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Sample records for glycine receptor channels

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

    PubMed

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

    2016-08-19

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

  2. Investigation of the alpha(1)-glycine receptor channel-opening kinetics in the submillisecond time domain.

    PubMed

    Grewer, C

    1999-08-01

    The activation and desensitization kinetics of the human alpha(1)-homooligomeric glycine receptor, which was transiently expressed in HEK 293 cells, were studied with a 100-microseconds time resolution to determine the rate and equilibrium constants of individual receptor reaction steps. Concentration jumps of the activating ligands glycine and beta-alanine were initiated by photolysis of caged, inactive precursors and were followed by neurotransmitter binding, receptor-channel opening, and receptor desensitization steps that were separated along the time axis. Analysis of the ligand concentration-dependence of these processes allows the determination of 1) the rate constants of glycine binding, k(+1) approximately 10(7) M(-1) s(-1), and dissociation, k(-1) = 1900 s(-1); 2) the rates of receptor-channel opening, k(op) = 2200 s(-1), and closing, k(cl) = 38 s(-1); 3) the receptor desensitization rate, alpha = 0.45 s(-1); 4) the number of occupied ligand binding sites necessary for receptor-channel activation and desensitization, n >/= 3; and 5) the maximum receptor-channel open probability, p(0) > 0.95. The kinetics of receptor-channel activation are insensitive to the transmembrane potential. A general model for glycine receptor activation explaining the experimental data consists of a sequential mechanism based on rapid ligand-binding steps preceding a rate-limiting receptor-channel opening reaction and slow receptor desensitization. PMID:10423421

  3. Expression of heteromeric glycine receptor-channels in rat spinal cultures and inhibition by neuroactive steroids.

    PubMed

    Fodor, László; Boros, András; Dezso, Péter; Maksay, Gábor

    2006-11-01

    Ionotropic glycine receptors were studied in cultured spinal cord neurons prepared from 17-day-old rat embryos, using whole-cell patch clamp electrophysiology. Glycine receptors of 3-17 days in vitro were characterized via subtype-specific channel blockade by micromolar picrotoxin and cyanotriphenylborate, as well as nanomolar strychnine. Potentiation by nanomolar tropisetron indicated coexpression of beta with alpha subunits. The neuroactive steroids pregnenolone sulfate and dehydroepiandrosterone sulfate, as well as alphaxalone and its 3beta epimer betaxalone inhibited the chloride current with IC(50) values of 19, 46, 16 and 208 microM, respectively, with no potentiation. Reverse transcription polymerase chain reaction and immunocytochemistry demonstrated mRNAs and proteins of alpha1, alpha2, alpha3 and beta subunits in rat spinal cord cultures. In conclusion, neuroactive steroids, both positive and negative modulators of gamma-aminobutyric-acid(A) receptors, inhibited heteromeric glycine receptors at micromolar concentrations. PMID:16784797

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed

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

    2015-07-10

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

  6. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

    PubMed Central

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K.; Mayer, Mark L.

    2015-01-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species. PMID:26460032

  7. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes.

    PubMed

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K; Mayer, Mark L

    2015-11-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species. PMID:26460032

  8. Zinc Modulation of Glycine Receptors

    PubMed Central

    Trombley, Paul Q.; Blakemore, Laura J.; Hill, Brook J.

    2011-01-01

    Glycine receptors are widely expressed in the mammalian central nervous system, and previous studies have demonstrated that glycine receptors are modulated by endogenous zinc. Zinc is concentrated in synaptic vesicles in several brain regions but is particularly abundant in the hippocampus and olfactory bulb. In the present study, we used patch-clamp electrophysiology of rat hippocampal and olfactory bulb neurons in primary culture to examine the effects of zinc on glycine receptors. Although glycine has been reported to reach millimolar concentrations during synaptic transmission, most previous studies on the effects of zinc on glycine receptors have used relatively low concentrations of glycine. High concentrations of glycine cause receptor desensitization. Our current results extend our previous demonstration that the modulatory actions of zinc are largely prevented when co-applied with desensitizing concentrations of glycine (300 μM), suggesting that the effects of zinc are dependent on the state of the receptor. In contrast, pre-application of 300 μM zinc, prior to glycine (300 μM) application, causes a slowly developing inhibition with a slow rate of recovery, suggesting that the timing of zinc and glycine release also influences the effects of zinc. Furthermore, previous evidence suggests that synaptically released zinc can gain intracellular access, and we provide the first demonstration that low concentrations of intracellular zinc can potentiate glycine receptors. These results support the notion that zinc has complex effects on glycine receptors and multiple factors may interact to influence the efficacy of glycinergic transmission. PMID:21530619

  9. A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABAA receptor chloride channels.

    PubMed

    Kruger, Wade; Gilbert, Daniel; Hawthorne, Rebecca; Hryciw, Deanne H; Frings, Stephan; Poronnik, Philip; Lynch, Joseph W

    2005-06-01

    There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an external I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels. PMID:15862914

  10. Identification and characterization of heptapeptide modulators of the glycine receptor.

    PubMed

    Cornelison, Garrett L; Pflanz, Natasha C; Tipps, Megan E; Mihic, S John

    2016-06-01

    The glycine receptor is a member of the Cys-loop receptor superfamily of ligand-gated ion channels and is implicated as a possible therapeutic target for the treatment of diseases such as alcoholism and inflammatory pain. In humans, four glycine receptor subtypes (α1, α2, α3, and β) co-assemble to form pentameric channel proteins as either α homomers or αβ heteromers. To date, few agents have been identified that can selectively modulate the glycine receptor, especially those possessing subtype specificity. We used a cell-based method of phage display panning, coupled with two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, to identify novel heptapeptide modulators of the α1β glycine receptor. This involved a panning procedure in which the phage library initially underwent subtractive panning against Human Embryonic Kidney (HEK) 293 cells expressing alternative glycine receptor subtypes before panning the remaining library over HEK 293 cells expressing the target, the α1β glycine receptor. Peptides were identified that act with selectivity on α1β and α3β, compared to α2β, glycine receptors. In addition, peptide activity at the glycine receptor decreased when zinc was chelated by tricine, similar to previous observations of a decrease in ethanol's enhancing actions at the receptor in the absence of zinc. Comparisons of the amino acid sequences of heptapeptides capable of potentiating glycine receptor function revealed several consensus sequences that may be predictive of a peptide's enhancing ability. PMID:27038522

  11. Glycine receptor mechanism elucidated by electron cryo-microscopy.

    PubMed

    Du, Juan; Lü, Wei; Wu, Shenping; Cheng, Yifan; Gouaux, Eric

    2015-10-01

    The strychnine-sensitive glycine receptor (GlyR) mediates inhibitory synaptic transmission in the spinal cord and brainstem and is linked to neurological disorders, including autism and hyperekplexia. Understanding of molecular mechanisms and pharmacology of glycine receptors has been hindered by a lack of high-resolution structures. Here we report electron cryo-microscopy structures of the zebrafish α1 GlyR with strychnine, glycine, or glycine and ivermectin (glycine/ivermectin). Strychnine arrests the receptor in an antagonist-bound closed ion channel state, glycine stabilizes the receptor in an agonist-bound open channel state, and the glycine/ivermectin complex adopts a potentially desensitized or partially open state. Relative to the glycine-bound state, strychnine expands the agonist-binding pocket via outward movement of the C loop, promotes rearrangement of the extracellular and transmembrane domain 'wrist' interface, and leads to rotation of the transmembrane domain towards the pore axis, occluding the ion conduction pathway. These structures illuminate the GlyR mechanism and define a rubric to interpret structures of Cys-loop receptors. PMID:26344198

  12. Mutations within the agonist-binding site convert the homomeric alpha1 glycine receptor into a Zn2+-activated chloride channel.

    PubMed

    Grudzinska, Joanna; Schumann, Tanja; Schemm, Rudolf; Betz, Heinrich; Laube, Bodo

    2008-01-01

    The divalent cation Zn2+ has been shown to regulate inhibitory neurotransmission in the mammalian CNS by affecting the activation of the strychnine-sensitive glycine receptor (GlyR). In spinal neurons and cells expressing recombinant GlyRs, low micromolar (<10 microM) concentrations of Zn2+ enhance glycine currents, whereas higher concentrations (>10 microM) have an inhibitory effect. Mutational studies have localized the Zn2+ binding sites mediating allosteric potentiation and inhibition of GlyRs in distinct regions of the N-terminal extracellular domain of the GlyR alpha-subunits. Here, we examined the Zn2+ sensitivity of different mutations within the agonist binding site of the homomeric alpha(1)-subunit GlyR upon heterologous expression in Xenopus oocytes. This revealed that six substitutions within the ligand-binding pocket result in a total loss of Zn2+ inhibition. Furthermore, substitution of the positively charged residues arginine 65 and arginine 131 by alanine (alpha(1)(R65A), alpha(1)(R131A), or of the aromatic residue phenylalanine 207 by histidine (alpha(1)(F207H)), converted the alpha(1) GlyR into a chloride channel that was activated by Zn2+ alone. Dose-response analysis of the alpha(1)(F207H) GlyR disclosed an EC(50) value of 1.2 microM for Zn2+ activation; concomitantly the apparent glycine affinity was 1000-fold reduced. Thus, single point mutations within the agonist-binding site of the alpha(1) subunit convert the inhibitory GlyR from a glycine-gated into a selectively Zn2+-activated chloride channel. This might be exploited for the design of metal-specific biosensors by modeling-assisted mutagenesis. PMID:18690053

  13. Glycine receptor mechanism illuminated by electron cryo-microscopy

    PubMed Central

    Du, Juan; Lü, Wei; Wu, Shenping; Cheng, Yifan; Gouaux, Eric

    2015-01-01

    Summary The strychnine-sensitive glycine receptor (GlyR) mediates inhibitory synaptic transmission in the spinal cord and brainstem and is linked to neurological disorders including autism and hyperekplexia. Understanding of molecular mechanisms and pharmacology of GlyRs has been hindered by a dearth of high-resolution structures. Here we report electron cryo-microscopy structures of the α1 GlyR with strychnine, glycine, or glycine/ivermectin. Strychnine arrests the receptor in an antagonist-bound, closed ion channel state, glycine stabilizes the receptor in an agonist-bound open channel state, and the glycine/ivermectin complex adopts a potentially desensitized or partially open state. Relative to the glycine-bound state, strychnine expands the agonist-binding pocket via outward movement of the C loop, promotes rearrangement of the extracellular and transmembrane domain ‘wrist’ interface, and leads to rotation of the transmembrane domain toward the pore axis, occluding the ion conduction pathway. These structures illuminate GlyR mechanism and define a rubric to interpret structures of Cys-loop receptors. PMID:26344198

  14. Glutamate and glycine modulation of 3H-MK801 binding to the NMDA receptor-ion channel complex in the vitamin B-6 deficient neonatal rat brain

    SciTech Connect

    Guilarte, T.R. )

    1990-02-26

    The authors have previously shown that the concentrations of the neuroactive amino acids glutamate (GLU) and glycine (GLY) are significantly altered in the seizure-prone vitamin B-6 deficient neonatal rat brain. Recently, it has been shown that GLU and GLY modulate the binding of {sup 3}H-MK801 to the ion channel associated with the N-methyl-D-aspartate (NMDA)-glutamate receptor subtype. The present investigation was undertaken to determine if GLU or GLY modulation of {sup 3}H-MK801 binding was altered in B-6 deficient neonatal rat brain. Preparation of cortical membranes from control and deficient 14 day old rats and {sup 3}H-MK801 binding assay were done as described by Ransom and Stec. The results show a significant reduction in the potency and efficacy of GLU modulation of {sup 3}H-MK801 binding, as well as a reduction in the efficacy of GLY, in membrane preparations from deficient rats compared to controls. These results indicate a reduced ability of GLU and GLY to potentiate the binding of {sup 3}H-MK801 to the NMDA receptor-ion channel in the B-6 deficient neonatal rat brain.

  15. [Molecular physiology of glycine receptors in nervous system of vertebrates].

    PubMed

    2014-03-01

    Glycine receptor is the anion-selective channel, providing fast synaptic transmission in the central nervous system of vertebrates. Together with the nicotinic acetylcholine, GABA and serotonin (5-HT3R) receptors, it belongs to the superfamily of pentameric cys-loop receptors. It has been cloned one beta and four alpha subunits of glycine receptor, which are specifically distributed in different areas of the nervous system. Due to their specific molecular properties and distribution, different subunits ensure important physiological functions: from control of motor activity and regulation of neuronal differentiation to sensory information processing and modulation of pain sensitivity. In this review we briefly describe main functions of these transmembrane proteins, their distribution and molecular architecture. Special attention is paid to recent studies on the molecular physiology of these receptors, as well as on presenting of molecular domains responsible for their modulation and dysfunction. PMID:25508361

  16. [Molecular physiology of glycine receptors in nervous system of vertebrates].

    PubMed

    Maleeva, G V; Brezhestovskiĭ, P D

    2014-03-01

    Glycine receptor is the anion-selective channel, providing fast synaptic transmission in the central nervous system of vertebrates. Together with the nicotinic acetylcholine, GABA and serotonin (5-HT3R) receptors, it belongs to the superfamily of pentameric cys-loop receptors. It has been cloned one beta and four alpha subunits of glycine receptor, which are specifically distributed in different areas of the nervous system. Due to their specific molecular properties and distribution, different subunits ensure important physiological functions: from control of motor activity and regulation of neuronal differentiation to sensory information processing and modulation of pain sensitivity. In this review we briefly describe main functions of these transmembrane proteins, their distribution and molecular architecture. Special attention is paid to recent studies on the molecular physiology of these receptors, as well as on presenting of molecular domains responsible for their modulation and dysfunction. PMID:25464730

  17. Principal role of NR3 subunits in NR1/NR3 excitatory glycine receptor function.

    PubMed

    Madry, Christian; Mesic, Ivana; Bartholomäus, Ingo; Nicke, Annette; Betz, Heinrich; Laube, Bodo

    2007-03-01

    Calcium-permeable N-methyl-d-aspartate (NMDA) receptors are tetrameric cation channels composed of glycine-binding NR1 and glutamate-binding NR2 subunits, which require binding of both glutamate and glycine for efficient channel gating. In contrast, receptors assembled from NR1 and NR3 subunits function as calcium-impermeable excitatory glycine receptors that respond to agonist application only with low efficacy. Here, we show that antagonists of and substitutions within the glycine-binding site of NR1 potentiate NR1/NR3 receptor function up to 25-fold, but inhibition or mutation of the NR3 glycine binding site reduces or abolishes receptor activation. Thus, glycine bound to the NR1 subunit causes auto-inhibition of NR1/NR3 receptors whereas glycine binding to the NR3 subunits is required for opening of the ion channel. Our results establish differential roles of the high-affinity NR3 and low-affinity NR1 glycine-binding sites in excitatory glycine receptor function. PMID:17214961

  18. Structure and Pharmacologic Modulation of Inhibitory Glycine Receptors.

    PubMed

    Burgos, Carlos F; Yévenes, Gonzalo E; Aguayo, Luis G

    2016-09-01

    Glycine receptors (GlyR) are inhibitory Cys-loop ion channels that contribute to the control of excitability along the central nervous system (CNS). GlyR are found in the spinal cord and brain stem, and more recently they were reported in higher regions of the CNS such as the hippocampus and nucleus accumbens. GlyR are involved in motor coordination, respiratory rhythms, pain transmission, and sensory processing, and they are targets for relevant physiologic and pharmacologic modulators. Several studies with protein crystallography and cryoelectron microscopy have shed light on the residues and mechanisms associated with the activation, blockade, and regulation of pentameric Cys-loop ion channels at the atomic level. Initial studies conducted on the extracellular domain of acetylcholine receptors, ion channels from prokaryote homologs-Erwinia chrysanthemi ligand-gated ion channel (ELIC), Gloeobacter violaceus ligand-gated ion channel (GLIC)-and crystallized eukaryotic receptors made it possible to define the overall structure and topology of the Cys-loop receptors. For example, the determination of pentameric GlyR structures bound to glycine and strychnine have contributed to visualizing the structural changes implicated in the transition between the open and closed states of the Cys-loop receptors. In this review, we summarize how the new information obtained in functional, mutagenesis, and structural studies have contributed to a better understanding of the function and regulation of GlyR. PMID:27401877

  19. Activity and Structural Comparisons of Solution Associating and Monomeric Channel-Forming Peptides Derived from the Glycine Receptor M2 Segment

    PubMed Central

    Cook, Gabriel A.; Prakash, Om; Zhang, Ke; Shank, Lalida P.; Takeguchi, Wade A.; Robbins, Ashley; Gong, Yu-Xi; Iwamoto, Takeo; Schultz, Bruce D.; Tomich, John M.

    2004-01-01

    A number of channel-forming peptides derived from the second transmembrane (TM) segment (M2) of the glycine receptor α1 subunit (M2GlyR), including the 22-residue sequence NK4-M2GlyR p22 wild type (WT) (KKKKPARVGLGITTVLTMTTQS), induce anion permeation across epithelial cell monolayers. In vitro assays suggest that this peptide or related sequences might function as a candidate for ion channel replacement therapy in treating channelopathies such as cystic fibrosis (CF). The wild-type sequence forms soluble associations in water that diminish its efficacy. Introduction of a single substitution S22W at the C-terminus, NK4-M2GlyR p22 S22W, eliminates the formation of higher molecular weight associations in solution. The S22W peptide also reduces the concentration of peptide required for half-maximal anion transport induced across Madin-Darby canine kidney cells (MDCK) monolayers. A combination of 2D double quantum filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame nuclear Overhauser effect spectroscopy (ROESY) data were recorded for both the associating WT and nonassociating S22W peptides and used to compare the primary structures and to assign the secondary structures. High-resolution structural studies were recorded in the solvent system (40% 2,2,2-Trifluoroethanol (TFE)/water), which gave the largest structural difference between the two peptides. Nuclear Overhauser effect crosspeak intensity provided interproton distances and the torsion angles were measured by spin-spin coupling constants. These constraints were put into the DYANA modeling program to generate a group of structures. These studies yielded energy-minimized structures for this mixed solvent environment. Structure for both peptides is confined to the 15-residue transmembrane segments. The energy-minimized structure for the WT peptide shows a partially helical extended structure. The S22W peptide

  20. Positive Modulation of the Glycine Receptor by Means of Glycine Receptor–Binding Aptamers

    PubMed Central

    Aneiros, Eduardo; Blank, Michael; Mueller, Johan; Nyman, Eva; Blind, Michael; Dabrowski, Michael A.; Andersson, Christin V.; Sandberg, Kristian

    2015-01-01

    According to the gate control theory of pain, the glycine receptors (GlyRs) are putative targets for development of therapeutic analgesics. A possible approach for novel analgesics is to develop a positive modulator of the glycine-activated Cl− channels. Unfortunately, there has been limited success in developing drug-like small molecules to study the impact of agonists or positive modulators on GlyRs. Eight RNA aptamers with low nanomolar affinity to GlyRα1 were generated, and their pharmacological properties analyzed. Cytochemistry using fluorescein-labeled aptamers demonstrated GlyRα1-dependent binding to the plasma membrane but also intracellular binding. Using a fluorescent membrane potential assay, we could identify five aptamers to be positive modulators. The positive modulation of one of the aptamers was confirmed by patch-clamp electrophysiology on L(tk) cells expressing GlyRα1 and/or GlyRα1β. This aptamer potentiated whole-cell Cl− currents in the presence of low concentrations of glycine. To our knowledge, this is the first demonstration ever of RNA aptamers acting as positive modulators for an ion channel. We believe that these aptamers are unique and valuable tools for further studies of GlyR biology and possibly also as tools for assay development in identifying small-molecule agonists and positive modulators. PMID:26071243

  1. Pharmacological characterisation of strychnine and brucine analogues at glycine and alpha7 nicotinic acetylcholine receptors.

    PubMed

    Jensen, Anders A; Gharagozloo, Parviz; Birdsall, Nigel J M; Zlotos, Darius P

    2006-06-01

    Strychnine and brucine from the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors, including some members of the superfamily of ligand-gated ion channels. In this study, we have characterised the pharmacological properties of tertiary and quaternary analogues as well as bisquaternary dimers of strychnine and brucine at human alpha1 and alpha1beta glycine receptors and at a chimera consisting of the amino-terminal domain of the alpha7 nicotinic receptor (containing the orthosteric ligand binding site) and the ion channel domain of the 5-HT3A serotonin receptor. Although the majority of the analogues displayed significantly increased Ki values at the glycine receptors compared to strychnine and brucine, a few retained the high antagonist potencies of the parent compounds. However, mirroring the pharmacological profiles of strychnine and brucine, none of the analogues displayed significant selectivity between the alpha1 and alpha1beta subtypes. The structure-activity relationships for the compounds at the alpha7/5-HT3 chimera were significantly different from those at the glycine receptors. Most strikingly, quaternization of strychnine and brucine with substituents possessing different steric and electronic properties completely eliminated the activity at the glycine receptors, whereas binding affinity to the alpha7/5-HT3 chimera was retained for the majority of the quaternary analogues. This study provides an insight into the structure-activity relationships for strychnine and brucine analogues at these ligand-gated ion channels. PMID:16687139

  2. Selective potentiation of alpha 1 glycine receptors by ginkgolic acid

    PubMed Central

    Maleeva, Galyna; Buldakova, Svetlana; Bregestovski, Piotr

    2015-01-01

    Glycine receptors (GlyRs) belong to the superfamily of pentameric cys-loop receptor-operated channels and are involved in numerous physiological functions, including movement, vision, and pain. In search for compounds performing subunit-specific modulation of GlyRs we studied action of ginkgolic acid, an abundant Ginkgo biloba product. Using patch-clamp recordings, we analyzed the effects of ginkgolic acid in concentrations from 30 nM to 25 μM on α1–α3 and α1/β, α2/β configurations of GlyR and on GABAARs expressed in cultured CHO-K1 cells and mouse neuroblastoma (N2a) cells. Ginkgolic acid caused an increase in the amplitude of currents mediated by homomeric α1 and heteromeric α1/β GlyRs and provoked a left-shift of the concentration-dependent curves for glycine. Even at high concentrations (10–25 μM) ginkgolic acid was not able to augment ionic currents mediated by α2, α2/β, and α3 GlyRs, or by GABAAR consisting of α1/β2/γ2 subunits. Mutation of three residues (T59A/A261G/A303S) in the α2 GlyR subunit to the corresponding ones from the α1 converted the action of ginkgolic acid to potentiation with a distinct decrease in EC50 for glycine, suggesting an important role for these residues in modulation by ginkgolic acid. Our results suggest that ginkgolic acid is a novel selective enhancer of α1 GlyRs. PMID:26578878

  3. Glycine receptor antibodies in PERM and related syndromes: characteristics, clinical features and outcomes

    PubMed Central

    Carvajal-González, Alexander; Leite, M. Isabel; Waters, Patrick; Woodhall, Mark; Coutinho, Ester; Balint, Bettina; Lang, Bethan; Pettingill, Philippa; Carr, Aisling; Sheerin, Una-Marie; Press, Raomand; Lunn, Michael P.; Lim, Ming; Maddison, Paul; Meinck, H.-M.; Vandenberghe, Wim

    2014-01-01

    The clinical associations of glycine receptor antibodies have not yet been described fully. We identified prospectively 52 antibody-positive patients and collated their clinical features, investigations and immunotherapy responses. Serum glycine receptor antibody endpoint titres ranged from 1:20 to 1:60 000. In 11 paired samples, serum levels were higher than (n = 10) or equal to (n = 1) cerebrospinal fluid levels; there was intrathecal synthesis of glycine receptor antibodies in each of the six pairs available for detailed study. Four patients also had high glutamic acid decarboxylase antibodies (>1000 U/ml), and one had high voltage-gated potassium channel-complex antibody (2442 pM). Seven patients with very low titres (<1:50) and unknown or alternative diagnoses were excluded from further study. Three of the remaining 45 patients had newly-identified thymomas and one had a lymphoma. Thirty-three patients were classified as progressive encephalomyelitis with rigidity and myoclonus, and two as stiff person syndrome; five had a limbic encephalitis or epileptic encephalopathy, two had brainstem features mainly, two had demyelinating optic neuropathies and one had an unclear diagnosis. Four patients (9%) died during the acute disease, but most showed marked improvement with immunotherapies. At most recent follow-up, (2–7 years, median 3 years, since first antibody detection), the median modified Rankin scale scores (excluding the four deaths) decreased from 5 at maximal severity to 1 (P < 0.0001), but relapses have occurred in five patients and a proportion are on reducing steroids or other maintenance immunotherapies as well as symptomatic treatments. The glycine receptor antibodies activated complement on glycine receptor-transfected human embryonic kidney cells at room temperature, and caused internalization and lysosomal degradation of the glycine receptors at 37°C. Immunoglobulin G antibodies bound to rodent spinal cord and brainstem co-localizing with

  4. Agonist action of taurine on glycine receptors in rat supraoptic magnocellular neurones: possible role in osmoregulation.

