular pattern molecules and triggering some pathogen responses in plants, some of these LRR-RLKs might have cyclic nucleotide cyclase activity. The recognition of pathogens may be connected to cyclic nucleotide generation and the activation of CNGCs, followed by cytosolic Ca(2+) increase and downstream signaling events (possibly involving nitric oxide, reactive oxygen species (ROS), calmodulin (CaM), CaM-like protein (CML) and protein kinases). Notably, CaM or CML could be the crucial sensor downstream from the early Ca(2+) signal leading to nitric oxide (NO) production during plant innate immune responses.

  7. 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

  8. 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

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

    PubMed Central

    1991-01-01

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

  10. Proline-induced hinges in transmembrane helices: possible roles in ion channel gating.

    PubMed

    Tieleman, D P; Shrivastava, I H; Ulmschneider, M R; Sansom, M S

    2001-08-01

    A number of ion channels contain transmembrane (TM) alpha-helices that contain proline-induced molecular hinges. These TM helices include the channel-forming peptide alamethicin (Alm), the S6 helix from voltage-gated potassium (Kv) channels, and the D5 helix from voltage-gated chloride (CLC) channels. For both Alm and KvS6, experimental data implicate hinge-bending motions of the helix in an aspect of channel gating. We have compared the hinge-bending motions of these TM helices in bilayer-like environments by multi-nanosecond MD simulations in an attempt to describe motions of these helices that may underlie possible modes of channel gating. Alm is an alpha-helical channel-forming peptide, which contains a central kink associated with a Gly-x-x-Pro motif in its sequence. Simulations of Alm in a TM orientation for 10 ns in an octane slab indicate that the Gly-x-x-Pro motif acts as a molecular hinge. The S6 helix from Shaker Kv channels contains a Pro-Val-Pro motif. Modeling studies and recent experimental data suggest that the KvS6 helix may be kinked in the vicinity of this motif. Simulations (10 ns) of an isolated KvS6 helix in an octane slab and in a POPC bilayer reveal hinge-bending motions. A pattern-matching approach was used to search for possible hinge-bending motifs in the TM helices of other ion channel proteins. This uncovered a conserved Gly-x-Pro motif in TM helix D5 of CLC channels. MD simulations of a model of hCLC1-D5 spanning an octane slab suggest that this channel also contains a TM helix that undergoes hinge-bending motion. In conclusion, our simulations suggest a model in which hinge-bending motions of TM helices may play a functional role in the gating mechanisms of several different families of ion channels.

  11. Voltage-gated channel properties of epithelial cells in porcine vomeronasal organ.

    PubMed

    Ishida, Tetsuya; Takei, Ryo; Gautam, Shree Hari; Otsuguro, Ken-Ichi; Ohta, Toshio; Ito, Shigeo; Habara, Yoshiaki; Saito, Toshiyuki

    2008-08-29

    Bipolar vomeronasal sensory neurons (VSNs) in the vomeronasal organ (VNO) are believed to detect pheromones in most mammals. The vomeronasal sensory epithelium (VSE) is composed of VSNs and supporting cells. There are morphological differences in VNOs between species. Many electrophysiological experiments have been performed on rodent VSEs but few on other mammals. We therefore investigated voltage-gated channel properties of cells in the porcine VSE using slice whole-cell voltage-clamp techniques. In immunohistochemical study of the porcine VSE, most PGP9.5-immunoreactive cells were found between the middle and basal region, and negative cells were distributed in the apical to middle region. Depolarizing pulses to epithelial cells from -90mV produced transient inward Na+ channel currents and sustained outward K+ channel currents with various amplitudes. The distribution of cells having high and low Na+ current densities was mostly consistent with the histological distribution of VSNs and supporting cells, respectively. The half-inactivation voltage of voltage-gated Na+ channels in supporting cells was 26mV more negative than that in VSNs. Voltage-gated K+ channel currents in both cell types were suppressed by tetraethylammonium to the same extent. VSNs possessed TTX-sensitive voltage-gated Na+ channels and Ni2+ -sensitive T-type Ca2+ channels. These results suggest that the histological distribution of porcine vomeronasal epithelial cells is more similar to the dog and goat than to rodents, and that the electrophysiological characteristics of porcine vomeronasal epithelial cells are similar to those of rodents. It is also suggested that porcine VSNs detecting pheromones generate action potentials through these channels.

  12. Gating characteristics of a steeply voltage-dependent gap junction channel in rat Schwann cells

    PubMed Central

    1993-01-01

    The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, Gss was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary junctional currents was detected corresponding to an unitary channel conductance of approximately 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic Gss voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states. PMID:8301264

  13. Utrophin suppresses low frequency oscillations and coupled gating of mechanosensitive ion channels in dystrophic skeletal muscle.

    PubMed

    Lansman, Jeffry B

    2015-01-01

    An absence of utrophin in muscle from mdx mice prolongs the open time of single mechanosensitive channels. On a time scale much longer than the duration of individual channel activations, genetic depletion of utrophin produces low frequency oscillations of channel open probability. Oscillatory channel opening occurred in the dystrophin/utrophin mutants, but was absent in wild-type and mdx fibers. By contrast, small conductance channels showed random gating behavior when present in the same patch. Applying a negative pressure to a patch on a DKO fiber produced a burst of mode II activity, but channels subsequently closed and remained silent for tens of seconds during the maintained pressure stimulus. In addition, simultaneous opening of multiple MS channels could be frequently observed in recordings from patches on DKO fibers, but only rarely in wild-type and mdx muscle. A model which accounts for the single-channel data is proposed in which utrophin acts as gating spring which maintains the mechanical stability a caveolar-like compartment. The state of this compartment is suggested to be dynamic; its continuity with the extracellular surface varying over seconds to minutes. Loss of the mechanical stability of this compartment contributes to pathogenic Ca(2+) entry through MS channels in Duchenne dystrophy.

  14. 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

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

    PubMed

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

    2013-12-05

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

  16. Fast and Slow Gating Relaxations in the Muscle Chloride Channel Clc-1

    PubMed Central

    Accardi, Alessio; Pusch, Michael

    2000-01-01

    Gating of the muscle chloride channel CLC-1 involves at least two processes evidenced by double-exponential current relaxations when stepping the voltage to negative values. However, there is little information about the gati