    PubMed

    Hussy, N; Deleuze, C; Pantaloni, A; Desarménien, M G; Moos, F

    1997-08-01

    1. To evaluate the implication of taurine in the physiology of supraoptic neurones, we (i) investigated the agonist properties of taurine on glycine and GABAA receptors of supraoptic magnocellular neurones acutely dissociated from adult rats, using whole-cell voltage clamp, (ii) studied the effects of taurine and strychnine in vivo by extracellular recordings of supraoptic vasopressin neurones in anaesthetized rats, and (iii) measured the osmolarity-dependent release of endogenous taurine from isolated supraoptic nuclei by HPLC. 2. GABA, glycine and taurine evoked rapidly activating currents that all reversed close to the equilibrium potential for Cl-, indicating activation of Cl(-)-selective channels. Glycine-activated currents were reversibly blocked by strychnine (IC50 of 35 nM with 100 microM glycine), but were unaffected by the GABAA antagonist gabazine (1-3 microM). GABA-activated currents were reversibly antagonized by 3 microM gabazine, but not by strychnine (up to 1 microM). 3. Responses to 1 mM taurine were blocked by strychnine but not by gabazine and showed no additivity with glycine-induced currents, indicating selective activation of glycine receptors. Responses to 10 mM taurine were partially antagonized by gabazine, the residual current being blocked by strychnine. Thus, taurine is also a weak agonist of GABAA receptors. 4. In the presence of gabazine, taurine activated glycine receptors with an EC50 of 406 microM. Taurine activated at most 70% of maximal glycine currents, suggesting that it is a partial agonist of glycine receptors. 5. In vivo, locally applied strychnine (300 nM) increased and taurine (1 mM) decreased the basal electrical activity of vasopressin neurones in normally hydrated rats. The effect of strychnine was markedly more pronounced in water-loaded rats. 6. Taurine, which is concentrated in supraoptic glial cells, could be released from isolated supraoptic nuclei upon hyposmotic stimulation. Decreases in osmolarity of 15 and 30

  5. Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors

    PubMed Central

    Farroni, Jeffrey S; McCool, Brian A

    2004-01-01

    Background Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. Results While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The β-amino acid taurine possessed 30–50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for β-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. Conclusions Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In

  6. Kinetic determinants of agonist action at the recombinant human glycine receptor

    PubMed Central

    Lewis, Trevor M; Schofield, Peter R; McClellan, Annette M L

    2003-01-01

    The amino acids glycine, β-alanine and taurine are all endogenous agonists of the glycine receptor. In this study, a combination of rapid agonist application onto macropatches and steady-state single-channel recordings was used to compare the actions of glycine, β-alanine and taurine upon homomeric α1 human glycine receptors transiently expressed in human embryonic kidney (HEK 293) cells. The 10–90 % rise times determined from rapid application of 100 μm of each agonist were indistinguishable, indicating each agonist has a similar association rate. At saturating concentrations (30 mm) the rise time for glycine (0.26 ms) was 1.8-fold faster than that for β-alanine (0.47 ms) and 3.9-fold faster than that for taurine (1.01 ms), indicating clear differences in the maximum opening rate between agonists. The relaxation following rapid removal of agonist was fitted with a single exponential for β-alanine (3.0 ms) and taurine (2.2 ms), and two exponential components for glycine with a weighted mean time constant of 27.1 ms. This was consistent with differences in dissociation rates estimated from analysis of bursts, with taurine > β-alanine > glycine. Exponential fits to the open period distributions gave time constants that did not differ between agonists and the geometric distribution for the number of openings per burst indicated that all three agonists had a significant component of single-opening bursts. Based upon these data, we propose a kinetic scheme with three independent open states, where the opening rates are dependent upon the activating agonist, while the closing rates are an intrinsic characteristic of the receptor. PMID:12679369

  7. MOLECULAR TARGETS AND MECHANISMS FOR ETHANOL ACTION IN GLYCINE RECEPTORS

    PubMed Central

    Perkins, Daya I.; Trudell, James R.; Crawford, Daniel K.; Alkana, Ronald L.; Davies, Daryl L.

    2010-01-01

    Glycine receptors (GlyRs) are recognized as the primary mediators of neuronal inhibition in the spinal cord, brain stem and higher brain regions known to be sensitive to ethanol. Building evidence supports the notion that ethanol acting on GlyRs causes at least a subset of its behavioral effects and may be involved in modulating ethanol intake. For over two decades, GlyRs have been studied at the molecular level as targets for ethanol action. Despite the advances in understanding the effects of ethanol in vivo and in vitro, the precise molecular sites and mechanisms of action for ethanol in ligand-gated ion channels in general, and in GlyRs specifically, are just now starting to become understood. The present review focuses on advances in our knowledge produced by using molecular biology, pressure antagonism, electrophysiology and molecular modeling strategies over the last two decades to probe, identify and model the initial molecular sites and mechanisms of ethanol action in GlyRs. The molecular targets on the GlyR are covered on a global perspective, which includes the intracellular, transmembrane and extracellular domains. The latter has received increasing attention in recent years. Recent molecular models of the sites of ethanol action in GlyRs and their implications to our understanding of possible mechanism of ethanol action and novel targets for drug development in GlyRs are discussed. PMID:20399807

  8. A Role for Accumbal Glycine Receptors in Modulation of Dopamine Release by the Glycine Transporter-1 Inhibitor Org25935

    PubMed Central

    Lidö, Helga Höifödt; Ericson, Mia; Marston, Hugh; Söderpalm, Bo

    2010-01-01

    Accumbal glycine modulates basal and ethanol-induced dopamine levels in the nucleus accumbens (nAc) as well as voluntary ethanol consumption. Also, systemic administration of the glycine transporter-1 inhibitor Org25935 elevates dopamine levels in nAc, prevents a further ethanol-induced dopamine elevation and robustly and dose-dependently decreases ethanol consumption in rats. Here we investigated whether Org25935 applied locally in nAc modulates dopamine release, and whether accumbal glycine receptors or NMDA receptors are involved in this tentative effect. We also addressed whether Org25935 and ethanol applied locally in nAc interact with dopamine levels, as seen after systemic administration. We used in vivo microdialysis coupled to HPLC-ED in freely moving male Wistar rats to monitor dopamine output in nAc after local perfusion of Org25935 alone, with ethanol, or Org25935-perfusion after pre-treatment with the glycine receptor antagonist strychnine or the NMDA receptor glycine site antagonist L-701.324. Local Org25935 increased extracellular dopamine levels in a subpopulation of rats. Local strychnine, but not systemic L-701.324, antagonized the dopamine-activating effect of Org25935. Ethanol failed to induce a dopamine overflow in the subpopulation responding to Org25935 with a dopamine elevation. The study supports a role for accumbal glycine receptors rather than NMDA receptor signaling in the dopamine-activating effect of Org25935. The results further indicate that the previously reported systemic Org25935–ethanol interaction with regard to accumbal dopamine is localized to the nAc. This adds to the growing evidence for the glycine receptor as an important player in the dopamine reward circuitry and in ethanol's effects within this system. PMID:21556278

  9. Activation of presynaptic glycine receptors facilitates glycine release from presynaptic terminals synapsing onto rat spinal sacral dorsal commissural nucleus neurons

    PubMed Central

    Jeong, Hyo-Jin; Jang, Il-Sung; Moorhouse, Andrew J; Akaike, Norio

    2003-01-01

    Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Here we report the novel finding that presynaptic glycine autoreceptors modulate release from terminals synapsing onto rat spinal sacral dorsal commissural nucleus (SDCN) neurons. In mechanically dissociated SDCN neurons, in which functional presynaptic nerve terminals remain adherent to the isolated neurons, exogenously applied glycine (3 μM) increased the frequency of glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) without affecting their amplitudes or decay times. This suggests that glycine acts presynaptically to increase glycine release probability. Picrotoxin, at a concentration that had little direct effect on sIPSC frequency and amplitude (30 μM), significantly attenuated glycine-induced presynaptic sIPSC facilitation. The glycine-induced sIPSC frequency facilitation was completely abolished either in a Ca2+-free external solution or in the presence of 100 μM Cd2+, suggesting the involvement of extracellular Ca2+ influx into the nerve terminals. The glycine action was also completely occluded in the presence of 300 nM tetrodotoxin. In recordings from SDCN neurons in spinal cord slices, glycine (10 μM) increased evoked IPSC (eIPSC) amplitude and decreased the extent of paired-pulse facilitation. In response to brief high frequency stimulus trains the eIPSCs displayed a profound frequency-dependent facilitation that was greatly reduced by picrotoxin (30 μM). These results indicate that glycine acts at presynaptic autoreceptors, causing depolarization of the glycinergic nerve terminals, the subsequent activation of voltage-dependent Na+ and Ca2+ channels, and facilitation of glycine release. Furthermore, this presynaptic facilitation was observed under more physiological conditions, suggesting that these glycinergic autoreceptors may contribute to the integration of local inhibitory inputs to SDCN neurons. PMID:12754315

  10. Structural difference between heteromeric somatic and homomeric axonal glycine receptors in the hypothalamo-neurohypophysial system.

    PubMed

    Deleuze, C; Runquist, M; Orcel, H; Rabié, A; Dayanithi, G; Alonso, G; Hussy, N

    2005-01-01

    Glycine receptors are ionotropic receptors formed by either the homomeric assembly of ligand-binding alpha subunits or the heteromeric combination of an alpha subunit and the auxiliary beta subunit. Glycine receptors in the brain are found at either pre- or post-synaptic sites. Rat supraoptic nucleus neurons express glycine receptors on the membrane of both their soma and dendrites within the supraoptic nucleus, and their axon terminals in the neurohypophysis. Taking advantage of the well-separated cellular compartments of this system, we correlated the structural properties of the receptors to their subcellular localization. Immunohistochemical study using the generic mAb4a antibody revealed that somatodendritic receptors were clustered, whereas axonal glycine receptors showed a more diffuse distribution. This was paralleled by the presence of clusters of the glycine receptor aggregating protein gephyrin in the supraoptic nucleus and its complete absence in the neurohypophysis. Moreover, another antibody recognizing the alpha1/alpha2 subunits similarly labeled the axonal glycine receptors, but did not recognize the somatodendritic receptor clusters of supraoptic nucleus neurons, indicative of structural differences between somatic and axonal glycine receptors. Furthermore, the subunits composing the somatic and axonal receptors have different molecular weight. Functional study further differentiated the two types of glycine receptors on the basis of their sensitivity to picrotoxin, identifying somatic receptors as alpha/beta heteromers, and axonal receptors as alpha homomers. These results indicate that targeting of glycine receptors to axonal or somatodendritic compartment is directly related to their subunit composition, and set the hypothalamo-neurohypophysial system as an excellent model to study the mechanisms of targeting of proteins to various neuronal cellular compartments. PMID:16125853

  11. Kainate receptor activation induces glycine receptor endocytosis through PKC deSUMOylation.

    PubMed

    Sun, Hao; Lu, Li; Zuo, Yong; Wang, Yan; Jiao, Yingfu; Zeng, Wei-Zheng; Huang, Chao; Zhu, Michael X; Zamponi, Gerald W; Zhou, Tong; Xu, Tian-Le; Cheng, Jinke; Li, Yong

    2014-01-01

    Surface expression and regulated endocytosis of glycine receptors (GlyRs) play a critical function in balancing neuronal excitability. SUMOylation (SUMO modification) is of critical importance for maintaining neuronal function in the central nervous system. Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices. This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC. SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels. Altogether, we have identified a SUMOylation-dependent regulatory pathway for GlyR endocytosis, which may have important physiological implications for proper neuronal excitability. PMID:25236484

  12. Purification and characterization of the glycine receptor of pig spinal cord

    SciTech Connect

    Graham, D.; Pfeiffer, F.; Simler, R.; Betz, H.

    1985-02-12

    A large-scale purification procedure was developed to isolate the glycine receptor of pig spinal cord by affinity chromatography on aminostrychnine agarose. After an overall purification of about 10,000-fold, the glycine receptor preparations contained three major polypeptides of Mr 48,000, 58,000, and 93,000. Photoaffinity labeling with (/sup 3/H)strychnine showed that the (/sup 3/H)strychnine binding site is associated with the Mr 48,000 and, to a much lesser extent, the Mr 58,000 polypeptides. (/sup 3/H)Strychnine binding to the purified receptor exhibited a dissociation constant K /sub D/ of 13.8 nM and was inhibited by the agonists glycine, taurine, and beta-alanine. Gel filtration and sucrose gradient centrifugation gave a Stokes radius of 7.1 nm and an apparent sedimentation coefficient of 9.6 S. Peptide mapping of the (/sup 3/H)strychnine-labeled Mr 48,000 polypeptides of purified pig and rat glycine receptor preparations showed that the strychnine binding region of this receptor subunit is highly conserved between these species. Also, three out of six monoclonal antibodies against the glycine receptor of rat spinal cord significantly cross-reacted with their corresponding polypeptides of the pig glycine receptor. These results show that the glycine receptor of pig spinal cord is very similar to the well-characterized rat receptor protein and can be purified in quantities sufficient for protein chemical analysis.

  13. Blockade of glycine transporter 1 by SSR-504734 promotes cognitive flexibility in glycine/NMDA receptor-dependent manner.

    PubMed

    Nikiforuk, Agnieszka; Kos, Tomasz; Rafa, Dominik; Behl, Berthold; Bespalov, Anton; Popik, Piotr

    2011-01-01

    Accumulating evidence suggests that cognitive processes may be regulated by glycine concentration in the local environment of glutamate N-methyl-d-aspartate receptor (NMDAR). The concentration of glycine is controlled, among other factors, by the glycine transporter 1 (GlyT1). While GlyT1 inhibitors are developed for a number of indications including cognitive improvement, little is known about their effects in tasks depending on prefrontal cortical function. We examined the effect of GlyT1 inhibitor SSR-504734 on cognitive flexibility assessed in the attentional set-shifting task in rats (ASST). The second goal was to elucidate whether SSR-504734 effect has been due to the compound's action at glycine/NMDAR site. Rats treated with SSR-504734 (3 and 10 mg/kg, IP) required significantly less trials to criteria during extra-dimensional shift (EDs) phase of the ASST. The effect of SSR-504734 (3 mg/kg) was completely prevented by the glycine/NMDAR site antagonist, L-687,414 (30 mg/kg, IP) that by itself exerted no effect on cognitive flexibility. Present study demonstrates that the elevation of glycine concentration through the blockade of its reuptake facilitates cognitive flexibility. As this effect was fully blocked by glycine/NMDAR antagonist, SSR-504734-induced cognitive improvement is likely mediated through glycine action at NMDAR. It is suggested that GlyT1 inhibitors like SSR-504734 may represent a useful pharmacological approach for cognitive enhancement, especially in domains critically affected in schizophrenia. PMID:21530555

  14. Glycine receptors: recent insights into their structural organization and functional diversity.

    PubMed

    Betz, Heinrich; Laube, Bodo

    2006-06-01

    Strychnine-sensitive glycine receptors (GlyRs) are known to mediate synaptic inhibition in spinal cord, brainstem and other regions of the CNS. During the past 5 years, considerable progress has been made in delineating structural determinants of ligand binding and channel activation in recombinant GlyRs. Furthermore, immunohistochemical and gene inactivation studies have disclosed distinct distributions and functions of differentially expressed GlyR subtypes in retina, hippocampus and the dorsal horn of the spinal cord. Accordingly, GlyRs regulate not only the excitability of motor and sensory neurones, but are also essential for the processing of photoreceptor signals, neuronal development and inflammatory pain sensitization. Hence, these receptors constitute promising targets for the development of clinically useful compounds. PMID:16805771

  15. Structural basis of dynamic glycine receptor clustering by gephyrin

    PubMed Central

    Sola, Maria; Bavro, Vassiliy N; Timmins, Joanna; Franz, Thomas; Ricard-Blum, Sylvie; Schoehn, Guy; Ruigrok, Rob W H; Paarmann, Ingo; Saiyed, Taslimarif; O'Sullivan, Gregory A; Schmitt, Bertram; Betz, Heinrich; Weissenhorn, Winfried

    2004-01-01

    Gephyrin is a bi-functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full-length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C-terminal region (gephyrin-E), which binds a GlyR β-subunit-derived peptide with high and low affinity. The crystal structure of the tetra-domain gephyrin-E in complex with the β-peptide bound to domain IV indicates how membrane-embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full-length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full-length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity. PMID:15201864

  16. Activation of glycine receptors modulates spontaneous epileptiform activity in the immature rat hippocampus

    PubMed Central

    Chen, Rongqing; Okabe, Akihito; Sun, Haiyan; Sharopov, Salim; Hanganu-Opatz, Ileana L; Kolbaev, Sergei N; Fukuda, Atsuo; Luhmann, Heiko J; Kilb, Werner

    2014-01-01

    While the expression of glycine receptors in the immature hippocampus has been shown, no information about the role of glycine receptors in controlling the excitability in the immature CNS is available. Therefore, we examined the effect of glycinergic agonists and antagonists in the CA3 region of an intact corticohippocampal preparation of the immature (postnatal days 4–7) rat using field potential recordings. Bath application of 100 μm taurine or 10 μm glycine enhanced the occurrence of recurrent epileptiform activity induced by 20 μm 4-aminopyridine in low Mg2+ solution. This proconvulsive effect was prevented by 3 μm strychnine or after incubation with the loop diuretic bumetanide (10 μm), suggesting that it required glycine receptors and an active NKCC1-dependent Cl− accumulation. Application of higher doses of taurine (≥1 mm) or glycine (100 μm) attenuated recurrent epileptiform discharges. The anticonvulsive effect of taurine was also observed in the presence of the GABAA receptor antagonist gabazine and was attenuated by strychnine, suggesting that it was partially mediated by glycine receptors. Bath application of the glycinergic antagonist strychnine (0.3 μm) induced epileptiform discharges. We conclude from these results that in the immature hippocampus, activation of glycine receptors can mediate both pro- and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress epileptiform activity. In summary, our study elucidated the important role of glycine receptors in the control of neuronal excitability in the immature hippocampus. PMID:24665103

  17. Signaling mechanism for modulation by ATP of glycine receptors on rat retinal ganglion cells.

    PubMed

    Zhang, Ping-Ping; Zhang, Gong; Zhou, Wei; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2016-01-01

    ATP modulates voltage- and ligand-gated channels in the CNS via the activation of ionotropic P2X and metabotropic P2Y receptors. While P2Y receptors are expressed in retinal neurons, the function of these receptors in the retina is largely unknown. Using whole-cell patch-clamp techniques in rat retinal slice preparations, we demonstrated that ATP suppressed glycine receptor-mediated currents of OFF type ganglion cells (OFF-GCs) dose-dependently, and the effect was in part mediated by P2Y1 and P2Y11, but not by P2X. The ATP effect was abolished by intracellular dialysis of a Gq/11 protein inhibitor and phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor, but not phosphatidylcholine (PC)-PLC inhibitor. The ATP effect was accompanied by an increase in [Ca(2+)]i through the IP3-sensitive pathway and was blocked by intracellular Ca(2+)-free solution. Furthermore, the ATP effect was eliminated in the presence of PKC inhibitors. Neither PKA nor PKG system was involved. These results suggest that the ATP-induced suppression may be mediated by a distinct Gq/11/PI-PLC/IP3/Ca(2+)/PKC signaling pathway, following the activation of P2Y1,11 and other P2Y subtypes. Consistently, ATP suppressed glycine receptor-mediated light-evoked inhibitory postsynaptic currents of OFF-GCs. These results suggest that ATP may modify the ON-to-OFF crossover inhibition, thus changing action potential patterns of OFF-GCs. PMID:27357477

  18. Signaling mechanism for modulation by ATP of glycine receptors on rat retinal ganglion cells

    PubMed Central

    Zhang, Ping-Ping; Zhang, Gong; Zhou, Wei; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2016-01-01

    ATP modulates voltage- and ligand-gated channels in the CNS via the activation of ionotropic P2X and metabotropic P2Y receptors. While P2Y receptors are expressed in retinal neurons, the function of these receptors in the retina is largely unknown. Using whole-cell patch-clamp techniques in rat retinal slice preparations, we demonstrated that ATP suppressed glycine receptor-mediated currents of OFF type ganglion cells (OFF-GCs) dose-dependently, and the effect was in part mediated by P2Y1 and P2Y11, but not by P2X. The ATP effect was abolished by intracellular dialysis of a Gq/11 protein inhibitor and phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor, but not phosphatidylcholine (PC)-PLC inhibitor. The ATP effect was accompanied by an increase in [Ca2+]i through the IP3-sensitive pathway and was blocked by intracellular Ca2+-free solution. Furthermore, the ATP effect was eliminated in the presence of PKC inhibitors. Neither PKA nor PKG system was involved. These results suggest that the ATP-induced suppression may be mediated by a distinct Gq/11/PI-PLC/IP3/Ca2+/PKC signaling pathway, following the activation of P2Y1,11 and other P2Y subtypes. Consistently, ATP suppressed glycine receptor-mediated light-evoked inhibitory postsynaptic currents of OFF-GCs. These results suggest that ATP may modify the ON-to-OFF crossover inhibition, thus changing action potential patterns of OFF-GCs. PMID:27357477

  19. Differentiated Human Midbrain-Derived Neural Progenitor Cells Express Excitatory Strychnine-Sensitive Glycine Receptors Containing α2β Subunits

    PubMed Central

    Wegner, Florian; Kraft, Robert; Busse, Kathy; Härtig, Wolfgang; Ahrens, Jörg; Leffler, Andreas; Dengler, Reinhard; Schwarz, Johannes

    2012-01-01

    Background Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson’s disease. While glutamate and GABAA receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. Methodology/Principal Findings Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na+-K+-Cl− co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. Conclusions/Significance These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro. PMID:22606311

  20. Anti-Glycine Receptor Antibody Mediated Progressive Encephalomyelitis with Rigidity and Myoclonus Associated with Breast Cancer

    PubMed Central

    De Blauwe, Sofie N.; Santens, Patrick

    2013-01-01

    We describe a 66-year-old woman who presented with a dramatic course of PERM. Anti-glycine receptor antibodies were found. She stabilized after plasma-exchange and partly recovered. Eighteen months later, a diagnosis of smouldering breast cancer with bone marrow metastasis was made. There are indications that this tumor was already present at first presentation. An overview of PERM and anti-glycine receptor antibodies is given. PMID:23936697

  1. The N-terminal domain of the GluN3A subunit determines the efficacy of glycine-activated NMDA receptors.

    PubMed

    Mesic, Ivana; Madry, Christian; Geider, Kirsten; Bernhard, Max; Betz, Heinrich; Laube, Bodo

    2016-06-01

    N-methyl-d-aspartate (NMDA) receptors composed of glycine-binding GluN1 and GluN3 subunits function as excitatory glycine receptors that respond to agonist application only with a very low efficacy. Binding of glycine to the high-affinity GluN3 subunits triggers channel opening, whereas glycine binding to the low-affinity GluN1 subunits causes an auto-inhibition of the maximal glycine-inducible receptor current (Imax). Hence, competitive antagonists of the GluN1 subunit strongly potentiate glycine responses of wild type (wt) GluN1/GluN3 receptors. Here, we show that co-expression of N-terminal domain (NTD) deleted GluN1 (GluN1(ΔNTD)) and GluN3 (GluN3(ΔNTD)) subunits in Xenopus oocytes generates GluN1/GluN3 receptors with a large increase in the glycine-inducible Imax accompanied by a strongly impaired GluN1 antagonist-mediated potentiation. Affinity purification after metabolic or surface labeling revealed no differences in subunit stoichiometry and surface expression between wt GluN1/GluN3A and mutant GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, indicating a specific effect of NTD deletions on the efficacy of receptor opening. Notably, GluN1/GluN3A(ΔNTD) receptors showed a similar increase in Imax and a greatly reduced GluN1 antagonist-mediated current potentiation as GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, whereas the glycine-induced currents of GluN1(ΔNTD)/GluN3A receptors resembled those of wt GluN1/GluN3A receptors. Furthermore, oxidative crosslinking of the homophilic GluN3A NTD intersubunit interface in mutant GluN1/GluN3A(R319C) receptors caused both a decrease in the glycine-induced Imax concomitantly with a marked increase in GluN1 antagonist-mediated current potentiation, whilst mutations within the intrasubunit region linking the GluN3A NTD to the ligand binding domain had opposite effects. Together these results show that the GluN3A NTD constitutes a crucial regulatory determinant of GluN1/GluN3A receptor function. PMID:26777280

  2. Contaminating levels of zinc found in commonly-used labware and buffers affect glycine receptor currents

    PubMed Central

    Cornelison, Garrett L.; Mihic, S. John

    2013-01-01

    Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nM to low μM concentrations, and inhibiting its effects at higher concentrations. Because of zinc’s high potency at the glycine receptor, there exists a possibility that effects attributed solely to exogenously-applied glycine in fact contain an undetected contribution of zinc acting as an allosteric modulator. We found that glycine solutions made up in standard buffers and using deionized distilled water produced effects that could be decreased by the zinc chelator tricine. This phenomenon was observed in three different vials tested and persisted even if vials were extensively washed, suggesting the zinc was probably present in the buffer constituents. In addition, polystyrene, but not glass, pipets bore a contaminant that enhanced glycine receptor function and that could also be antagonized by tricine. Our findings suggest that without checking for this effect using a chelator such as tricine, one cannot assume that responses elicited by glycine applied alone are not necessarily also partially due to some level of allosteric modulation by zinc. PMID:24177173

  3. Contaminating levels of zinc found in commonly-used labware and buffers affect glycine receptor currents.

    PubMed

    Cornelison, Garrett L; Mihic, S John

    2014-01-01

    Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nM to low μM concentrations, and inhibiting its effects at higher concentrations. Because of zinc's high potency at the glycine receptor, there exists a possibility that effects attributed solely to exogenously-applied glycine in fact contain an undetected contribution of zinc acting as an allosteric modulator. We found that glycine solutions made up in standard buffers and using deionized distilled water produced effects that could be decreased by the zinc chelator tricine. This phenomenon was observed in three different vials tested and persisted even if vials were extensively washed, suggesting the zinc was probably present in the buffer constituents. In addition, polystyrene, but not glass, pipets bore a contaminant that enhanced glycine receptor function and that could also be antagonized by tricine. Our findings suggest that without checking for this effect using a chelator such as tricine, one cannot assume that responses elicited by glycine applied alone are not necessarily also partially due to some level of allosteric modulation by zinc. PMID:24177173

  4. Enhancement of homomeric glycine receptor function by long-chain alcohols and anaesthetics.

    PubMed Central

    Mascia, M. P.; Machu, T. K.; Harris, R. A.

    1996-01-01

    1. The effects of n-alcohols (ethanol to dodecanol) and anaesthetics on strychnine-sensitive glycine receptors were studied in Xenopus oocytes expressing homomeric alpha 1 or alpha 2 glycine receptor subunits, with the two electrode voltage-clamp recording technique. 2. The glycine-induced chloride conductance of homomeric alpha glycine receptors was potentiated by all the alcohols tested when an EC2 concentration of glycine was used. Homomeric alpha 1 and alpha 2 receptors were potentiated similarly by the n-alcohols, except that low concentrations of ethanol produced greater potentiation with alpha 1, as previously reported. 3. Increasing the n-alcohol carbon number has been shown to increase the potency of the alcohols up to decanol at concentrations corresponding to EC50s for producing loss of righting reflex in tadpoles. However, dodecanol was no more potent than decanol, and only modest potentiation (30-60%) was obtained with dodecanol, in contrast to marked (150-200%) potentiation with the other alcohols. Thus, a "cut-off' occurred at about dodecanol. 4. Propofol, alphaxalone, pentobarbitone, halothane and enflurane, reversibly potentiated the function of homomeric alpha 1 glycine receptors at concentrations which represent approximately twice the EC50 for production of anaesthesia in mammals, but ketamine and etomidate were ineffective. 5. Two novel cyclobutane compounds were tested; the anaesthetic compound (1-chloro-1,2,2-trifluorocyclobutane) from 0.5 to 5 mM potentiated the action of glycine in a concentration-dependent manner; however, the non-anaesthetic analogue (1,2-dichloro-hexfluorocyclobutane) had no effect on glycine receptor function at concentrations (25 to 80 microM) predicted to be anaesthetic, based on the lipid solubility of this compound. 6. These results suggest that the alpha subunits of strychnine-sensitive glycine receptors contain sites of action for n-alcohols, propofol, alphaxalone, pentobarbitone and volatile anaesthetics, but not

  5. Activation-induced structural change in the GluN1/GluN3A excitatory glycine receptor

    SciTech Connect

    Balasuriya, Dilshan; Takahashi, Hirohide; Srivats, Shyam; Edwardson, J. Michael

    2014-08-08

    Highlights: • We studied the response of the GluN1/GluN3A excitatory glycine receptor to activation. • GluN1 and GluN3A subunits interacted within transfected cells. • The GluN1/GluN3A receptor was functionally active. • Glycine or D-serine caused a ∼1 nm height reduction in bilayer-integrated receptors. • This height reduction was abolished by the glycine antagonist DCKA. - Abstract: Unlike GluN2-containing N-methyl-D-aspartate (NMDA) receptors, which require both glycine and glutamate for activation, receptors composed of GluN1 and GluN3 subunits are activated by glycine alone. Here, we used atomic force microscopy (AFM) imaging to examine the response to activation of the GluN1/GluN3A excitatory glycine receptor. GluN1 and GluN3A subunits were shown to interact intimately within transfected tsA 201 cells. Isolated GluN1/GluN3A receptors integrated into lipid bilayers responded to addition of either glycine or D-serine, but not glutamate, with a ∼1 nm reduction in height of the extracellular domain. The height reduction in response to glycine was abolished by the glycine antagonist 5,7-dichlorokynurenic acid. Our results represent the first demonstration of the effect of activation on the conformation of this receptor.

  6. Disturbed Neuronal ER-Golgi Sorting of Unassembled Glycine Receptors Suggests Altered Subcellular Processing Is a Cause of Human Hyperekplexia

    PubMed Central

    Schaefer, Natascha; Kluck, Christoph J.; Price, Kerry L.; Meiselbach, Heike; Vornberger, Nadine; Schwarzinger, Stephan; Hartmann, Stephanie; Langlhofer, Georg; Schulz, Solveig; Schlegel, Nadja; Brockmann, Knut; Lynch, Bryan; Becker, Cord-Michael; Lummis, Sarah C.R.

    2015-01-01

    Recent studies on the pathogenic mechanisms of recessive hyperekplexia indicate disturbances in glycine receptor (GlyR) α1 biogenesis. Here, we examine the properties of a range of novel glycine receptor mutants identified in human hyperekplexia patients using expression in transfected cell lines and primary neurons. All of the novel mutants localized in the large extracellular domain of the GlyR α1 have reduced cell surface expression with a high proportion of receptors being retained in the ER, although there is forward trafficking of glycosylated subpopulations into the ER-Golgi intermediate compartment and cis-Golgi compartment. CD spectroscopy revealed that the mutant receptors have proportions of secondary structural elements similar to wild-type receptors. Two mutants in loop B (G160R, T162M) were functional, but none of those in loop D/β2–3 were. One nonfunctional truncated mutant (R316X) could be rescued by coexpression with the lacking C-terminal domain. We conclude that a proportion of GlyR α1 mutants can be transported to the plasma membrane but do not necessarily form functional ion channels. We suggest that loop D/β2–3 is an important determinant for GlyR trafficking and functionality, whereas alterations to loop B alter agonist potencies, indicating that residues here are critical elements in ligand binding. PMID:25568133

  7. The Intracellular Loop of the Glycine Receptor: It's not all about the Size.

    PubMed

    Langlhofer, Georg; Villmann, Carmen

    2016-01-01

    The family of Cys-loop receptors (CLRs) shares a high degree of homology and sequence identity. The overall structural elements are highly conserved with a large extracellular domain (ECD) harboring an α-helix and 10 β-sheets. Following the ECD, four transmembrane domains (TMD) are connected by intracellular and extracellular loop structures. Except the TM3-4 loop, their length comprises 7-14 residues. The TM3-4 loop forms the largest part of the intracellular domain (ICD) and exhibits the most variable region between all CLRs. The ICD is defined by the TM3-4 loop together with the TM1-2 loop preceding the ion channel pore. During the last decade, crystallization approaches were successful for some members of the CLR family. To allow crystallization, the intracellular loop was in most structures replaced by a short linker present in prokaryotic CLRs. Therefore, no structural information about the large TM3-4 loop of CLRs including the glycine receptors (GlyRs) is available except for some basic stretches close to TM3 and TM4. The intracellular loop has been intensively studied with regard to functional aspects including desensitization, modulation of channel physiology by pharmacological substances, posttranslational modifications, and motifs important for trafficking. Furthermore, the ICD interacts with scaffold proteins enabling inhibitory synapse formation. This review focuses on attempts to define structural and functional elements within the ICD of GlyRs discussed with the background of protein-protein interactions and functional channel formation in the absence of the TM3-4 loop. PMID:27330534

  8. The Intracellular Loop of the Glycine Receptor: It’s not all about the Size

    PubMed Central

    Langlhofer, Georg; Villmann, Carmen

    2016-01-01

    The family of Cys-loop receptors (CLRs) shares a high degree of homology and sequence identity. The overall structural elements are highly conserved with a large extracellular domain (ECD) harboring an α-helix and 10 β-sheets. Following the ECD, four transmembrane domains (TMD) are connected by intracellular and extracellular loop structures. Except the TM3–4 loop, their length comprises 7–14 residues. The TM3–4 loop forms the largest part of the intracellular domain (ICD) and exhibits the most variable region between all CLRs. The ICD is defined by the TM3–4 loop together with the TM1–2 loop preceding the ion channel pore. During the last decade, crystallization approaches were successful for some members of the CLR family. To allow crystallization, the intracellular loop was in most structures replaced by a short linker present in prokaryotic CLRs. Therefore, no structural information about the large TM3–4 loop of CLRs including the glycine receptors (GlyRs) is available except for some basic stretches close to TM3 and TM4. The intracellular loop has been intensively studied with regard to functional aspects including desensitization, modulation of channel physiology by pharmacological substances, posttranslational modifications, and motifs important for trafficking. Furthermore, the ICD interacts with scaffold proteins enabling inhibitory synapse formation. This review focuses on attempts to define structural and functional elements within the ICD of GlyRs discussed with the background of protein-protein interactions and functional channel formation in the absence of the TM3–4 loop. PMID:27330534

  9. Computer Simulator of Glycine Receptor Activity: A New Window into a Virtual World.

    PubMed

    Boronovsky, S E; Nartsissov, Y R

    2016-07-01

    Our study reports computer software that simulates the work of a single glycine receptor (GlyR). GlyRs have been found in various types of tissues, but their most important role seems to be in neurons, where they hyperpolarise membranes by opening chloride transmembrane channels. The software is based on a combination of two blocks. One block describes the Brownian dynamics of charged particle motion in a dielectric medium, and the other block determines the probability and timing of receptor activation. Using this software, the voltage-current dependencies and time curves of the transmembrane current were obtained. The mean value of the simulated anion current (4.5 ± 0.3 pA) is in good agreement with measured values under identical conditions ([Formula: see text] pA). It was shown that there is a condition under which the GlyR anion channel remains active despite a negligible chloride gradient. Virtual experiments allow evaluation of the value of half maximal effective concentration (EC[Formula: see text]) of the GlyR ([Formula: see text] [Formula: see text]M) and confirm that this receptor activates according to a mechanism involving three ligand binding sites. The advantage of the model is the ability to adjust parameters to the precise demands of experimental researchers. Moreover, the introduced algorithm has low computational power demands; therefore, it can be used as a research tool for assistance with structural experiments and applied aspects of neurophysiology. PMID:27412156

  10. Disturbances of Ligand Potency and Enhanced Degradation of the Human Glycine Receptor at Affected Positions G160 and T162 Originally Identified in Patients Suffering from Hyperekplexia

    PubMed Central

    Atak, Sinem; Langlhofer, Georg; Schaefer, Natascha; Kessler, Denise; Meiselbach, Heike; Delto, Carolyn; Schindelin, Hermann; Villmann, Carmen

    2015-01-01

    Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GlyR) undergoes direct interaction with the incoming ligand via a cation-π interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GlyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GlyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, T162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof. PMID:26733802

  11. Multifunctional basic motif in the glycine receptor intracellular domain induces subunit-specific sorting.

    PubMed

    Melzer, Nima; Villmann, Carmen; Becker, Kristina; Harvey, Kirsten; Harvey, Robert J; Vogel, Nico; Kluck, Christoph J; Kneussel, Matthias; Becker, Cord-Michael

    2010-02-01

    The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated ion channel that mediates fast synaptic inhibition in the vertebrate central nervous system. As a member of the family of Cys-loop receptors, it assembles from five homologous subunits (GlyRalpha1-4 and -beta). Each subunit contains an extracellular ligand binding domain, four transmembrane domains (TM), and an intracellular domain, formed by the loop connecting TM3 and TM4 (TM3-4 loop). The TM3-4 loops of the subunits GlyRalpha1 and -alpha3 harbor a conserved basic motif, which is part of a potential nuclear localization signal. When tested for functionality by live cell imaging of green fluorescent protein and beta-galactosidase-tagged domain constructs, the TM3-4 loops of GlyRalpha1 and -alpha3, but not of GlyRalpha2 and -beta, exhibited nuclear sorting activity. Subunit specificity may be attributed to slight amino acid alterations in the basic motif. In yeast two-hybrid screening and GST pulldown assays, karyopherin alpha3 and alpha4 were found to interact with the TM3-4 loop, providing a molecular mechanism for the observed intracellular trafficking. These results indicate that the multifunctional basic motif of the TM3-4 loop is capable of mediating a karyopherin-dependent intracellular sorting of full-length GlyRs. PMID:19959465

  12. Modulation of inhibitory glycine receptors in cultured embryonic mouse hippocampal neurons by zinc, thiol containing redox agents and carnosine.

    PubMed

    Thio, L L; Zhang, H X

    2006-01-01

    Modulation of inhibitory glycine receptors by zinc (Zn(2+)) and endogenous redox agents such as glutathione may alter inhibition in the mammalian brain. Despite the abundance of Zn(2+) in the hippocampus and its ability to modulate glycine receptors, few studies have examined Zn(2+) modulation of hippocampal glycine receptors. Whether redox agents modulate hippocampal glycine receptors also remains unknown. This study examined Zn(2+) and redox modulation of glycine receptor-mediated currents in cultured embryonic mouse hippocampal neurons using whole-cell recordings. Zn(2+) concentrations below 10 microM potentiated currents elicited by low glycine, beta-alanine, and taurine concentrations by 300-400%. Zn(2+) concentrations above 300 microM produced nearly complete inhibition. Potentiating Zn(2+) concentrations shifted the dose-response curves for the three agonists to the left and decreased the Hill coefficient for glycine and beta-alanine but not taurine. Inhibiting Zn(2+) concentrations shifted the dose-response curves for glycine and beta-alanine to the right but reduced the maximum taurine response. Histidine residues may participate in potentiation because diethyl pyrocarbonate and pH 5.4 diminished Zn(2+) enhancement of glycine currents. pH 5.4 diminished Zn(2+) block of glycine currents, but diethyl pyrocarbonate did not. These findings indicate that separate sites mediate Zn(2+) potentiation and inhibition. The redox agents glutathione, dithiothreitol, tris(2-carboxyethyl)phosphine, and 5,5'-dithiobis(2-nitrobenzoic acid) did not alter glycine currents by a redox mechanism. However, glutathione and dithiothreitol interfered with the effects of Zn(2+) on glycine currents by chelating it. Carnosine had similar effects. Thus, Zn(2+) and thiol containing redox agents that chelate Zn(2+) modulate hippocampal glycine receptors with the mechanism of Zn(2+) modulation being agonist dependent. PMID:16515845

  13. Colocalization of synaptic GABA(C)-receptors with GABA (A)-receptors and glycine-receptors in the rodent central nervous system.

    PubMed

    Frazao, Renata; Nogueira, Maria Ines; Wässle, Heinz

    2007-10-01

    Fast inhibition in the nervous system is preferentially mediated by GABA- and glycine-receptors. Two types of ionotropic GABA-receptor, the GABA(A)-receptor and GABA(C)-receptor, have been identified; they have specific molecular compositions, different sensitivities to GABA, different kinetics, and distinct pharmacological profiles. We have studied, by immunocytochemistry, the synaptic localization of glycine-, GABA(A)-, and GABA(C)-receptors in rodent retina, spinal cord, midbrain, and brain-stem. Antibodies specific for the alpha1 subunit of the glycine-receptor, the gamma2 subunit of the GABA(A)-receptor, and the rho subunits of the GABA(C)-receptor have been applied. Using double-immunolabeling, we have determined whether these receptors are expressed at the same postsynaptic sites. In the retina, no such colocalization was observed. However, in the spinal cord, we found the colocalization of glycine-receptors with GABA(A)- or GABA(C)-receptors and the colocalization of GABA(A)- and GABA(C)-receptors in approximately 25% of the synapses. In the midbrain and brain-stem, GABA(A)- and GABA(C)-receptors were colocalized in 10%-15% of the postsynaptic sites. We discuss the possible expression of heteromeric (hybrid) receptors assembled from GABA(A)- and GABA(C)-receptor subunits. Our results suggest that GABA(A)- and GABA(C)-receptors are colocalized in a minority of synapses of the central nervous system. PMID:17610086

  14. Osmoregulation of vasopressin secretion via activation of neurohypophysial nerve terminals glycine receptors by glial taurine.

    PubMed

    Hussy, N; Brès, V; Rochette, M; Duvoid, A; Alonso, G; Dayanithi, G; Moos, F C

    2001-09-15

    Osmotic regulation of supraoptic nucleus (SON) neuron activity depends in part on activation of neuronal glycine receptors (GlyRs), most probably by taurine released from adjacent astrocytes. In the neurohypophysis in which the axons of SON neurons terminate, taurine is also concentrated in and osmo-dependently released by pituicytes, the specialized glial cells ensheathing nerve terminals. We now show that taurine release from isolated neurohypophyses is enhanced by hypo-osmotic and decreased by hyper-osmotic stimulation. The high osmosensitivity is shown by the significant increase on only 3.3% reduction in osmolarity. Inhibition of taurine release by 5-nitro-2-(3-phenylpropylamino)benzoic acid, niflumic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid suggests the involvement of volume-sensitive anion channels. On purified neurohypophysial nerve endings, activation of strychnine-sensitive GlyRs by taurine or glycine primarily inhibits the high K(+)-induced rise in [Ca(2+)](i) and subsequent release of vasopressin. Expression of GlyRs in vasopressin and oxytocin terminals is confirmed by immunohistochemistry. Their implication in the osmoregulation of neurohormone secretion was assessed on isolated whole neurohypophyses. A 6.6% hypo-osmotic stimulus reduces by half the depolarization-evoked vasopressin secretion, an inhibition totally prevented by strychnine. Most importantly, depletion of taurine by a taurine transport inhibitor also abolishes the osmo-dependent inhibition of vasopressin release. Therefore, in the neurohypophysis, an osmoregulatory system involving pituicytes, taurine, and GlyRs is operating to control Ca(2+) influx in and neurohormone release from nerve terminals. This elucidates the functional role of glial taurine in the neurohypophysis, reveals the expression of GlyRs on axon terminals, and further defines the role of glial cells in the regulation of neuroendocrine function. PMID:11549721

  15. Mechanism of anion permeation through channels gated by glycine and gamma-aminobutyric acid in mouse cultured spinal neurones.

    PubMed Central

    Bormann, J; Hamill, O P; Sakmann, B

    1987-01-01

    1. The ion-selective and ion transport properties of glycine receptor (GlyR) and gamma-aminobutyric acid receptor (GABAR) channels in the soma membrane of mouse spinal cord neurones were investigated using the whole-cell, cell-attached and outside-out patch versions of the patch-clamp technique. 2. Current-voltage (I-V) relations of transmitter-activated currents obtained from whole-cell measurements with 145 mM-Cl- intracellularly and extracellularly, showed outward rectification. In voltage-jump experiments, the instantaneous I-V relations were linear, and the steady-state I-V relations were rectifying outwardly indicating that the gating of GlyR and GABAR channels is voltage sensitive. 3. The reversal potential of whole-cell currents shifted 56 mV per tenfold change in internal Cl- activity indicating activation of Cl(-)-selective channels. The permeability ratio of K+ to Cl- (PK/PCl) was smaller than 0.05 for both channels. 4. The permeability sequence for large polyatomic anions was formate greater than bicarbonate greater than acetate greater than phosphate greater than propionate for GABAR channels; phosphate and propionate were not measurably permeant in GlyR channels. This indicates that open GlyR and GABAR channels have effective pore diameters of 5.2 and 5.6 A, respectively. The sequence of relative permeabilities for small anions was SCN- greater than I- greater than Br- greater than Cl- greater than F- for both channels. 5. GlyR and GABAR channels are multi-conductance-state channels. In cell-attached patches the single-channel slope conductances close to 0 mV membrane potential were 29, 18 and 10 pS for glycine, and 28, 17 and 10 pS for GABA-activated channels. The most frequently observed (main) conductance states were 29 and 17 pS for the GlyR and GABAR channel, respectively. 6. In outside-out patches with equal extracellular and intracellular concentrations of 145 mM-Cl-, the conductance states were 46, 30, 20 and 12 pS for GlyR channels and 44, 30

  16. Single channel kinetics of a glutamate receptor.

    PubMed Central

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

    1987-01-01

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

  17. Single Channel Kinetics of a Glutamate Receptor

    PubMed Central

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

    1986-01-01

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

  18. Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure

    PubMed Central

    Moraga-Cid, Gustavo; Sauguet, Ludovic; Huon, Christèle; Malherbe, Laurie; Girard-Blanc, Christine; Petres, Stéphane; Murail, Samuel; Taly, Antoine; Baaden, Marc; Delarue, Marc; Corringer, Pierre-Jean

    2015-01-01

    The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α1GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α1GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α1GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a pore-opening mechanism for the α1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor. PMID:25730860

  19. Molecular cloning and characterization of a glycine-like receptor gene from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

    PubMed Central

    Flores-Fernández, José Miguel; Gutiérrez-Ortega, Abel; Padilla-Camberos, Eduardo; Rosario-Cruz, Rodrigo; Hernández-Gutiérrez, Rodolfo; Martínez-Velázquez, Moisés

    2014-01-01

    The cattle tick Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR) gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus. PMID:25174962

  20. Molecular cloning and characterization of a glycine-like receptor gene from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

    PubMed

    Flores-Fernández, José Miguel; Gutiérrez-Ortega, Abel; Padilla-Camberos, Eduardo; Rosario-Cruz, Rodrigo; Hernández-Gutiérrez, Rodolfo; Martínez-Velázquez, Moisés

    2014-01-01

    The cattle tick Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR) gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus. PMID:25174962

  1. Conformational Transitions in the Glycine-Bound GluN1 NMDA Receptor LBD via Single-Molecule FRET

    PubMed Central

    Cooper, David R.; Dolino, Drew M.; Jaurich, Henriette; Shuang, Bo; Ramaswamy, Swarna; Nurik, Caitlin E.; Chen, Jixin; Jayaraman, Vasanthi; Landes, Christy F.

    2015-01-01

    The N-methyl-D-aspartate receptor (NMDAR) is a member of the glutamate receptor family of proteins and is responsible for excitatory transmission. Activation of the receptor is thought to be controlled by conformational changes in the ligand binding domain (LBD); however, glutamate receptor LBDs can occupy multiple conformations even in the activated form. This work probes equilibrium transitions among NMDAR LBD conformations by monitoring the distance across the glycine-bound LBD cleft using single-molecule Förster resonance energy transfer (smFRET). Recent improvements in photoprotection solutions allowed us to monitor transitions among the multiple conformations. Also, we applied a recently developed model-free algorithm called “step transition and state identification” to identify the number of states, their smFRET efficiencies, and their interstate kinetics. Reversible interstate conversions, corresponding to transitions among a wide range of cleft widths, were identified in the glycine-bound LBD, on much longer timescales compared to channel opening. These transitions were confirmed to be equilibrium in nature by shifting the distribution reversibly via denaturant. We found that the NMDAR LBD proceeds primarily from one adjacent smFRET state to the next under equilibrium conditions, consistent with a cleft-opening/closing mechanism. Overall, by analyzing the state-to-state transition dynamics and distributions, we achieve insight into specifics of long-lived LBD equilibrium structural dynamics, as well as obtain a more general description of equilibrium folding/unfolding in a conformationally dynamic protein. The relationship between such long-lived LBD dynamics and channel function in the full receptor remains an open and interesting question. PMID:26153703

  2. Conformational transitions in the glycine-bound GluN1 NMDA receptor LBD via single-molecule FRET.

    PubMed

    Cooper, David R; Dolino, Drew M; Jaurich, Henriette; Shuang, Bo; Ramaswamy, Swarna; Nurik, Caitlin E; Chen, Jixin; Jayaraman, Vasanthi; Landes, Christy F

    2015-07-01

    The N-methyl-D-aspartate receptor (NMDAR) is a member of the glutamate receptor family of proteins and is responsible for excitatory transmission. Activation of the receptor is thought to be controlled by conformational changes in the ligand binding domain (LBD); however, glutamate receptor LBDs can occupy multiple conformations even in the activated form. This work probes equilibrium transitions among NMDAR LBD conformations by monitoring the distance across the glycine-bound LBD cleft using single-molecule Förster resonance energy transfer (smFRET). Recent improvements in photoprotection solutions allowed us to monitor transitions among the multiple conformations. Also, we applied a recently developed model-free algorithm called "step transition and state identification" to identify the number of states, their smFRET efficiencies, and their interstate kinetics. Reversible interstate conversions, corresponding to transitions among a wide range of cleft widths, were identified in the glycine-bound LBD, on much longer timescales compared to channel opening. These transitions were confirmed to be equilibrium in nature by shifting the distribution reversibly via denaturant. We found that the NMDAR LBD proceeds primarily from one adjacent smFRET state to the next under equilibrium conditions, consistent with a cleft-opening/closing mechanism. Overall, by analyzing the state-to-state transition dynamics and distributions, we achieve insight into specifics of long-lived LBD equilibrium structural dynamics, as well as obtain a more general description of equilibrium folding/unfolding in a conformationally dynamic protein. The relationship between such long-lived LBD dynamics and channel function in the full receptor remains an open and interesting question. PMID:26153703

  3. Taurine release by astrocytes modulates osmosensitive glycine receptor tone and excitability in the adult supraoptic nucleus.

    PubMed

    Choe, Katrina Y; Olson, James E; Bourque, Charles W

    2012-09-01

    Cells can release the free amino acid taurine through volume-regulated anion channels (VRACs), and it has been hypothesized that taurine released from glial cells is capable of inhibiting action potential (AP) firing by activating neuronal glycine receptors (GlyRs) (Hussy et al., 1997). Although an inhibitory GlyR tone is widely observed in the brain, it remains unknown whether this specifically reflects gliotransmission because most neurons also express VRACs and other endogenous molecules can activate GlyRs. We found that VRACs are absent in neurons of the rat supraoptic nucleus (SON), suggesting that glial cells are the exclusive source of taurine in this nucleus. Application of strychnine to rat hypothalamic explants caused a depolarization of SON neurons associated with a decrease of chloride conductance and could excite these cells in the absence of fast synaptic transmission. This inhibitory GlyR tone was eliminated by pharmacological blockade of VRACs, by cellular taurine depletion, by metabolic inactivation of glia with fluorocitrate, and after retraction of astrocytic processes that intercalate neuronal somata and dendrites. Finally, GlyR tone varied inversely with extracellular fluid tonicity to mediate the osmotic control of AP firing by SON neurons. These findings establish taurine as a physiological gliotransmitter and show that gliotransmission is a spatially constrained process that can be modulated by the morphological rearrangement of astrocytes. PMID:22956842

  4. A GLRA1 null mutation in recessive hyperekplexia challenges the functional role of glycine receptors

    SciTech Connect

    Brune, W.; Saul, M.; Becker, C.M.

    1996-05-01

    Dominant missense mutations in the human glycine receptor (GlyR) {alpha}1 subunit gene (GLRA1) give rise to hereditary hyperekplexia. These mutations impair agonist affinities and change conductance states of expressed mutant channels, resulting in a partial loss of function. In a recessive case of hyperekplexia, we found a deletion of exons 1-6 of the GLRA1 gene. Born to consanguineous parents, the affected child is homozygous for this GLRA1{sup null} allele consistent with a complete loss of gene function. The child displayed exaggerated startle responses and pronounced head-retraction jerks reflecting a disinhibition of vestigial brain-stem reflexes. In contrast, proprio- and exteroceptive inhibition of muscle activity previously correlated to glycinergic mechanisms were not affected. This case demonstrates that, in contrast to the lethal effect of a null allele in the recessive mouse mutant oscillator (Glra1{sup spd-ot}), the loss of the GlyR {alpha}1 subunit is effectively compensated in man. 38 refs.

  5. A GLRA1 null mutation in recessive hyperekplexia challenges the functional role of glycine receptors.

    PubMed Central

    Brune, W.; Weber, R. G.; Saul, B.; von Knebel Doeberitz, M.; Grond-Ginsbach, C.; Kellerman, K.; Meinck, H. M.; Becker, C. M.

    1996-01-01

    Dominant missense mutations in the human glycine receptor (GlyR) alpha 1 subunit gene (GLRA1) give rise to hereditary hyperekplexia. These mutations impair agonist affinities and change conductance states of expressed mutant channels, resulting in a partial loss of function. In a recessive case of hyperekplexia, we found a deletion of exons 1-6 of the GLRA1 gene. Born to consanguineous parents, the affected child is homozygous for this GLRA1(null) allele consistent with a complete loss of gene function. The child displayed exaggerated startle responses and pronounced head-retraction jerks reflecting a disinhibition of vestigial brain-stem reflexes. In contrast, proprio- and exteroceptive inhibition of muscle activity previously correlated to glycinergic mechanisms were not affected. This case demonstrates that, in contrast to the lethal effect of a null allele in the recessive mouse mutant oscillator (Glra1 spd-ot), the loss of the GlyR alpha 1 subunit is effectively compensated in man. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8651283

  6. Modulation of Recombinant Human α1 Glycine Receptors by Mono- and Disaccharides: A Kinetic Study.

    PubMed

    Breitinger, Ulrike; Sticht, Heinrich; Breitinger, Hans-Georg

    2016-08-17

    Glycine receptors (GlyRs) mediate fast synaptic inhibition in spinal cord, brainstem, and higher brain centers. Recently, glucose was identified as a positive modulator of GlyR-mediated currents. Here, we investigated extent and kinetics of the positive modulation of recombinant human α1 glycine receptors by different mono- and disaccharides and sorbitol using patch-clamp recording techniques. Glucose and fructose augmented glycine-mediated whole-cell currents with an EC50 of 6-7 mM. At concentrations > 10 mM, the maximum of current enhancement was reached within ∼30 min. Kinetics of GlyR modulation resemble those of protein glycation. On-rates were <0.5 h for saturating concentrations of monosaccharides and ∼1.5 h for disaccharides. Off-rates were considerably slower (>24 h). Galactose, the C4-epimer of glucose, and the sugar alcohol sorbitol had no effect on GlyR currents. Recent cryoelectron microscopy structures were used to identify a potential binding site for saccharides near the ivermectin binding pocket with lysine 143 as possible attachment site. The GlyR mutant α1(K143A) was not potentiated by glucose, suggesting an involvement of this residue in glycine receptor modulation by saccharides. PMID:27227552

  7. Activation of Strychnine-Sensitive Glycine Receptors by Shilajit on Preoptic Hypothalamic Neurons of Juvenile Mice.

    PubMed

    Bhattarai, Janardhan Prasad; Cho, Dong Hyu; Han, Seong Kyu

    2016-02-29

    Shilajit, a mineral pitch, has been used in Ayurveda and Siddha system of medicine to treat many human ailments, and is reported to contain at least 85 minerals in ionic form. This study examined the possible mechanism of Shilajit action on preoptic hypothalamic neurons using juvenile mice. The hypothalamic neurons are the key regulator of many hormonal systems. In voltage clamp mode at a holding potential of -60 mV, and under a high chloride pipette solution, Shilajit induced dose-dependent inward current. Shilajit-induced inward currents were reproducible and persisted in the presence of 0.5 μM tetrodotoxin (TTX) suggesting a postsynaptic action of Shilajit on hypothalamic neurons. The currents induced by Shilajit were almost completely blocked by 2 μM strychnine (Stry), a glycine receptor antagonist. In addition, Shilajit-induced inward currents were partially blocked by bicuculline. Under a gramicidin-perforated patch clamp mode, Shilajit induced membrane depolarization on juvenile neurons. These results show that Shilajit affects hypothalamic neuronal activities by activating the Stry-sensitive glycine receptor with α₂/α₂β subunit. Taken together, these results suggest that Shilajit contains some ingredients with possible glycine mimetic activities and might influence hypothalamic neurophysiology through activation of Stry-sensitive glycine receptor-mediated responses on hypothalamic neurons postsynaptically. PMID:26875561

  8. Transient receptor potential-like channels mediate metabotropic glutamate receptor EPSCs in rat dopamine neurones.

    PubMed

    Bengtson, C Peter; Tozzi, Alessandro; Bernardi, Giorgio; Mercuri, Nicola B

    2004-03-01

    Transient receptor potential (TRP) channels form cationic channels activated by diverse factors including mechanical stimuli, changes in osmolarity, pH and temperature, as well as the exogenous irritant, capsaicin. Metabotropic glutamate receptors have also recently been linked to TRP channel activation in neurones of the substantia nigra, hippocampus and cerebellum, suggesting a novel role for such channels in synaptic communication via endogenous neurotransmitters. We tested this for dopamine neurones in rat brain slices by characterizing the current-voltage relationship and pharmacology of EPSCs mediated by group I metabotropic glutamate receptor subtype 1 (mGluR1). Slow inward currents (273 +/- 35 pA peak amplitude, 381 +/- 25 ms latency, holding potential (V(h)) =-73 mV) representing evoked mGluR1 EPSCs were isolated in the presence of antagonists of AMPA, NMDA, GABA(A), GABA(B), muscarinic and glycine receptors. CPCCOEt (100 microM), an mGluR1 antagonist, blocked the residual EPSC in all recordings. mGluR1-activated EPSCs reversed polarity near -10 mV, consistent with the involvement of a cationic channel. Extracellular application of the non-selective TRP channel blockers SKF 96365, flufenamic acid and ruthenium red caused reversible inhibition of mGluR1-activated EPSCs. These characteristics parallel those of mGluR1 activation with an agonist and indicate the involvement of a TRP-like channel in mGluR1-mediated EPSCs. PMID:14724196

  9. The N-terminal domains of both NR1 and NR2 subunits determine allosteric Zn2+ inhibition and glycine affinity of N-methyl-D-aspartate receptors.

    PubMed

    Madry, Christian; Mesic, Ivana; Betz, Heinrich; Laube, Bodo

    2007-12-01

    The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors (iGluRs) is a tetrameric protein composed of homologous NR1 and NR2 subunits, which require the binding of glycine and glutamate, respectively, for efficient channel gating. The extracellular N-terminal domains (NTDs) of iGluR subunits show sequence homology to the bacterial periplasmic leucine/isoleucine/valine binding protein (LIVBP) and have been implicated in iGluR assembly, trafficking, and function. Here, we investigated how deletion of the NR1- and NR2-NTDs affects the expression and function of NMDA receptors. Both proteolytic cleavage of the NR1-NTD from assembled NR1/NR2 receptors and coexpression of the NTD-deleted NR1 subunit with wild-type or NTD-deleted NR2 subunits resulted in agonist-gated channels that closely resembled wild-type receptors. This indicates that the NTDs of both NMDA receptor subunits are not essential for receptor assembly and function. However, deletion of either the NR1 or the NR2 NTD eliminated high-affinity, allosteric inhibition of agonist-induced currents by Zn2+ and ifenprodil, consistent with the idea that interdomain interactions between these domains are important for allosteric receptor modulation. Furthermore, by replacing the NR2A-NTD with the NR2B NTD, and vice versa, the different glycine affinities of NR1/NR2A and NR1/NR2B receptors were found to be determined by their respective NR2-NTDs. Together, these data show that the NTDs of both the NR1 and NR2 subunits determine allosteric inhibition and glycine potency but are not required for NMDA receptor assembly. PMID:17878266

  10. N-Arachidonyl glycine does not activate G protein-coupled receptor 18 signaling via canonical pathways.

    PubMed

    Lu, Van B; Puhl, Henry L; Ikeda, Stephen R

    2013-01-01

    Recent studies propose that N-arachidonyl glycine (NAGly), a carboxylic analogue of anandamide, is an endogenous ligand of the Gα(i/o) protein-coupled receptor 18 (GPR18). However, a high-throughput β-arrestin-based screen failed to detect activation of GPR18 by NAGly (Yin et al., 2009; JBC, 18:12328). To address this inconsistency, this study investigated GPR18 coupling in a native neuronal system with endogenous signaling pathways and effectors. GPR18 was heterologously expressed in rat sympathetic neurons, and the modulation of N-type (Ca(v)2.2) calcium channels was examined. Proper expression and trafficking of receptor were confirmed by the "rim-like" fluorescence of fluorescently tagged receptor and the positive staining of external hemagglutinin-tagged GPR18-expressing cells. Application of NAGly on GPR18-expressing neurons did not inhibit calcium currents but instead potentiated currents in a voltage-dependent manner, similar to what has previously been reported (Guo et al., 2008; J Neurophysiol, 100:1147). Other proposed agonists of GPR18, including anandamide and abnormal cannabidiol, also failed to induce inhibition of calcium currents. Mutants of GPR18, designed to constitutively activate receptors, did not tonically inhibit calcium currents, indicating a lack of GPR18 activation or coupling to endogenous G proteins. Other downstream effectors of Gα(i/o)-coupled receptors, G protein-coupled inwardly rectifying potassium channels and adenylate cyclase, were not modulated by GPR18 signaling. Furthermore, GPR18 did not couple to other G proteins tested: Gα(s), Gα(z), and Gα(15). These results suggest NAGly is not an agonist for GPR18 or that GPR18 signaling involves noncanonical pathways not examined in these studies. PMID:23104136

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

    PubMed

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

    1992-10-01

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

  12. Cellular Functions of Transient Receptor Potential channels

    PubMed Central

    Dadon, Daniela; Minke, Baruch

    2010-01-01

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

  13. Glycine and GABAA Ultra-Sensitive Ethanol Receptors as Novel Tools for Alcohol and Brain Research

    PubMed Central

    Naito, Anna; Muchhala, Karan H.; Asatryan, Liana; Trudell, James R.; Homanics, Gregg E.; Perkins, Daya I.; Alkana, Ronald L.

    2014-01-01

    A critical obstacle to developing effective medications to prevent and/or treat alcohol use disorders is the lack of specific knowledge regarding the plethora of molecular targets and mechanisms underlying alcohol (ethanol) action in the brain. To identify the role of individual receptor subunits in ethanol-induced behaviors, we developed a novel class of ultra-sensitive ethanol receptors (USERs) that allow activation of a single receptor subunit population sensitized to extremely low ethanol concentrations. USERs were created by mutating as few as four residues in the extracellular loop 2 region of glycine receptors (GlyRs) or γ-aminobutyric acid type A receptors (GABAARs), which are implicated in causing many behavioral effects linked to ethanol abuse. USERs, expressed in Xenopus oocytes and tested using two-electrode voltage clamp, demonstrated an increase in ethanol sensitivity of 100-fold over wild-type receptors by significantly decreasing the threshold and increasing the magnitude of ethanol response, without altering general receptor properties including sensitivity to the neurosteroid, allopregnanolone. These profound changes in ethanol sensitivity were observed across multiple subunits of GlyRs and GABAARs. Collectively, our studies set the stage for using USER technology in genetically engineered animals as a unique tool to increase understanding of the neurobiological basis of the behavioral effects of ethanol. PMID:25245406

  14. Functional glycine receptor maturation in the absence of glycinergic input in dopaminergic neurones of the rat substantia nigra

    PubMed Central

    Mangin, J M; Guyon, A; Eugène, D; Paupardin-Tritsch, D; Legendre, P

    2002-01-01

    The postnatal maturation pattern of glycine receptor channels (GlyRs) expressed by dopaminergic (DA) neurones of the rat substantia nigra pars compacta (SNc) was investigated using single-channel and whole-cell patch-clamp recordings in brain slices from rats aged 7–21 postnatal days (P). In neonatal rats (P7-P10), GlyRs exhibited a main conductance state of 100–110 pS with a mean open time of 16 ms. In juvenile rats (P19-P22), both the GlyR main conductance state (46-55 pS) and the mean open time (6.8 ms) were decreased. In neonatal rats, application of 30 μm picrotoxin, which is known to block homomeric GlyRs, strongly reduced glycine-evoked responses, while it was much less effective in juvenile rats. These results suggest that these GlyRs correspond functionally to α2 homomeric GlyRs in neonatal rats and α1/β heteromeric GlyRs in juvenile rats. A drastic but transient decrease in the glycine responsiveness of DA neurones occurred around P17 concomitant to the functional switch from the homomeric state to the heteromeric state. This age corresponds to a maturation phase for DA neurones. The application of 1 μm gabazine blocked spontaneous or evoked inhibitory synaptic current, while the addition of 1 μm strychnine had no effect, suggesting a lack of functional glycinergic synapses on DA neurones. Although it has been proposed that taurine is co-released with GABA at GABAergic synapses on DA neurones, in the present study the stimulation of GABAergic fibres failed to activate GlyRs. Blockade of taurine transporters and applications of high K+ and hyposmotic solutions were also unable to induce any strychnine-sensitive current. We conclude that functional maturation of GlyRs can occur in the absence of any detectable GlyR activation in DA neurones of the SNc. PMID:12154171

  15. Acid-sensitive ion channels and receptors.

    PubMed

    Holzer, Peter

    2009-01-01

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

  16. Acid-sensitive ion channels and receptors

    PubMed Central

    Holzer, Peter

    2015-01-01

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

  17. Propofol restores the function of "hyperekplexic" mutant glycine receptors in Xenopus oocytes and mice.

    PubMed

    O'Shea, Sean Michael; Becker, Lore; Weiher, Hans; Betz, Heinrich; Laube, Bodo

    2004-03-01

    Human hereditary hyperekplexia ("startle disease") is a neurological disorder characterized by exaggerated, convulsive movements in response to unexpected stimuli. Molecular genetic studies have shown that this disease is often caused by amino acid substitutions at arginine 271 to glutamine or leucine of the alpha1 subunit of the inhibitory glycine receptor (GlyR). When exogenously expressed in Xenopus oocytes, agonist responses of mutant alpha1(R271Q) and alpha1(R271L) GlyRs show higher EC50 values and lower maximal inducible responses (relative efficacies) compared with oocytes expressing wild-type alpha1 GlyR subunits. Here, we report that the maximal glycine-induced currents (I(max)) of mutant alpha1(R271Q) and alpha1(R271L) GlyRs were dramatically potentiated in the presence of the anesthetic propofol (PRO), whereas the I(max) of wild-type alpha(1) receptors was not affected. Quantitative analysis of the agonist responses of the isofunctionally substituted alpha1(R271K) mutant GlyR revealed that saturating concentrations of PRO decreased the EC50 values of both glycine and the partial agonist beta-alanine by >10-fold, with relative efficacies increasing by 4- and 16-fold, respectively. Transgenic (tg) mice carrying the alpha1(R271Q) mutation (tg271Q-300) have both spontaneous and induced tremor episodes that closely resemble the movements of startled hyperekplexic patients. After treatment with subanesthetic doses of PRO, the tg271Q-300 mutant mice showed temporary reflexive and locomotor improvements that made them indistinguishable from wild-type mice. Together, these results demonstrate that the functional and behavioral effects of hyperekplexia mutations can be effectively reversed by drugs that potentiate GlyR responses. PMID:14999083

  18. Cannabinoids suppress inflammatory and neuropathic pain by targeting α3 glycine receptors

    PubMed Central

    Xiong, Wei; Cui, Tanxing; Cheng, Kejun; Yang, Fei; Chen, Shao-Rui; Willenbring, Dan; Guan, Yun; Pan, Hui-Lin; Ren, Ke; Xu, Yan

    2012-01-01

    Certain types of nonpsychoactive cannabinoids can potentiate glycine receptors (GlyRs), an important target for nociceptive regulation at the spinal level. However, little is known about the potential and mechanism of glycinergic cannabinoids for chronic pain treatment. We report that systemic and intrathecal administration of cannabidiol (CBD), a major nonpsychoactive component of marijuana, and its modified derivatives significantly suppress chronic inflammatory and neuropathic pain without causing apparent analgesic tolerance in rodents. The cannabinoids significantly potentiate glycine currents in dorsal horn neurons in rat spinal cord slices. The analgesic potency of 11 structurally similar cannabinoids is positively correlated with cannabinoid potentiation of the α3 GlyRs. In contrast, the cannabinoid analgesia is neither correlated with their binding affinity for CB1 and CB2 receptors nor with their psychoactive side effects. NMR analysis reveals a direct interaction between CBD and S296 in the third transmembrane domain of purified α3 GlyR. The cannabinoid-induced analgesic effect is absent in mice lacking the α3 GlyRs. Our findings suggest that the α3 GlyRs mediate glycinergic cannabinoid-induced suppression of chronic pain. These cannabinoids may represent a novel class of therapeutic agents for the treatment of chronic pain and other diseases involving GlyR dysfunction. PMID:22585736

  19. Activation of glycine receptor phase-shifts the circadian rhythm in neuronal activity in the mouse suprachiasmatic nucleus

    PubMed Central

    Mordel, Jérôme; Karnas, Diana; Inyushkin, Alexey; Challet, Etienne; Pévet, Paul; Meissl, Hilmar

    2011-01-01

    Abstract In mammals, the master clock in the suprachiasmatic nucleus (SCN) of the hypothalamus is composed of numerous synchronized oscillating cells that drive daily behavioural and physiological processes. Several entrainment pathways, afferent inputs to the SCN with their neurotransmitter and neuromodulator systems, can reset the circadian system regularly and also modulate neuronal activity within the SCN. In the present study, we investigated the function of the inhibitory neurotransmitter glycine on neuronal activity in the mouse SCN and on resetting of the circadian clock. The effects of glycine on the electrical activity of SCN cells from C57Bl/6 mice were studied either by patch-clamp recordings from acute brain slices or by long-term recordings from organotypic brain slices using multi-microelectrode arrays (MEA). Voltage-clamp recordings confirmed the existence of glycine-induced, chloride-selective currents in SCN neurons. These currents were reversibly suppressed by strychnine, phenylbenzene ω-phosphono-α-amino acid (PMBA) or ginkgolide B, selective blockers of glycine receptors (GlyRs). Long-term recordings of the spontaneous activity of SCN neurons revealed that glycine application induces a phase advance during the subjective day and a phase delay during the early subjective night. Both effects were suppressed by strychnine or by PMBA. These results suggest that glycine is able to modulate circadian activity by acting directly on its specific receptors in SCN neurons. PMID:21486797

  20. Activation of glycine receptor phase-shifts the circadian rhythm in neuronal activity in the mouse suprachiasmatic nucleus.

    PubMed

    Mordel, Jérôme; Karnas, Diana; Inyushkin, Alexey; Challet, Etienne; Pévet, Paul; Meissl, Hilmar

    2011-05-01

    In mammals, the master clock in the suprachiasmatic nucleus (SCN) of the hypothalamus is composed of numerous synchronized oscillating cells that drive daily behavioural and physiological processes. Several entrainment pathways, afferent inputs to the SCN with their neurotransmitter and neuromodulator systems, can reset the circadian system regularly and also modulate neuronal activity within the SCN. In the present study, we investigated the function of the inhibitory neurotransmitter glycine on neuronal activity in the mouse SCN and on resetting of the circadian clock. The effects of glycine on the electrical activity of SCN cells from C57Bl/6 mice were studied either by patch-clamp recordings from acute brain slices or by long-term recordings from organotypic brain slices using multi-microelectrode arrays(MEA). Voltage-clamp recordings confirmed the existence of glycine-induced, chloride-selective currents in SCN neurons. These currents were reversibly suppressed by strychnine, phenylbenzeneω-phosphono-α-amino acid (PMBA) or ginkgolide B, selective blockers of glycine receptors(GlyRs). Long-term recordings of the spontaneous activity of SCN neurons revealed that glycine application induces a phase advance during the subjective day and a phase delay during the early subjective night. Both effects were suppressed by strychnine or by PMBA. These results suggest that glycine is able to modulate circadian activity by acting directly on its specific receptors in SCN neurons. PMID:21486797

  1. Augmentation of glycine receptor alpha3 currents suggests a mechanism for glucose-mediated analgesia.

    PubMed

    Breitinger, Ulrike; Breitinger, Hans-Georg

    2016-01-26

    The inhibitory glycine receptor (GlyR) mediates rapid synaptic inhibition in the mammalian central nervous system. Recently, glucose was identified as a positive modulator of α1 GlyRs. Here, recombinant human α3GlyRs with and without glucose treatment were studied using patch clamp methods. Similar to α1GlyRs, receptor variants α3L and α3K were potentiated by sugar. Glucose treatment reduced EC50 values of GlyR α3L and α3K by a factor of 4.5 and 3.3, respectively, without affecting maximum currents or desensitization. The high-activity mutant α3L(P185L) was not further potentiated by glucose. Potentiation of glycinergic signalling may underlie some of the analgetic effects of glucose. PMID:26656729

  2. Kinetics of unliganded acetylcholine receptor channel gating.

    PubMed Central

    Jackson, M B

    1986-01-01

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

  3. Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors.

    PubMed

    Guo, Xiaoli; Chronis, Demosthenis; De La Torre, Carola M; Smeda, John; Wang, Xiaohong; Mitchum, Melissa G

    2015-08-01

    CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE-receptor kinase-WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant-parasitic cyst nematodes secrete CLE-like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode-induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock-down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants. PMID:25581705

  4. Isovaleric, methylmalonic, and propionic acid decrease anesthetic EC50 in tadpoles, modulate glycine receptor function, and interact with the lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

    PubMed Central

    Weng, Yun; Hsu, Tienyi Theresa; Zhao, Jing; Nishimura, Stefanie; Fuller, Gerald G.; Sonner, James M.

    2010-01-01

    Introduction Elevated concentrations of isovaleric, methylmalonic, and propionic acid are associated with impaired consciousness in genetic diseases (organic acidemias). We conjectured that part of the central nervous system depression observed in these disorders was due to anesthetic effects of these metabolites. We tested three hypotheses. First, that these metabolites would have anesthetic-sparing effects, possibly being anesthetics by themselves. Second, that these compounds would modulate glycine and GABAA receptor function, increasing chloride currents through these channels as potent clinical inhaled anesthetics do. Third, that these compounds would affect physical properties of lipids. Methods Anesthetic EC50’s were measured in Xenopus laevis tadpoles. Glycine and GABAA receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. Pressure-area isotherms of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers were measured with and without added organic acids. Results Isovaleric acid was an anesthetic in tadpoles, while methylmalonic and propionic acid decreased isoflurane’s EC50 by half. All three organic acids concentration-dependently increased current through α1 glycine receptors. There were minimal effects on α1β2γ2s GABAA receptors. The organic acids increased total lateral pressure (surface pressure) of DPPC monolayers, including at mean molecular areas typical of bilayers. Conclusion Isovaleric, methylmalonic, and propionic acid have anesthetic affects in tadpoles, positively modulate glycine receptor fuction, and affect physical properties of DPPC monolayers. PMID:19372333

  5. The N-methyl D-aspartate receptor glycine site and D-serine metabolism: an evolutionary perspective.

    PubMed Central

    Schell, Michael J

    2004-01-01

    The N-methyl D-aspartate (NMDA) type of glutamate receptor requires two distinct agonists to operate. Glycine is assumed to be the endogenous ligand for the NMDA receptor glycine site, but this notion has been challenged by the discovery of high levels of endogenous d-serine in the mammalian forebrain. I have outlined an evolutionary framework for the appearance of a glycine site in animals and the metabolic events leading to high levels of D-serine in brain. Sequence alignments of the glycine-binding regions, along with the scant experimental data available, suggest that the properties of invertebrate NMDA receptor glycine sites are probably different from those in vertebrates. The synthesis of D-serine in brain is due to a pyridoxal-5'-phosphate (B(6))-requiring serine racemase in glia. Although it remains unknown when serine racemase first evolved, data concerning the evolution of B(6) enzymes, along with the known occurrences of serine racemases in animals, point to D-serine synthesis arising around the divergence time of arthropods. D-Serine catabolism occurs via the ancient peroxisomal enzyme d-amino acid oxidase (DAO), whose ontogenetic expression in the hindbrain of mammals is delayed until the postnatal period and absent from the forebrain. The phylogeny of D-serine metabolism has relevance to our understanding of brain ontogeny, schizophrenia and neurotransmitter dynamics. PMID:15306409

  6. 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

  7. Glycine receptor mutants of the mouse: what are possible routes of inhibitory compensation?

    PubMed Central

    Schaefer, Natascha; Vogel, Nicolas; Villmann, Carmen

    2012-01-01

    Defects in glycinergic inhibition result in a complex neuromotor disorder in humans known as hyperekplexia (OMIM 149400) with similar phenotypes in rodents characterized by an exaggerated startle reflex and hypertonia. Analogous to genetic defects in humans single point mutations, microdeletions, or insertions in the Glra1 gene but also in the Glrb gene underlie the pathology in mice. The mutations either localized in the α (spasmodic, oscillator, cincinnati, Nmf11) or the β (spastic) subunit of the glycine receptor (GlyR) are much less tolerated in mice than in humans, leaving the question for the existence of different regulatory elements of the pathomechanisms in humans and rodents. In addition to the spontaneous mutations, new insights into understanding of the regulatory pathways in hyperekplexia or glycine encephalopathy arose from the constantly increasing number of knock-out as well as knock-in mutants of GlyRs. Over the last five years, various efforts using in vivo whole cell recordings provided a detailed analysis of the kinetic parameters underlying glycinergic dysfunction. Presynaptic compensation as well as postsynaptic compensatory mechanisms in these mice by other GlyR subunits or GABAA receptors, and the role of extra-synaptic GlyRs is still a matter of debate. A recent study on the mouse mutant oscillator displayed a novel aspect for compensation of functionality by complementation of receptor domains that fold independently. This review focuses on defects in glycinergic neurotransmission in mice discussed with the background of human hyperekplexia en route to strategies of compensation. PMID:23118727

  8. Spontaneous openings of the acetylcholine receptor channel.

    PubMed Central

    Jackson, M B

    1984-01-01

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

  9. Insulin-like growth factor-1 inhibits adult supraoptic neurons via complementary modulation of mechanoreceptors and glycine receptors.

    PubMed

    Ster, Jeanne; Colomer, Claude; Monzo, Cécile; Duvoid-Guillou, Anne; Moos, Françoise; Alonso, Gérard; Hussy, Nicolas

    2005-03-01

    In the CNS, insulin-like growth factor-1 (IGF-1) is mainly known for its trophic effect both during development and in adulthood. Here, we show than in adult rat supraoptic nucleus (SON), IGF-1 receptor immunoreactivity is present in neurons, whereas IGF-1 immunoreactivity is found principally in astrocytes and more moderately in neurons. In vivo application of IGF-1 within the SON acutely inhibits the activity of both vasopressin and oxytocin neurons, the two populations of SON neuroendocrine cells. Recordings of acutely isolated SON neurons showed that this inhibition occurs through two rapid and reversible mechanisms, both involving the neuronal IGF-1 receptor but different intracellular messengers. IGF-1 inhibits Gd3+-sensitive and osmosensitive mechanoreceptor cation current via phosphatidylinositol-3 (PI3) kinase activation. IGF-1 also potentiates taurine-activated glycine receptor (GlyR) Cl- currents by increasing the agonist sensitivity through a extremely rapid (within a second) PI3 kinase-independent mechanism. Both mechanoreceptor channels and GlyR, which form the excitatory and inhibitory components of SON neuron osmosensitivity, are active at rest, and their respective inhibition and potentiation will both be inhibitory, leading to strong decrease in neuronal activity. It will be of interest to determine whether IGF-1 is released by neurons, thus participating in an inhibitory autocontrol, or astrocytes, then joining the growing family of glia-to-neuron transmitters that modulate neuronal and synaptic activity. Through the opposite and complementary acute regulation of mechanoreceptors and GlyR, IGF-1 appears as a new important neuromodulator in the adult CNS, participating in the complex integration of neural messages that regulates the level of neuronal excitability. PMID:15745952

  10. A NMDA receptor glycine site partial agonist, GLYX-13, that simultaneously enhances LTP and reduces LTD at Schaffer collateral-CA1 synapses in hippocampus

    PubMed Central

    Zhang, Xiao-lei; Sullivan, John A.; Moskal, Joseph R.; Stanton, Patric K.

    2008-01-01

    N-methyl-D-aspartate glutamate receptors (NMDAR) are a key route for Ca2+ influx into neurons important to both activity-dependent synaptic plasticity and, when uncontrolled, triggering events that cause neuronal degeneration and death. Among regulatory binding sites on the NMDAR complex is a glycine binding site, distinct from the glutamate binding site, which must be co-activated for NMDAR channel opening. We developed a novel glycine site partial agonist, GLYX-13, which is both nootropic and neuroprotective in vivo. Here, we assessed the effects of GLYX-13 on long-term synaptic plasticity and NMDAR transmission at Schaffer collateral-CA1 synapses in hippocampal slices in vitro. GLYX-13 simultaneously enhanced the magnitude of long-term potentiation (LTP) of synaptic transmission, while reducing long-term depression (LTD). GLYX-13 reduced NMDA receptor-mediated synaptic currents in CA1 pyramidal neurons evoked by low-frequency Schaffer collateral stimulation, but enhanced NMDAR currents during high-frequency bursts of activity, and these actions were occluded by a saturating concentration of the glycine site agonist D-serine. Direct two-photon imaging of Schaffer collateral burst-evoked increases in [Ca2+] in individual dendritic spines revealed that GLYX-13 selectively enhanced burst-induced NMDAR-dependent spine Ca2+ influx. Examining the rate of MK-801 block of synaptic versus extrasynaptic NMDAR-gated channels revealed that GLYX-13 selectively enhanced activation of burst-driven extrasynaptic NMDARs, with an action that was blocked by the NR2B-selective NMDAR antagonist ifenprodil. Our data suggest that GLYX-13 may have unique therapeutic potential as a learning and memory enhancer because of its ability to simultaneously enhance LTP and suppress LTD. PMID:18796308

  11. Generation of Functional Inhibitory Synapses Incorporating Defined Combinations of GABA(A) or Glycine Receptor Subunits

    PubMed Central

    Dixon, Christine L.; Zhang, Yan; Lynch, Joseph W.

    2015-01-01

    Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR) and glycine receptor (GlyR) isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of individual isoforms under synaptic stimulation conditions in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2–5 weeks. PMID:26778954

  12. Isolation and characterization of the brassinosteroid receptor gene (GmBRI1) from Glycine max.

    PubMed

    Wang, Miao; Sun, Shi; Wu, Cunxiang; Han, Tianfu; Wang, Qingyu

    2014-01-01

    Brassinosteroids (BRs) constitute a group of steroidal phytohormones that contribute to a wide range of plant growth and development functions. The genetic modulation of BR receptor genes, which play major roles in the BR signaling pathway, can create semi-dwarf plants that have great advantages in crop production. In this study, a brassinosteroid insensitive gene homologous with AtBRI1 and other BRIs was isolated from Glycine max and designated as GmBRI1. A bioinformatic analysis revealed that GmBRI1 shares a conserved kinase domain and 25 tandem leucine-rich repeats (LRRs) that are characteristic of a BR receptor for BR reception and reaction and bear a striking similarity in protein tertiary structure to AtBRI1. GmBRI1 transcripts were more abundant in soybean hypocotyls and could be upregulated in response to exogenous BR treatment. The transformation of GmBRI1 into the Arabidopsis dwarf mutant bri1-5 restored the phenotype, especially regarding pod size and plant height. Additionally, this complementation is a consequence of a restored BR signaling pathway demonstrated in the light/dark analysis, root inhibition assay and BR-response gene expression. Therefore, GmBRI1 functions as a BR receptor to alter BR-mediated signaling and is valuable for improving plant architecture and enhancing the yield of soybean. PMID:24599079

  13. Pulsed Electron Spin Resonance Resolves the Coordination Site of Cu2+ Ions in α1-Glycine Receptor

    PubMed Central

    Ruthstein, Sharon; Stone, Katherine M.; Cunningham, Timothy F.; Ji, Ming; Cascio, Michael; Saxena, Sunil

    2010-01-01

    Herein, we identify the coordination environment of Cu2+ in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn2+ and Cu2+ significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo. Zn2+ can either potentiate or inhibit GlyR activity depending on its concentration, while Cu2+ is inhibitory. To better understand the molecular basis of the inhibitory effect we have used electron spin resonance to directly examine Cu2+ coordination and stoichiometry. We show that Cu2+ has one binding site per α1 subunit, and that five Cu2+ can be coordinated per GlyR. Cu2+ binds to E192 and H215 in each subunit of GlyR with a 40 μM apparent dissociation constant, consistent with earlier functional measurements. However, the coordination site does not include several residues of the agonist/antagonist binding site that were previously suggested to have roles in Cu2+ coordination by functional measurements. Intriguingly, the E192/H215 site has been proposed as the potentiating Zn2+ site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR. PMID:20959090

  14. Mutations M287L and Q266I in the Glycine Receptor α1 Subunit Change Sensitivity to Volatile Anesthetics in Oocytes and Neurons, but Not the Minimal Alveolar Concentration in Knockin Mice

    PubMed Central

    Borghese, Cecilia M.; Xiong, Wei; Oh, S. Irene; Ho, Angel; Mihic, S. John; Zhang, Li; Lovinger, David M.; Homanics, Gregg E.; Eger, Edmond I; Harris, R. Adron

    2012-01-01

    Background Volatile anesthetics (VAs) alter the function of key central nervous system proteins but it is not clear which, if any, of these targets mediates the immobility produced by VAs in the face of noxious stimulation. A leading candidate is the glycine receptor, a ligand-gated ion channel important for spinal physiology. VAs variously enhance such function, and blockade of spinal GlyRs with strychnine affects the minimal alveolar concentration (an anesthetic EC50) in proportion to the degree of enhancement. Methods We produced single amino acid mutations into the glycine receptorα1 subunit that increased (M287L, third transmembrane region) or decreased (Q266I, second transmembrane region) sensitivity to isoflurane in recombinant receptors, and introduced such receptors into mice. The resulting knockin mice presented impaired glycinergic transmission, but heterozygous animals survived to adulthood, and we determined the effect of isoflurane on glycine-evoked responses of brain stem neurons from the knockin mice, and the minimal alveolar concentration for isoflurane and other VAs in the immature and mature knockin mice. Results Studies of glycine-evoked currents in brain stem neurons from knock-in mice confirmed the changes seen with recombinant receptors. No increases in the minimal alveolar concentration were found in knockin mice, but the minimal alveolar concentration for isoflurane and enflurane (but not halothane) decreased in 2-week-old Q266I mice. This change is opposite to the one expected for a mutation that decreases the sensitivity to volatile anesthetics. Conclusion Taken together, these results indicate that glycine receptors containing the α1 subunit are not likely to be crucial for the action of isoflurane and other VAs. PMID:22885675

  15. Cochlear nucleus neurons redistribute synaptic AMPA and glycine receptors in response to monaural conductive hearing loss

    PubMed Central

    Whiting, Brittany; Moiseff, Andrew; Rubio, María E.

    2009-01-01

    Neurons restore their function in response to external or internal perturbations and maintain neuronal or network stability through a homeostatic scaling mechanism. Homeostatic responses at synapses along the auditory system would be important for adaptation to normal and abnormal fluctuations in the sensory environment. We investigated at the electron microscopic level and after postembedding immunogold labeling whether projection neurons in the cochlear nucleus responded to modifications of auditory nerve activity. After unilaterally reducing the level of auditory inputs by ~ 20 dB by monaural earplugging, auditory nerve synapses on bushy cells somata and basal dendrites of fusiform cells of the ventral and dorsal cochlear nucleus, respectively, upregulated GluR3 AMPA receptor subunit, while inhibitory synapses decreased the expression of GlyRα1 subunit. These changes in expression levels were fully reversible once the earplug was removed, indicating that activity affects the trafficking of receptors at synapses. Excitatory synapses on apical dendrites of fusiform cells (parallel fibers) with different synaptic AMPA receptor subunit composition, were not affected by sound attenuation, as the expression levels of AMPA receptor subunits were the same as in normal hearing littermates. GlyRα1 subunit expression at inhibitory synapses on apical dendrites of fusiform cells was also found unaffected. Furthermore, fusiform and bushy cells of the contralateral side to the earplugging upregulated the GluR3 subunit at auditory nerve synapses. These results show that cochlear nucleus neurons innervated by the auditory nerve, are able to respond to small changes in sound levels by redistributing specific AMPA and glycine receptor subunits. PMID:19646510

  16. GABA and glycine in the developing brain.

    PubMed

    Ito, Susumu

    2016-09-01

    GABA and glycine are major inhibitory neurotransmitters in the CNS and act on receptors coupled to chloride channels. During early developmental periods, both GABA and glycine depolarize membrane potentials due to the relatively high intracellular Cl(-) concentration. Therefore, they can act as excitatory neurotransmitters. GABA and glycine are involved in spontaneous neural network activities in the immature CNS such as giant depolarizing potentials (GDPs) in neonatal hippocampal neurons, which are generated by the synchronous activity of GABAergic interneurons and glutamatergic principal neurons. GDPs and GDP-like activities in the developing brains are thought to be important for the activity-dependent functiogenesis through Ca(2+) influx and/or other intracellular signaling pathways activated by depolarization or stimulation of metabotropic receptors. However, if GABA and glycine do not shift from excitatory to inhibitory neurotransmitters at the birth and in maturation, it may result in neural disorders including autism spectrum disorders. PMID:26951057

  17. Reduction of spinal glycine receptor-mediated miniature inhibitory postsynaptic currents in streptozotocin-induced diabetic neuropathic pain.

    PubMed

    Chiu, Yu-Chi; Liao, Wen-Tzu; Liu, Chia-Kai; Wu, Chih-Hsien; Lin, Chung-Ren

    2016-01-12

    Diabetic neuropathic pain (DNP) is a common clinical problem, and the mechanisms underlying the onset and progression of this complication are poorly understood. The present study examined the glycine receptors (GlyR) in the control of synaptic input to dorsal horn neurons in diabetes. Male Sprague-Dawley rats with or without streptozotocin (STZ) intraperitoneal injections were used. Tactile sensitivities were assessed by measuring paw withdrawal thresholds to von Frey filaments for four weeks. The extent of GlyR-mediated inhibition controlling primary afferent-evoked excitation in dorsal horn neurons was examined by using the whole cell patch clamp recording technique in isolated adult rat spinal cord slices. The content of the spinal dorsal horn glycine levels was measured by microdialysis. An intrathecal glycine agonist injection was used to test whether mimicking endogenous glycine-receptor-mediated inhibition reduces DNP. We found that persistent hyperglycemia induced by the administration of STZ caused a decrease in the paw withdrawal latency to mechanical stimuli. The miniature inhibitory post-synaptic current (mIPSC) rise, decay kinetics and mean GlyR-mediated mIPSC amplitude were not affected in DNP. The mean frequency of GlyR-mediated mIPSC of lamina I neurons from DNP rats was, however, significantly reduced when compared with neurons from control rats. Principal passive and active membrane properties and the firing patterns of spinal lamina I neurons were not changed in DNP rats. Spinal microdialysis rats had a significantly decreased glycine level following its initial elevation. The intrathecal administration of glycine diminished tactile pain hypersensitivity in DNP rats. In conclusion, these results indicate that long-lasting hyperglycemia induced by STZ injections leads to a reduced glycinergic inhibitory control of spinal lamina I neurons through a presynaptic mechanism. PMID:26598022

  18. Conserved Glycine Residues in the Cytoplasmic Domain of the Aspartate Receptor Play Essential Roles in Kinase Coupling and On–Off Switching†

    PubMed Central

    Coleman, Matthew D.; Bass, Randal B.; Mehan, Ryan S.; Falke, Joseph J.

    2010-01-01

    The aspartate receptor of the bacterial chemotaxis pathway serves as a scaffold for the formation of a multiprotein signaling complex containing the receptor and the cytoplasmic pathway components. Within this complex, the receptor regulates the autophosphorylation activity of histidine kinase CheA, thereby controlling the signals sent to the flagellar motor and the receptor adaptation system. The receptor cytoplasmic domain, which controls the on–off switching of CheA, possesses 14 glycine residues that are highly conserved in related receptors. In principle, these conserved glycines could be required for static turns, bends, or close packing in the cytoplasmic domain, or they could be required for conformational dynamics during receptor on–off switching. To determine which glycines are essential and to probe their functional roles, we have substituted each conserved glycine with both alanine and cysteine, and then measured the effects on receptor function in vivo and in vitro. The results reveal a subset of six glycines which are required for receptor function during cellular chemotaxis. Two of these essential glycines (G388 and G391) are located at a hairpin turn at the distal end of the folded cytoplasmic domain, where they are required for the tertiary fold of the signaling subdomain and for CheA kinase activation. Three other essential glycines (G338, G339, and G437) are located at the border between the adaptation and signaling subdomains, where they play key roles in CheA kinase activation and on–off switching. These three glycines form a ring around the four-helix bundle that comprises the receptor cytoplasmic domain, yielding a novel architectural feature termed a bundle hinge. The final essential glycine (G455) is located in the adaptation subdomain where it is required for on–off switching. Overall, the findings confirm that six of the 14 conserved cytoplasmic glycines are essential for receptor function because they enable helix turns and bends

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

    PubMed Central

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

    2007-01-01

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

  20. Early expression of glycine and GABA(A) receptors in developing spinal cord neurons. Effects on neurite outgrowth.

    PubMed

    Tapia, J C; Mentis, G Z; Navarrete, R; Nualart, F; Figueroa, E; Sánchez, A; Aguayo, L G

    2001-01-01

    Using fluorometric and immunocytochemical techniques, we found that high glycine concentrations or blockade of glycine receptors increases neurite outgrowth in developing mouse spinal cord neurons. Glycine- and GABA(A)-activated currents were demonstrated during applications of glycine and GABA (50-100 microM) in 5 days in vitro (DIV) neurons. Long application (> or =10 min) of 100 microM glycine desensitized the membrane response by more than 95%. Application of glutamate in the absence of external Mg(2+), at several membrane potentials, did not produce any detectable membrane response in these cells. Immunocytochemical studies with NR1 and GluR1 antibodies showed a delayed appearance of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors respectively. Spontaneous synaptic activity was readily observed in 5 DIV neurons. The use of various receptor antagonists (strychnine, bicuculline, DL-2-amino-5-phosphonovalerate [APV], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) revealed that this activity was predominantly glycinergic, and to a smaller extent, GABAergic. In the presence of bicuculline, APV and CNQX, we detected abundant spontaneous depolarizing potentials which often reached the action potential threshold. Further evidence for functional synaptic activity was provided by the detection of co-localization of gephyrin and synaptophysin at 5 DIV using confocal microscopy. Fluorometric studies with Fluo-3, a Ca(2+) indicator, in 5 DIV cultures showed the presence of spontaneous fluctuations associated with tetrodotoxin-sensitive synaptic events. The number of neurons displaying these fluctuations was significantly increased (>100%) when the cells were bathed in a strychnine-containing solution. On the other hand, these synaptically mediated Ca(2+) events were blocked by the co-application of strychnine and bicuculline. This suggests that glycine and GABA(A) receptors provide a fundamental regulation of both

  1. The 4′lysine in the putative channel lining domain affects desensitization but not the single-channel conductance of recombinant homomeric 5-HT3A receptors

    PubMed Central

    Gunthorpe, Martin J; Peters, John A; Gill, Catherine H; Lambert, Jeremy J; Lummis, Sarah C R

    2000-01-01

    The 5-HT3 receptor is a transmitter-gated ion channel of the Cys-loop superfamily. Uniquely, 5-HT3 receptor subunits (5-HT3A and 5-HT3B) possess a positively charged lysine residue within the putative channel lining M2 domain (4′ position). Using whole cell recording techniques, we examined the role of this residue in receptor function using wild-type (WT) and mutant 5-HT3A receptor subunits of murine origin transiently expressed in human embryonic kidney (HEK 293) cells. WT 5-HT3A receptors mediated rapidly activating currents in response to 5-HT (10–90 % rise time, 103 ms; EC50, 2.34 μm; Hill coefficient, nH, 2.87). The currents rectified inwardly, reversed in sign at a potential of −9 mV and desensitized in the continuous presence of agonist (half-time of desensitization, t1/2, 2.13 s). 5-HT3A receptor subunits in which the 4′lysine was mutated to arginine, glutamine, serine or glycine formed functional receptors. 5-HT EC50 values were approximately 2-fold lower than for WT 5-HT3A receptors, but Hill coefficients, kinetics of current activation, rectification, and reversal potentials were unaltered. Each of the mutants desensitized more slowly than the WT 5-HT3A receptor, with the arginine and glycine mutations exhibiting the greatest effect (5-fold reduction). The rank order of effect was arginine > glycine > serine > glutamine. The single-channel conductance of the WT 5-HT3A receptor, as assessed by fluctuation analysis of macroscopic currents, was 390 fS. A similar value was obtained for the 4′lysine mutant receptors. Thus it appears unlikely that 4′lysine is exposed to the channel lumen. Mutation of residues immediately adjacent to 4′lysine to glutamate or lysine resulted in lack of receptor expression or function. We conclude that 4′lysine does not form part of the channel lining, but may play an important role in 5-HT3 receptor desensitization. PMID:10639097

  2. A single glycine-alanine exchange directs ligand specificity of the elephant progestin receptor.

    PubMed

    Wierer, Michael; Schrey, Anna K; Kühne, Ronald; Ulbrich, Susanne E; Meyer, Heinrich H D

    2012-01-01

    The primary gestagen of elephants is 5α-dihydroprogesterone (DHP), which is unlike all other mammals studied until now. The level of DHP in elephants equals that of progesterone in other mammals, and elephants are able to bind DHP with similar affinity to progesterone indicating a unique ligand-binding specificity of the elephant progestin receptor (PR). Using site-directed mutagenesis in combination with in vitro binding studies we here report that this change in specificity is due to a single glycine to alanine exchange at position 722 (G722A) of PR, which specifically increases DHP affinity while not affecting binding of progesterone. By conducting molecular dynamics simulations comparing human and elephant PR ligand-binding domains (LBD), we observed that the alanine methyl group at position 722 is able to push the DHP A-ring into a position similar to progesterone. In the human PR, the DHP A-ring position is twisted towards helix 3 of PR thereby disturbing the hydrogen bond pattern around the C3-keto group, resulting in a lower binding affinity. Furthermore, we observed that the elephant PR ligand-binding pocket is more rigid than the human analogue, which probably explains the higher affinity towards both progesterone and DHP. Interestingly, the G722A substitution is not elephant-specific, rather it is also present in five independent lineages of mammalian evolution, suggesting a special role of the substitution for the development of distinct mammalian gestagen systems. PMID:23209719

  3. Glycine receptors control the generation of projection neurons in the developing cerebral cortex

    PubMed Central

    Avila, A; Vidal, P M; Tielens, S; Morelli, G; Laguesse, S; Harvey, R J; Rigo, J-M; Nguyen, L

    2014-01-01

    The development of the cerebral cortex requires coordinated regulation of proliferation, specification, migration and differentiation of cortical progenitors into functionally integrated neurons. The completion of the neurogenic program requires a dynamic interplay between cell intrinsic regulators and extrinsic cues, such as growth factor and neurotransmitters. We previously demonstrated a role for extrasynaptic glycine receptors (GlyRs) containing the α2 subunit in cerebral cortical neurogenesis, revealing that endogenous GlyR activation promotes interneuron migration in the developing cortical wall. The proliferative compartment of the cortex comprises apical progenitors that give birth to neurons directly or indirectly through the generation of basal progenitors, which serve as amplification step to generate the bulk of cortical neurons. The present work shows that genetic inactivation of Glra2, the gene coding the α2 subunit of GlyRs, disrupts dorsal cortical progenitor homeostasis with an impaired capability of apical progenitors to generate basal progenitors. This defect results in an overall reduction of projection neurons that settle in upper or deep layers of the cerebral cortex. Overall, the depletion of cortical neurons observed in Glra2-knockout embryos leads to moderate microcephaly in newborn Glra2-knockout mice. Taken together, our findings support a contribution of GlyR α2 to early processes in cerebral cortical neurogenesis that are required later for the proper development of cortical circuits. PMID:24926615

  4. Characteristics of glycine receptors expressed by embryonic rat brain mRNAs.

    PubMed

    García-Alcocer, G; García-Colunga, J; Martínez-Torres, A; Miledi, R

    2001-02-27

    A study was made of glycine (Gly) and gamma-aminobutyric acid (GABA) receptors expressed in Xenopus oocytes injected with rat mRNAs isolated from the encephalon, midbrain, and brainstem of 18-day-old rat embryos. In oocytes injected with encephalon, midbrain, or brainstem mRNAs, the Gly-current amplitudes (membrane current elicited by Gly; 1 mM Gly) were respectively 115 +/- 35, 346 +/- 28, and 389 +/- 22 nA, whereas the GABA-currents (1 mM GABA) were all < or =40 nA. Moreover, the Gly-currents desensitized faster in oocytes injected with encephalon or brainstem mRNAs. The EC(50) for Gly was 611 +/- 77 microM for encephalon, 661 +/- 28 microM for midbrain, and 506 +/- 18 microM for brainstem mRNA-injected oocytes, and the corresponding Hill coefficients were all approximately 2. Strychnine inhibited all of the Gly-currents, with an IC(50) of 56 +/- 3 nM for encephalon, 97 +/- 4 nM for midbrain, and 72 +/- 4 nM for brainstem mRNAs. During repetitive Gly applications, the Gly-currents were potentiated by 1.6-fold for encephalon, 2.1-fold for midbrain, and 1.3-fold for brainstem RNA-injected oocytes. Raising the extracellular Ca(2+) concentration significantly increased the Gly-currents in oocytes injected with midbrain and brainstem mRNAs. Reverse transcription-PCR studies showed differences in the Gly receptor (GlyR) alpha-subunits expressed, whereas the beta-subunit was present in all three types of mRNA. These results indicate differential expression of GlyR mRNAs in the brain areas examined, and these mRNAs lead to the expression of GlyRs that have different properties. The modulation of GlyRs by Ca(2+) could play important functions during brain development. PMID:11226317

  5. 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

  6. Presynaptic facilitation of glycinergic mIPSC is reduced in mice lacking α3 glycine receptor subunits.

    PubMed

    Kono, Y; Hülsmann, S

    2016-04-21

    Glycinergic neurons provide an important mechanism to control excitation of motoneurons in the brainstem and a reduction or loss of glycinergic inhibition can be deleterious by leading to hyperexcitation such as in hyperekplexia or neurodegeneration and neuronal death as in amyotrophic lateral sclerosis (ALS). Second messenger systems that change cyclic AMP and lead to phosphorylation of the α3 subunit of the glycine receptor (GlyR α3) have been shown to be potent modulators of synaptic inhibition in the spinal cord and brain stem. In this study we analyzed the role of GlyR α3 in synaptic inhibition to the hypoglossal nucleus using Glra3 (the gene encoding the glycine receptor α3 subunit) knockout mice. We observed that baseline glycinergic synaptic transmission to nucleus of hypoglossal motoneurons is rather normal in Glra3 knockout mice. Interestingly, we found that the modulation of synaptic transmission by cAMP-mediated pathways appeared to be reduced in Glra3 knockout mice. In the second postnatal week the forskolin-induced increase of miniature inhibitory postsynaptic potential (mIPSC) frequency was significantly larger in control as compared to Glra3 knockout mice suggesting that presynaptic glycine release in the hypoglossal nucleus is partially depending on GlyR α3. PMID:26851771

  7. Inter- and Intra-Subunit Butanol/Isoflurane Sites of Action in the Human Glycine Receptor

    PubMed Central

    McCracken, Mandy L.; Gorini, Giorgio; McCracken, Lindsay M.; Mayfield, R. Dayne; Harris, R. Adron; Trudell, James R.

    2016-01-01

    Glycine receptors (GlyRs) mediate inhibitory neurotransmission and are targets for alcohols and anesthetics in brain. GlyR transmembrane (TM) domains contain critical residues for alcohol/anesthetic action: amino acid A288 in TM3 forms crosslinks with TM1 (I229) in the adjacent subunit as well as TM2 (S267) and TM4 (Y406, W407, I409, Y410) in the same subunit. We hypothesized that these residues may participate in intra-subunit and inter-subunit sites of alcohol/anesthetic action. The following double and triple mutants of GLRA1 cDNA (encoding human glycine receptor alpha 1 subunit) were injected into Xenopus laevis oocytes: I229C/A288C, I229C/A288C/C290S, A288C/Y406C, A288C/W407C, A288C/I409C, and A288C/Y410C along with the corresponding single mutants and wild-type GLRA1. Butanol (22 mM) or isoflurane (0.6 mM) potentiation of GlyR-mediated currents before and after application of the cysteine crosslinking agent HgCl2 (10 μM) was measured using two-electrode voltage clamp electrophysiology. Crosslinking nearly abolished butanol and isoflurane potentiation in the I229C/A288C and I229C/A288C/C290S mutants but had no effect in single mutants or wild-type. Crosslinking also inhibited butanol and isoflurane potentiation in the TM3-4 mutants (A288C/Y406C, A288C/W407C, A288C/I409C, A288C/Y410C) with no effect in single mutants or wild-type. We extracted proteins from oocytes expressing I229C/288C, A288C/Y410C, or wild-type GlyRs, used mass spectrometry to verify their expression and possible inter-subunit dimerization, plus immunoblotting to investigate the biochemical features of proposed crosslinks. Wild-type GlyR subunits measured about 50 kDa; after crosslinking, the dimeric/monomeric 100:50 kDa band ratio was significantly increased in I229C/288C but not A288C/Y410C mutants or wild-type, providing support for TM1-3 inter-subunit and TM3-4 intra-subunit crosslinking. A GlyR homology model based on the GluCl template provides further evidence for a multi-site model

  8. Inter- and Intra-Subunit Butanol/Isoflurane Sites of Action in the Human Glycine Receptor.

    PubMed

    McCracken, Mandy L; Gorini, Giorgio; McCracken, Lindsay M; Mayfield, R Dayne; Harris, R Adron; Trudell, James R

    2016-01-01

    Glycine receptors (GlyRs) mediate inhibitory neurotransmission and are targets for alcohols and anesthetics in brain. GlyR transmembrane (TM) domains contain critical residues for alcohol/anesthetic action: amino acid A288 in TM3 forms crosslinks with TM1 (I229) in the adjacent subunit as well as TM2 (S267) and TM4 (Y406, W407, I409, Y410) in the same subunit. We hypothesized that these residues may participate in intra-subunit and inter-subunit sites of alcohol/anesthetic action. The following double and triple mutants of GLRA1 cDNA (encoding human glycine receptor alpha 1 subunit) were injected into Xenopus laevis oocytes: I229C/A288C, I229C/A288C/C290S, A288C/Y406C, A288C/W407C, A288C/I409C, and A288C/Y410C along with the corresponding single mutants and wild-type GLRA1. Butanol (22 mM) or isoflurane (0.6 mM) potentiation of GlyR-mediated currents before and after application of the cysteine crosslinking agent HgCl2 (10 μM) was measured using two-electrode voltage clamp electrophysiology. Crosslinking nearly abolished butanol and isoflurane potentiation in the I229C/A288C and I229C/A288C/C290S mutants but had no effect in single mutants or wild-type. Crosslinking also inhibited butanol and isoflurane potentiation in the TM3-4 mutants (A288C/Y406C, A288C/W407C, A288C/I409C, A288C/Y410C) with no effect in single mutants or wild-type. We extracted proteins from oocytes expressing I229C/288C, A288C/Y410C, or wild-type GlyRs, used mass spectrometry to verify their expression and possible inter-subunit dimerization, plus immunoblotting to investigate the biochemical features of proposed crosslinks. Wild-type GlyR subunits measured about 50 kDa; after crosslinking, the dimeric/monomeric 100:50 kDa band ratio was significantly increased in I229C/288C but not A288C/Y410C mutants or wild-type, providing support for TM1-3 inter-subunit and TM3-4 intra-subunit crosslinking. A GlyR homology model based on the GluCl template provides further evidence for a multi-site model

  9. Baicalin Activates Glycine and γ-Aminobutyric Acid Receptors on Substantia Gelatinosa Neurons of the Trigeminal Subsnucleus Caudalis in Juvenile Mice.

    PubMed

    Yin, Hua; Bhattarai, Janardhan Prasad; Oh, Sun Mi; Park, Soo Joung; Ahn, Dong Kuk; Han, Seong Kyu

    2016-01-01

    The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) receives nociceptive afferent inputs from thin-myelinated A[Formula: see text] fibers and unmyelinated C fibers and has been shown to be involved in the processing of orofacial nociceptive information. Scutellaria baicalensis Georgi (Huang-Qin, SbG), one of the 50 fundamental herbs of Chinese herbology, has been used historically as anti-inflammatory and antineoplastic medicine. Baicalin, one of the major compounds of SbG, has been reported to have neuroprotective, anti-inflammatory and analgesic effects. However, the receptor type activated by baicalin and its precise action mechanism on the SG neurons of Vc have not yet been studied. The whole-cell patch clamp technique was performed to examine the ion channels activated by baicalin on the SG neurons of Vc. In high Cl[Formula: see text] pipette solution, the baicalin (300[Formula: see text][Formula: see text]M) induced repeatable inward currents ([Formula: see text][Formula: see text]pA, [Formula: see text]) without desensitization on all the SG neurons tested. Further, the inward currents showed a concentration (0.1-3[Formula: see text]mM) dependent pattern. The inward current was sustained in the presence of tetrodotoxin (0.5[Formula: see text][Formula: see text]M), a voltage sensitive Na[Formula: see text] channel blocker. In addition, baicalin-induced inward currents were reduced in the presence of picrotoxin (50[Formula: see text][Formula: see text]M), a GABAA receptor antagonist, flumazenil (100[Formula: see text][Formula: see text]M), a benzodiazepine-sensitive GABAA receptor antagonist, and strychnine (2[Formula: see text][Formula: see text]M), a glycine receptor antagonist, respectively. These results indicate that baicalin has inhibitory effects on the SG neurons of the Vc, which are due to the activation of GABAA and/or the glycine receptor. Our results suggest that baicalin may be a potential target for orofacial pain modulation

  10. Proteomic Analysis of Glycine Receptor β Subunit (GlyRβ)-interacting Proteins

    PubMed Central

    del Pino, Isabel; Koch, Dennis; Schemm, Rudolf; Qualmann, Britta; Betz, Heinrich; Paarmann, Ingo

    2014-01-01

    Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378–455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs. PMID:24509844

  11. Unconventional pharmacology of a neuronal nicotinic receptor mutated in the channel domain.

    PubMed

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

    1992-02-15

    The putative channel-forming MII domains of the nicotinic, gamma-aminobutyric acid type A, and glycine receptors contain a highly conserved leucine residue. Mutation of this hydrophobic amino acid in the neuronal nicotinic receptor alpha 7 (Leu-247), reconstituted in Xenopus oocytes, modifies the ionic response to acetylcholine and alters desensitization. Furthermore, the Leu----Thr (L247T) mutant has two conducting states (46 pS and 80 pS), in contrast with the wild-type (WT) receptor, which has only one (45 pS). We now show that this mutant possesses a rather paradoxical pharmacology: antagonists of the WT receptor such as dihydro-beta-erythroidin, hexamethonium, or (+)-tubocurarine elicit ionic currents when applied to the L247T alpha 7 mutant and these responses are blocked by alpha-bungarotoxin. Furthermore, prolonged application of acetylcholine causes desensitization in the WT but leads to a potentiation of the responses to acetylcholine or dihydro-beta-erythroidin in the mutant. These data are consistent with a scheme in which mutation of Leu-247 renders a desensitized state in the WT channel a conducting state. They also strengthen the proposal that, in the WT, some competitive antagonists may stabilize desensitized states. Finally, these observations may shed light on properties of other ion channels, in particular the glutamate receptors, which display multiple conductance levels associated with various pharmacological agents. PMID:1741378

  12. (3H) 5,7-dichlorokynurenic acid, a high affinity ligand for the NMDA receptor glycine regulatory site

    SciTech Connect

    Hurt, S.D.; Baron, B.M. )

    1991-01-01

    The NMDA subtype of glutamate receptors is allosterically linked to a strychnine-insensitive glycine regulatory site. Kynurenic acid and its halogenated derivatives are non-competitive NMDA antagonists acting at the glycine site. The authors have prepared (3H) 5,7-dichlorokyrurenic acid (DCKA) as an antagonist radioligand and have characterized its binding. 3-Bromo-5,7-DCKA was catalytically dehalogenated in the presence of tritium gas and HPLC purified to yield (3H) 5,7-DCKA with a specific activity of 17.6 Ci/mmol. (3H) 5,7-DCKA bound to rat brain synaptosomes with a Kd of 69 {plus minus} 23 nM and Bmax = 14.5 {plus minus} 3.2 pmoles/mg protein. Binding was 65-70% specific at 10 nM (3H) 5,7-DCKA. This ligand is thus more selective and has higher affinity than (3H) glycine, in addition to being an antagonist.

  13. Effects of Salt Loading on the Regulation of Rat Hypothalamic Magnocellular Neurosecretory Cells by Ionotropic GABA and Glycine Receptors.

    PubMed

    Choe, K Y; Trudel, E; Bourque, C W

    2016-04-01

    Synaptic and extrasynaptic transmission mediated by ionotropic GABA and glycine receptors plays a critical role in shaping the action potential firing (spiking) activity of hypothalamic magnocellular neurosecretory cells and therefore determines the rate at which vasopressin and oxytocin are released from the neurohypophysis. The inhibitory effect of these transmitters relies on the maintenance of a low concentration of intracellular chloride ions such that, when activated by GABA or glycine, a hyperpolarisation of the neuronal membrane potential results. In this review, we highlight the various ways by which the two types of inhibitory receptors contribute to homeostasis by fine-tuning the spiking rate of vasopressin-releasing magnocellular neurosecretory cells in a manner dependent on the hydration state of the animal. In addition, we review the currently available evidence on how the strength of these inhibitory pathways can be regulated during chronic hypernatraemia via a form of activity-dependent depolarisation of the chloride reversal potential, leading to an abolition of these inhibitory pathways potentially causing sodium-dependent elevations in blood pressure. PMID:26833894

  14. Crystal structure of a heterotetrameric NMDA receptor ion channel

    PubMed Central

    Karakas, Erkan; Furukawa, Hiro

    2014-01-01

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

  15. Dual Allosteric Effect in Glycine/NMDA Receptor Antagonism: A Comparative QSAR Approach

    PubMed Central

    Sharma, Manish; Gupta, Vipin B.

    2010-01-01

    A comparative Hansch type QSAR study was conducted using multiple regression analysis on various sets of quinoxalines, quinoxalin-4-ones, quinazoline-2-carboxylates, 4-hydroxyquinolin-2(1H)-ones, 2-carboxytetrahydroquinolines, phenyl-hydroxy-quinolones, nitroquinolones and 4-substituted-3-phenylquinolin-2(1H)-ones as selective glycine/NMDA site antagonists. Ten statistically validated equations were developed, which indicated the importance of CMR, Verloop’s sterimol L1 and ClogP parameters in contributing towards biological activity. Interestingly, normal and inverse parabolic relationships were found with CMR in different series, indicating a dual allosteric binding mode in glycine/NMDA antagonism. Equations reveal an optimum CMR of 10 ± 10% is required for good potency of antagonists. Other equations indicate the presence of anionic functionality at 4-position of quinoline/quinolone ring system is not absolutely required for effective binding. The observations are laterally validated and in accordance with previous studies.

  16. Taurine activates excitatory non-synaptic glycine receptors on dopamine neurones in ventral tegmental area of young rats

    PubMed Central

    Wang, Fushun; Xiao, Cheng; Ye, Jiang Hong

    2005-01-01

    The physiological and pharmacological properties of taurine-induced responses were investigated in dopaminergic (DA) neurones from the ventral tegmental area (VTA) of young rats aged 1–13 postnatal days, either in acute brain slices or acutely dissociated neurones. When whole-cell responses were recorded from current-clamped neurones using the gramicidin-perforated technique, the application of taurine (0.01–30 mm) accelerated firings and induced membrane depolarization. In voltage-clamped neurones, taurine induced a current which was antagonized by strychnine and by picrotoxin, but not by bicuculline. In addition, taurine-induced current showed complete cross-desensitization with glycine-activated currents but not with γ-aminobutyric acid (GABA)-activated currents. Thus, taurine is a full agonist of the glycine receptors (GlyRs) in the VTA. Further studies found that taurine acted mainly on non-synaptic GlyRs. The application of 20 μm bicuculline abolished the spontaneous inhibitory post-synaptic currents (IPSCs) in 40/45 neurones, and 93% of the evoked IPSCs. The addition of 1 μm strychnine completely eliminated the remaining IPSCs. These results suggest that GABAergic IPSCs predominate, and that functional glycinergic synapses are present in a subset of the VTA neurones. The application of 1 μm strychnine alone induced an outward current, suggesting that these neurones were exposed to tonically released taurine/glycine. In conclusion, by activating non-synaptic GlyRs, taurine may act as an excitatory extra-synaptic neurotransmitter in the VTA during early development. PMID:15817633

  17. Dendritic NMDA receptors activate axonal calcium channels

    PubMed Central

    Christie, Jason M.; Jahr, Craig E.

    2008-01-01

    Summary NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca2+ and monovalent cations, they could alter release directly by increasing presynaptic Ca2+ or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca2+ channels (VSCCs). Using two-photon microscopy to measure Ca2+ excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca2+ transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca2+ entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca2+-dependent forms of presynaptic plasticity and release. PMID:18957221

  18. Three-dimensional autoradiographic localization of quench-corrected glycine receptor specific activity in the mouse brain using sup 3 H-strychnine as the ligand

    SciTech Connect

    White, W.F.; O'Gorman, S.; Roe, A.W. )

    1990-03-01

    The autoradiographic analysis of neurotransmitter receptor distribution is a powerful technique that provides extensive information on the localization of neurotransmitter systems. Computer methodologies are described for the analysis of autoradiographic material which include quench correction, 3-dimensional display, and quantification based on anatomical boundaries determined from the tissue sections. These methodologies are applied to the problem of the distribution of glycine receptors measured by 3H-strychnine binding in the mouse CNS. The most distinctive feature of this distribution is its marked caudorostral gradient. The highest densities of binding sites within this gradient were seen in somatic motor and sensory areas; high densities of binding were seen in branchial efferent and special sensory areas. Moderate levels were seen in nuclei related to visceral function. Densities within the reticular formation paralleled the overall gradient with high to moderate levels of binding. The colliculi had low and the diencephalon had very low levels of binding. No binding was seen in the cerebellum or the telencephalon with the exception of the amygdala, which had very low levels of specific binding. This distribution of glycine receptors correlates well with the known functional distribution of glycine synaptic function. These data are illustrated in 3 dimensions and discussed in terms of the significance of the analysis techniques on this type of data as well as the functional significance of the distribution of glycine receptors.

  19. Synthesis and biological evaluation of a new set of pyrazolo[1,5-c]quinazolines as glycine/N-methyl-D-aspartic acid receptor antagonists.

    PubMed

    Varano, Flavia; Catarzi, Daniela; Colotta, Vittoria; Poli, Daniela; Filacchioni, Guido; Galli, Alessandro; Costagli, Chiara

    2009-08-01

    Previous studies have shown that 8-chloro-5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-2-carboxylates (PQZ series) represent a family of glycine/N-methyl-D-aspartic acid (NMDA) and/or (R,S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) and/or kainic acid (KA) receptor antagonists. Moreover, some groups have been identified that introduced in suitable positions of the PQZ 2-carboxylate framework shift affinity and selectivity toward glycine/NMDA receptor. These substituents are a carboxylate function at position-1 and/or a chlorine atom at position-9. In this paper we report a study on some new 5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-1-carboxylates bearing at position-2 a lipophilic amide group or lacking substituent at this same position. All the newly synthesised compounds were evaluated for their binding at glycine/NMDA, AMPA and KA receptors. These studies led to the identification of some new PQZ derivatives endowed with good glycine/NMDA receptor affinity and selectivity and to better definition of the structure-activity relationship (SAR) of this class of compounds. PMID:19652407

  20. Effect of plasma protein binding on in vivo activity and brain penetration of glycine/NMDA receptor antagonists.

    PubMed

    Rowley, M; Kulagowski, J J; Watt, A P; Rathbone, D; Stevenson, G I; Carling, R W; Baker, R; Marshall, G R; Kemp, J A; Foster, A C; Grimwood, S; Hargreaves, R; Hurley, C; Saywell, K L; Tricklebank, M D; Leeson, P D

    1997-12-01

    A major issue in designing drugs as antagonists at the glycine site of the NMDA receptor has been to achieve good in vivo activity. A series of 4-hydroxyquinolone glycine antagonists was found to be active in the DBA/2 mouse anticonvulsant assay, but improvements in in vitro affinity were not mirrored by corresponding increases in anticonvulsant activity. Here we show that binding of the compounds to plasma protein limits their brain penetration. Relative binding to the major plasma protein, albumin, was measured in two different ways: by a radioligand binding experiment or using an HPLC assay, for a wide structural range of glycine/NMDA site ligands. These measures of plasma protein binding correlate well (r = 0.84), and the HPLC assay has been used extensively to quantify plasma protein binding. For the 4-hydroxyquinolone series, binding to plasma protein correlates (r = 0.92) with log P (octanol/pH 7.4 buffer) over a range of log P values from 0 to 5. The anticonvulsant activity increases with in vitro affinity, but the slope of a plot of pED50 versus pIC50 is low (0.40); taking plasma protein binding into account in this plot increases the slope to 0.60. This shows that binding to albumin in plasma reduces the amount of compound free to diffuse across the blood-brain barrier. Further evidence comes from three other experiments: (a) Direct measurements of brain/blood ratios for three compounds (2, 16, 26) show the ratio decreases with increasing log R. (b) Warfarin, which competes for albumin binding sites dose-dependently, decreased the ED50 of 26 for protection against seizures induced by NMDLA. (c) Direct measurements of brain penetration using an in situ brain perfusion model in rat to measure the amount of drug crossing the blood-brain barrier showed that compounds 2, 26, and 32 penetrate the brain well in the absence of plasma protein, but this is greatly reduced when the drug is delivered in plasma. In the 4-hydroxyquinolones glycine site binding affinity

  1. Low level expression of glycine receptor beta subunit transgene is sufficient for phenotype correction in spastic mice.

    PubMed Central

    Hartenstein, B; Schenkel, J; Kuhse, J; Besenbeck, B; Kling, C; Becker, C M; Betz, H; Weiher, H

    1996-01-01

    Mutations in inhibitory glycine receptor (GlyR) subunit genes are associated with neuromotor diseases in man and mouse. To use the potential of the mouse mutants as animal models of human disease, we altered GlyR levels in mutant mice and studied their phenotype. A transgene coding for the beta subunit of the rat GlyR was introduced into the genetic background of the spa mutation, which is characterized by low endogenous expression levels of the beta subunit and a dramatic neuromotor phenotype. The resulting transgenic mice expressed the beta subunit mRNA at intermediate levels, and their phenotype was rescued. This provides formal proof for the casual relationship between GlyR beta gene mutation and motor disease, and indicates that a low level of beta gene expression (25% of normal) is sufficient for proper functioning of glycinergic synapses. Images PMID:8635460

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

    PubMed Central

    Lynagh, Timothy; Lynch, Joseph W.

    2012-01-01

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

  3. Glycine release is regulated by metabotropic glutamate receptors sensitive to mGluR2/3 ligands and activated by N-acetylaspartylglutamate (NAAG).

    PubMed

    Romei, Cristina; Raiteri, Maurizio; Raiteri, Luca

    2013-03-01

    The presence of metabotropic glutamate receptors (mGluRs) of group II modulating glycine exocytosis from glycinergic nerve endings of mouse spinal cord was investigated. Purified synaptosomes were selectively prelabeled with [(3)H]glycine through the neuronal transporter GlyT2 and subsequently depolarized by superfusion with 12 mM KCl. The selective mGluR2/3 agonist LY379268 inhibited the K(+)-evoked overflow of [(3)H]glycine in a concentration-dependent manner (EC(50) about 0.2 nM). The effect of LY379268 was prevented by the selective mGluR2/3 antagonist LY341495 (IC(50) about 1 nM). N-acetylaspartylglutamate (NAAG) inhibited [(3)H]glycine overflow with extraordinary potency (EC(50) about 50 fmol). In contrast, glutamate was ineffective up to 0.1 nM, excluding that glutamate contamination of commercial NAAG samples is responsible for the reported activity of NAAG at mGluR3. LY341495 antagonized the NAAG inhibition of [(3)H]glycine release. The effect of a combination of maximally effective concentrations of LY379268 and NAAG exhibited no additivity. The non-hydrolysable NAAG analogue N-acetylaspartyl-β-linked glutamate (β-NAAG) antagonized NAAG and LY379268. In conclusion, our results show that glycinergic nerve endings in spinal cord are endowed with group II mGluRs mediating inhibition of glycine exocytosis. NAAG can activate these presynaptic receptors with extremely high affinity and with characteristics compatible with the reported mGluR3 pharmacology. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'. PMID:22659408

  4. Hyperekplexia phenotype of glycine receptor alpha1 subunit mutant mice identifies Zn(2+) as an essential endogenous modulator of glycinergic neurotransmission.

    PubMed

    Hirzel, Klaus; Müller, Ulrike; Latal, A Tobias; Hülsmann, Swen; Grudzinska, Joanna; Seeliger, Mathias W; Betz, Heinrich; Laube, Bodo

    2006-11-22

    Zn(2+) is thought to modulate neurotransmission by affecting currents mediated by ligand-gated ion channels and transmitter reuptake by Na(+)-dependent transporter systems. Here, we examined the in vivo relevance of Zn(2+) neuromodulation by producing knockin mice carrying the mutation D80A in the glycine receptor (GlyR) alpha1 subunit gene (Glra1). This substitution selectively eliminates the potentiating effect of Zn(2+) on GlyR currents. Mice homozygous for Glra1(D80A) develop a severe neuromotor phenotype postnatally that resembles forms of human hyperekplexia (startle disease) caused by mutations in GlyR genes. In spinal neurons and brainstem slices from Glra1(D80A) mice, GlyR expression, synaptic localization, and basal glycinergic transmission were normal; however, potentiation of spontaneous glycinergic currents by Zn(2+) was significantly impaired. Thus, the hyperekplexia phenotype of Glra1(D80A) mice is due to the loss of Zn(2+) potentiation of alpha1 subunit containing GlyRs, indicating that synaptic Zn(2+) is essential for proper in vivo functioning of glycinergic neurotransmission. PMID:17114051

  5. Functional Insights from Glutamate Receptor Ion Channel Structures

    PubMed Central

    Kumar, Janesh; Mayer, Mark L.

    2014-01-01

    X-ray crystal structures for the soluble amino terminal and ligand binding domains of glutamate receptor ion channels, combined with a 3.6 Å resolution structure of the full length AMPA receptor GluA2 homotetramer, provide unique insights into the mechanisms of iGluR assembly and function. Increasingly sophisticated biochemical, computational and electrophysiological experiments are beginning to reveal the mechanism of action of partial agonists, and yield new models for the mechanism of action of allosteric modulators. Newly identified NMDA receptor ligands acting at novel sites offer hope for development of subtype selective modulators. Many issues remain unsolved, including the role of the ATD in AMPA receptor signaling, and the mechanisms by which auxiliary proteins regulate receptor activity. The structural basis for ion permeation and ion channel block also remain areas of uncertainty, and despite substantial progress, molecular dynamics simulations have yet to reveal how binding of glutamate opens the ion channel pore. PMID:22974439

  6. The anticonvulsant and behavioural profile of L-687,414, a partial agonist acting at the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex.

    PubMed

    Tricklebank, M D; Bristow, L J; Hutson, P H; Leeson, P D; Rowley, M; Saywell, K; Singh, L; Tattersall, F D; Thorn, L; Williams, B J

    1994-11-01

    1. The anticonvulsant and behavioural effects of the glycine/NMDA receptor partial agonist, L-687,414 (R(+)-cis-beta-methyl-3-amino-1-hydroxypyrrolid-2-one) have been investigated in rodents. 2. L-687,414 dose-dependently antagonized seizures induced by N-methyl-D,L- aspartic acid (NMDLA, ED50 = 19.7 mg kg-1), pentylenetetrazol (PTZ, ED50 = 13.0 mg kg-1) and electroshock (ED50 = 26.1 mg kg-1) when given intravenously 15 min before test, in male Swiss Webster mice but was most potent against audiogenic seizures induced by a 120 dB bell in DBA/2 mice (ED50 = 5.1 mg kg-1, i.p., 30 min before test). 3. L-687,414 also induced impairments of performance in a rotarod test in both Swiss Webster and DBA/2 mice and the ratio [rotarod MED:anticonvulsant ED50] varied between 0.9 and 5, depending on the convulsant used. 4. Similar behaviours to those seen after administration of the non-competitive NMDA receptor antagonist, MK-801 (head weaving, body rolling, hyperlocomotion) were seen in the mouse after giving L-687,414, although the peak effect occurred at a dose (100 mg kg-1) which was 5-20 times the anticonvulsant ED50S, depending on the convulsant used. Unlike MK-801, however, doses of L-687,414 that were behaviourally stimulant did not increase dopamine turnover in the nucleus accumbens. 5. Consistent with the interaction of L-687,414 with the glycine/NMDA receptor, the anticonvulsant, ataxic and motor stimulant effects of the compound were significantly attenuated by the glycine/NMDA receptor agonist, D-serine (10-100 micrograms per mouse, i.c.v.). 6. The results show that L-687,414 is a potent, orally active anticonvulsant with a more benign pharmacological profile than antagonists acting at the ion channel of the NMDA receptor complex. The compound is a useful tool with which to probe the functional role of the glycine co-agonist site in vivo. PMID:7858861

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

    PubMed

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

    2016-07-01

    The molecular sensors that mediate temperature changes in living organisms are a large family of proteins known as thermosensitive transient receptor potential (TRP) ion channels. These membrane proteins are polymodal receptors that can be activated by cold or hot temperatures, depending on the channel subtype, voltage, and ligands. The stimuli sensors are allosterically coupled to a pore domain, increasing the probability of finding the channel in its ion conductive conformation. In this review we first discuss the allosteric coupling between the temperature and voltage sensor modules and the pore domain, and then discuss the thermodynamic foundations of thermo-TRP channel activation. We provide a structural overview of the molecular determinants of temperature sensing. We also posit an anisotropic thermal diffusion model that may explain the large temperature sensitivity of TRP channels. Additionally, we examine the effect of several ligands on TRP channel function and the evidence regarding their mechanisms of action. PMID:27297398

  8. Pharmacological properties and H+ sensitivity of excitatory amino acid receptor channels in rat cerebellar granule neurones.

    PubMed Central

    Traynelis, S F; Cull-Candy, S G

    1991-01-01

    1. N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate receptor channels have been examined in rat cerebellar granule neurones with whole-cell and single-channel patch-clamp methods. The whole-cell peak and steady-state aspartate and NMDA currents were reversibly inhibited by extracellular protons; the IC50 (concentration producing half-maximal inhibition) for the full H+ inhibition curve for NMDA receptors corresponded to pH 7.3, near to physiological pH. (S)-AMPA and kainate whole-cell currents were inhibited by protons with IC50 values that corresponded to pH 6.3 and 5.7, respectively; these receptors were, however, insensitive to H+ concentrations that inhibited NMDA receptor responses. 2. Proton inhibition of the NMDA, AMPA and kainate receptor-mediated responses was voltage insensitive, and did not involve a shift in reversal potential. 3. The EC50 (concentration producing half-maximal effect) for aspartate calculated from the whole-cell dose-response curve was similar at pH 6.8 and 7.6 (mean 11.2 microM). Although the EC50 for glycine potentiation of the aspartate response was marginally increased from 273 nM at pH 7.6 to 373 nM at pH 6.8, H+ inhibition was not overcome by up to 1 mM-external glycine. Inhibiting concentrations of H+ appropriate for AMPA and kainate receptors did not markedly alter the EC50 values determined for (S)-AMPA (3.4 microM) and kainate (114 microM) at pH 7.2. 4. Treatment of neurones with N-ethylmaleimide, iodoacetic acid, dithiothretiol or diethyl pyrocarbonate did not influence proton inhibition of NMDA receptor responses. However, treatment with diethyl pyrocarbonate, which potentiated aspartate responses, appeared to reduce the effectiveness of Zn2+ inhibition of NMDA receptors. 5. Desensitization of whole-cell NMDA and (S)-AMPA currents was studied with ionophoretic application of agonist to the cell soma. Whole-cell aspartate currents desensitized rapidly, irrespective of the

  9. Glycine and GABAA receptors mediate tonic and phasic inhibitory processes that contribute to prepulse inhibition in the goldfish startle network

    PubMed Central

    Curtin, Paul C. P.; Preuss, Thomas

    2015-01-01

    Prepulse inhibition (PPI) is understood as a sensorimotor gating process that attenuates sensory flow to the startle pathway during early stages (20–1000 ms) of information processing. Here, we applied in vivo electrophysiology and pharmacology to determine if PPI is mediated by glycine receptors (GlyRs) and/or GABAA receptors (GABAARs) in the goldfish auditory startle circuit. Specifically, we used selective antagonists to dissect the contributions of target receptors on sound-evoked postsynaptic potentials (PSPs) recorded in the neurons that initiate startle, the Mauthner-cells (M-cell). We found that strychnine, a GlyR antagonist, disrupted a fast-activated (5 ms) and rapidly (<50 ms) decaying (feed-forward) inhibitory process that contributes to PPI at 20 ms prepulse/pulse inter-stimulus intervals (ISI). Additionally we observed increases of the evoked postsynaptic potential (PSP) peak amplitude (+87.43 ± 21.53%, N = 9) and duration (+204 ± 48.91%, N = 9). In contrast, treatment with bicuculline, a GABAAR antagonist, caused a general reduction in PPI across all tested interstimulus intervals (ISIs) (20–500 ms). Bicuculline also increased PSP peak amplitude (+133.8 ± 10.3%, N = 5) and PSP duration (+284.95 ± 65.64%, N = 5). Treatment with either antagonist also tonically increased post-synaptic excitability in the M-cells, reflected by an increase in the magnitude of antidromically-evoked action potentials (APs) by 15.07 ± 3.21%, N = 7 and 16.23 ± 7.08%, N = 5 for strychnine and bicuculline, respectively. These results suggest that GABAARs and GlyRs are functionally segregated to short- and longer-lasting sound-evoked (phasic) inhibitory processes that contribute to PPI, with the mediation of tonic inhibition by both receptor systems being critical for gain control within the M-cell startle circuit. PMID:25852486

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

    PubMed

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

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

  11. Altered mnemonic functions and resistance to NMDA receptor antagonism by forebrain conditional knockout of glycine transporter 1

    PubMed Central

    Singer, Philipp; Yee, Benjamin K.; Feldon, Joram; Iwasato, Takuji; Itohara, Shigeyoshi; Grampp, Thomas; Prenosil, George; Benke, Dietmar; Möhler, Hanns; Boison, Detlev

    2009-01-01

    Converging evidence from pharmacological and molecular studies has led to the suggestion that inhibition of glycine transporter 1 (GlyT1) constitutes an effective means to boost N-methyl-D-aspartate receptor (NMDAR) activity by increasing the extra-cellular concentration of glycine in the vicinity of glutamatergic synapses. However, the precise extent and limitation of this approach to alter cognitive function, and therefore its potential as a treatment strategy against psychiatric conditions marked by cognitive impairments, remains to be fully examined. Here, we generated mutant mice lacking GlyT1 in the entire forebrain including neurons and glia. This conditional knockout system allows a more precise examination of GlyT1 down-regulation in the brain on behaviour and cognition. The mutation was highly effective in attenuating the motor-stimulating effect of acute NMDAR blockade by phencyclidine, although no appreciable elevation in NMDAR-mediated EPSC was observed in the hippocampus. Enhanced cognitive performance was observed in spatial working memory and object recognition memory while spatial reference memory and associative learning remained unaltered. These findings provide further credence for the potential cognitive enhancing effects of brain GlyT1 inhibition. At the same time, they indicated potential phenotypic differences when compared with other constitutive and conditional GlyT1 knockout lines, and highlighted the possibility of a functional divergence between the neuronal and glia subpopulations of GlyT1 in the regulation of learning and memory processes. The relevance of this distinction to the design of future GlyT1 blockers as therapeutic tools in the treatment of cognitive disorders remains to be further investigated. PMID:19332109

  12. Binding-site mutations in the alpha1 subunit of the inhibitory glycine receptor convert the inhibitory metal ion Cu2+ into a positive modulator.

    PubMed

    Schumann, Tanja; Grudzinska, Joanna; Kuzmin, Dmitry; Betz, Heinrich; Laube, Bodo

    2009-01-01

    The divalent cation copper (Cu2+) has been shown to inhibit chloride currents mediated by the inhibitory glycine receptor (GlyR). Here, we analyzed Cu2+ inhibition of homo- and hetero-oligomeric GlyRs expressed in Xenopus oocytes. No significant differences in Cu2+ inhibitory potency were found between alpha1, alpha2 and alpha3 GlyRs as well as heteromeric alpha1beta receptors. Furthermore, GlyR alpha1 mutations known to reduce inhibition or potentiation of GlyR currents by Zn2+ had no effect on Cu2+ inhibition. However, Cu2+ was found to competitively antagonize glycine binding, suggesting that Cu2+ binds at the agonist-binding site. Mutations within the glycine-binding site of the GlyR alpha1 subunit reduced the inhibitory potency of Cu2+ and led to an up to 4-fold potentiation of glycine-elicited currents by Cu2+. Molecular dynamics simulation suggests this to be due to increased Cu2+ binding energies. Our data show that GlyR binding-site mutations can convert inhibitors of agonist binding into highly effective positive modulators. PMID:18793654

  13. Combined stimulation of the glycine and polyamine sites of the NMDA receptor attenuates NMDA blockade-induced learning deficits of rats in a 14-unit T-maze.

    PubMed

    Meyer, R C; Knox, J; Purwin, D A; Spangler, E L; Ingram, D K

    1998-02-01

    The present study examined the effects of multi-site activation of the glycine and polyamine sites of the NMDA receptor on memory formation in rats learning a 14-unit T-maze task. The competitive NMDA receptor antagonist, (+/-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP, 9 mg/kg), was used to impair learning. The objectives were two-fold: (1) to investigate the effects of independent stimulation of the strychnine-insensitive glycine site or the polyamine site; (2) to investigate the effects of simultaneous activation of these two sites. Male, Fischer-344 rats were pretrained to a criterion of 13 out of 15 shock avoidances in a straight runway, and 24 h later were trained in a 14-unit T-maze that also required shock avoidance. Prior to maze training, rats received intraperitoneal (i.p.) injections of saline, saline plus CPP, CPP plus the glycine agonist, D-cycloserine (DCS, 30 or 40 mg/kg), CPP plus the polyamine agonist, spermine (SPM, 2.5 or 5 mg/kg), or CPP plus a combination of DCS (7.5 mg/kg) and SPM (0.625 mg/kg). Individual administration of either DCS or SPM attenuated the CPP-induced maze learning impairment in a dose-dependent manner. However, the combined treatment with both DCS and SPM completely reversed the learning deficit at doses five-fold less than either drug given alone. These findings provide additional evidence that the glycine and polyamine modulatory sites of the NMDA receptor are involved in memory formation. Furthermore, the potent synergistic effect resulting from combined activation of the glycine and polyamine sites would suggest a stronger interaction between these two sites than previously considered, and might provide new therapeutic approaches for enhancing glutamatergic function. PMID:9498733

  14. Presynaptic glycine receptors as a potential therapeutic target for hyperekplexia disease

    PubMed Central

    Xiong, Wei; Chen, Shao-Rui; He, Liming; Cheng, Kejun; Zhao, Yi-Lin; Chen, Hong; Li, De-Pei; Homanics, Gregg E.; Peever, John; Rice, Kenner C.; Wu, Ling-gang; Pan, Hui-Lin; Zhang, Li

    2014-01-01

    While postsynaptic GlyRs as α/β heteromers attract the most research attention, little is known about the role of presynaptic GlyRs, likely α homomers, in diseases. Here, we demonstrate that DH-CBD, a nonpsychoactive cannabinoid, can rescue GlyR functional deficiency and exaggerated acoustic and tactile startle responses in mice bearing the point-mutations in the α1 GlyRs responsible for a hereditary startle/hyperekplexia disease. The GlyRs expressed as α1 homomers either in HEK-293 cells or at presynaptic terminals of the calyceal synapses in auditory brainstem are most vulnerable to hyperekplexia mutation-induced impairment. Homomeric mutants are more sensitive than heteromers to DH-CBD, suggesting presynaptic GlyRs as a primary target. Consistent with this, DH-CBD selectively rescues impaired presynaptic GlyR activity and diminished glycine release in the brainstem and spinal cord of hyperekplexic mutant mice. Thus, presynaptic α GlyRs emerge as a potential therapeutic target for dominant hyperekplexia disease and other diseases with GlyR deficiency. PMID:24390226

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

    PubMed

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

    2016-09-01

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

  16. 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

  17. Glycine Transporters and Their Inhibitors

    NASA Astrophysics Data System (ADS)

    Gilfillan, Robert; Kerr, Jennifer; Walker, Glenn; Wishart, Grant

    Glycine plays a ubiquitous role in many biological processes. In the central nervous system it serves as an important neurotransmitter acting as an agonist at strychnine-sensitive glycine receptors and as an essential co-agonist with glutamate at the NMDA receptor complex. Control of glycine concentrations in the vicinity of these receptors is mediated by the specific glycine transporters, GlyT1 and GlyT2. Inhibition of these transporters has been postulated to be of potential benefit in several therapeutic indications including schizophrenia and pain. In this review we discuss our current knowledge of glycine transporters and focus on recent advances in the medicinal chemistry of GlyT1 and GlyT2 inhibitors.

  18. Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE-receptor kinase-WOX signaling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem), and vascular cambium are tightly controlled by CLE signaling pathway...

  19. Blockade of glutamatergic and GABAergic receptor channels by trimethyltin chloride

    PubMed Central

    Krüger, Katharina; Diepgrond, Victoria; Ahnefeld, Maria; Wackerbeck, Christina; Madeja, Michael; Binding, Norbert; Musshoff, Ulrich

    2005-01-01

    Organotin compounds such as trimethyltin chloride (TMT) are among the most toxic of the organometallics. As their main target for toxicity is the central nervous system, the aim of the present study was to investigate the effects of TMT on receptor channels involved in various processes of synaptic transmission. The Xenopus oocyte expression system was chosen for direct assessment of TMT effects on voltage-operated potassium channels and glutamatergic and GABAergic receptors, and hippocampal slices from rat brain for analyzing TMT effects on identified synaptic sites. TMT was found to be ineffective, at 100 μmol l−1, against several potassium- and sodium-operated ion channel functions as well as the metabotropic glutamate receptor. The functions of the ionotropic glutamate and the GABAA receptor channels were inhibited by TMT in micromolar concentrations. Thus, at a maximum concentration of 100 μmol l−1, around 20–30% of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and GABAA receptor-mediated ion currents and 35% of the N-methyl-D-aspartate receptor-mediated ion currents were blocked. In the hippocampal slice model, the inhibitory effects of TMT were much stronger than expected from the results on the ion channels. Bath application of TMT significantly reduced the amplitudes of evoked excitatory postsynaptic field potentials in a concentration-dependent and nonreversible manner.  Induction of long-term potentiation, recorded from the CA1 dendritic region, was inhibited by TMT and failed completely at a concentration of 10 μmol l−1. In general, TMT affects the excitatory and inhibitory synaptic processes in a receptor specific manner and is able to disturb the activity within a neuronal network. PMID:15655511

  20. Interaction of ion channels and receptors with PDZ domain proteins.

    PubMed

    Kornau, H C; Seeburg, P H; Kennedy, M B

    1997-06-01

    The complex anatomy of neurons demands a high degree of functional organization. Therefore, membrane receptors and ion channels are often localized to selected subcellular sites and coupled to specific signal transduction machineries. PDZ domains have come into focus as protein interaction modules that mediate the binding of a class of submembraneous proteins to membrane receptors and ion channels and thus subserve these organizational aspects. The structures of two PDZ domains have been resolved, which has led to a structural understanding of the specificity of interactions of various PDZ domains with their respective partners. The functional implications of PDZ domain interactions are now being addressed in vitro and in vivo. PMID:9232802

  1. 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

  2. T-cell receptor accessory and co-receptor molecules in channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    T cell receptor (TCR) associated invariant chains CD3gamma/delta,epsilon, and zeta as well as TCR co-receptors CD8alpha and CD8beta were isolated from the channel catfish, Ictalurus punctatus, at both the gene and cDNA levels. All of catfish CD3 sequences encode for proteins that resemble their resp...

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

    PubMed

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

    2001-06-01

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

  4. Transient Receptor Potential (TRP) channels in T cells.

    PubMed

    Bertin, Samuel; Raz, Eyal

    2016-05-01

    The transient receptor potential (TRP) family of ion channels is widely expressed in many cell types and plays various physiological roles. Growing evidence suggests that certain TRP channels are functionally expressed in the immune system. Indeed, an increasing number of reports have demonstrated the functional expression of several TRP channels in innate and adaptive immune cells and have highlighted their critical role in the activation and function of these cells. However, very few reviews have been entirely dedicated to this subject. Here, we will summarize the recent findings with regards to TRP channel expression in T cells and discuss their emerging role as regulators of T cell activation and functions. Moreover, these studies suggest that beyond their pharmaceutical interest in pain management, certain TRP channels may represent potential novel therapeutic targets for various immune-related diseases. PMID:26468011

  5. Halothane shortens acetylcholine receptor channel kinetics without affecting conductance.

    PubMed Central

    Lechleiter, J; Gruener, R

    1984-01-01

    The extracellular patch-clamp technique was used to examine how halothane, a general anesthetic, affects the properties of single nicotinic acetylcholine receptor channels of embryonic Xenopus skeletal muscle cells grown in culture. Under control conditions, single-channel events showed a bimodal distribution on the basis of current amplitudes. This distribution was maintained during exposure to halothane and its washout. In addition, the mean current value of the low-and high-amplitude channels was unaffected by the presence of the anesthetic at clinically relevant concentrations. In contrast, halothane shortened the burst durations of both channel types in a concentration-dependent manner. This shortening of burst durations may be an expression of the more rapid relaxation of the channel protein to the nonconducting state, possibly due to the disordering effect of the anesthetic on membrane lipids in which the receptor protein is embedded. This functional change, in the behavior of the synaptic receptor, provides further direct information on the mode of action of general anesthetics. Images PMID:6326154

  6. Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium

    PubMed Central

    Shabir, Saqib; Cross, William; Kirkwood, Lisa A.; Pearson, Joanna F.; Appleby, Peter A.; Walker, Dawn; Eardley, Ian

    2013-01-01

    In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca2+. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca2+ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca2+ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting. PMID:23720349

  7. A Pentasymmetric Open Channel Blocker for Cys-Loop Receptor Channels

    PubMed Central

    Baur, Roland; Puthenkalam, Roshan; Ernst, Margot; Trauner, Dirk; Sigel, Erwin

    2014-01-01

    γ-Aminobutyric acid type A receptors (GABAA receptors) are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. These receptors show near five-fold symmetry that is most pronounced in the second trans-membrane domain M2 lining the Cl− ion channel. To take advantage of this inherent symmetry, we screened a variety of aromatic anions with matched symmetry and found an inhibitor, pentacyanocyclopentdienyl anion (PCCP−) that exhibited all characteristics of an open channel blocker. Inhibition was strongly dependent on the membrane potential. Through mutagenesis and covalent modification, we identified the region α1V256-α1T261 in the rat recombinant GABAA receptor to be important for PCCP− action. Introduction of positive charges into M2 increased the affinity for PCCP− while PCCP− prevented the access of a positively charged molecule into M2. Interestingly, other anion selective cys-loop receptors were also inhibited by PCCP−, among them the Drosophila RDL GABAA receptor carrying an insecticide resistance mutation, suggesting that PCCP− could serve as an insecticide. PMID:25184303

  8. A pentasymmetric open channel blocker for Cys-loop receptor channels.

    PubMed

    Carta, Valentina; Pangerl, Michael; Baur, Roland; Puthenkalam, Roshan; Ernst, Margot; Trauner, Dirk; Sigel, Erwin

    2014-01-01

    γ-Aminobutyric acid type A receptors (GABAA receptors) are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. These receptors show near five-fold symmetry that is most pronounced in the second trans-membrane domain M2 lining the Cl- ion channel. To take advantage of this inherent symmetry, we screened a variety of aromatic anions with matched symmetry and found an inhibitor, pentacyanocyclopentdienyl anion (PCCP-) that exhibited all characteristics of an open channel blocker. Inhibition was strongly dependent on the membrane potential. Through mutagenesis and covalent modification, we identified the region α1V256-α1T261 in the rat recombinant GABAA receptor to be important for PCCP- action. Introduction of positive charges into M2 increased the affinity for PCCP- while PCCP- prevented the access of a positively charged molecule into M2. Interestingly, other anion selective cys-loop receptors were also inhibited by PCCP-, among them the Drosophila RDL GABAA receptor carrying an insecticide resistance mutation, suggesting that PCCP- could serve as an insecticide. PMID:25184303

  9. Lipid modulation of thermal transient receptor potential channels.

    PubMed

    Hernández-García, Enrique; Rosenbaum, Tamara

    2014-01-01

    There is a subgroup of transient receptor potential (TRP) ion channels that are responsive to temperature (thermo-TRP channels). These are important to a variety of sensory and physiological phenomena such as pain and taste perception. All thermo-TRP channels known to date are subject to modulation by lipidic molecules of many kinds, from the ubiquitous cholesterol to more specialized molecules such as prostaglandins. Although the mechanisms and sites of binding of lipids on thermo-TRPs are largely unknown, the explosion on research of lipids and ion channels has revealed previously unsuspected roles for them. Diacyl glycerol is a lipid produced by phospholipase C (PLC) and it was discovered to modulate TRP channels in the eye of the fly, and many mammal TRP channels have been found to interact with lipids. While most of the lipids acting on thermo-TRP channels have been found to activate them, there are a few capable of inhibition. Phosphatidylinositol 4,5-bisphosphate is even capable of both inhibition and activation on a couple of thermo-TRPs, depending on the cellular context. More data is required to assess the mechanism through which lipids affect thermo-TRP channel activity and the physiological importance of this interaction. PMID:25366236

  10. Loss of glycine receptors containing the α3 subunit compromises auditory nerve activity, but not outer hair cell function.

    PubMed

    Dlugaiczyk, Julia; Hecker, Dietmar; Neubert, Christian; Buerbank, Stefanie; Campanelli, Dario; Becker, Cord-Michael; Betz, Heinrich; Knipper, Marlies; Rüttiger, Lukas; Schick, Bernhard

    2016-07-01

    Inhibitory glycine receptors containing the α3 subunit (GlyRα3) regulate sensory information processing in the CNS and retina. In previous work, we demonstrated the presence of postsynaptic GlyRα3 immunoreactivity at efferent synapses of the medial and lateral olivocochlear bundle in the organ of Corti; however, the role of these α3-GlyRs in auditory signalling has remained elusive. The present study analyzes distortion-product otoacoustic emissions (DPOAEs) and auditory brainstem responses (ABRs) of knockout mice with a targeted inactivation of the Glra3 gene (Glra3(-/-)) and their wildtype littermates (Glra3(+/+)) before and seven days after acoustic trauma (AT; 4-16 kHz, 120 dB SPL, 1 h). Before AT, DPOAE thresholds were slightly, but significantly lower, and DPOAE amplitudes were slightly larger in Glra3(-/-) as compared to Glra3(+/+) mice. While click- and f-ABR thresholds were similar in both genotypes before AT, threshold-normalized click-ABR wave I amplitudes were smaller in Glra3(-/-) mice as compared to their wildtype littermates. Following AT, both the decrement of ABR wave I amplitudes and the delay of wave I latencies were more pronounced in Glra3(-/-) than Glra3(+/+) mice. Accordingly, correlation between early click-evoked ABR signals (0-2.5 ms from stimulus onset) before and after AT was significantly reduced for Glra3(-/-) as compared to Glra3(+/+) mice. In summary, these results show that loss of α3-GlyRs compromises suprathreshold auditory nerve activity, but not outer hair cell function. PMID:27208792

  11. Mutational and haplotype analysis of the {alpha}{sub 1} subunit of the glycine receptor in hyperekplexia patients

    SciTech Connect

    Shiang, R.; Zhu, Y.Z.; Wasmuth, J.J.

    1994-09-01

    Familial hyperekplexia or Startle disease (STHE) is a rare autosomal dominant neurologic disorder manifested by marked muscular hypertonia in infants and exaggerated startle response that persists throughout the lifetime of the patient. This disorder is caused by mutations in the {alpha}{sub 1} subunit of the receptor for the inhibitory neurotransmitter glycine (GLRA1). Previously, we have reported three mutations, two of which change arginine 271 (Arg 271) to uncharged amino acids and a third which changes a tyrosine at amino acid 279 to a cysteine. The most common mutation, detected in three of six original families, is a G to A transition mutation at Arg 271. Four new STHE patients have been screened and were found to have the most common Arg 271 mutation. Three of the new patients have a clear family history while family information on the fourth patient was unavailable. Four possible sporadic cases of STHE have been screened by DGGE in all exons of the GLRA1 gene and no mutations have been detected. These sporadic cases may represent defects from other causes. A new three-allele dinucleotide repeat polymorphism at the GLRA1 locus has been detected. Haplotype analysis of two polymorphisms at the GLRA1 locus and CA-repeat polymorphism, D5S119, suggests that the most common mutation arose at least two and most likely three independent times. Thus, it appears that at least five independent GLRA1 mutation events (two of which are identical) have occurred in ten STHE families. The fact that these mutations affect only two amino acids suggests that the dominant STHE phenotype can only be caused by abnormalities in a highly restricted region of GLRA1.

  12. Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor).

    PubMed Central

    Chen, S R; Leong, P; Imredy, J P; Bartlett, C; Zhang, L; MacLennan, D H

    1997-01-01

    We report transient expression of a full-length cDNA encoding the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in HEK-293 cells. The single-channel properties of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate-solubilized and sucrose gradient-purified recombinant Ca2+ release channels were investigated by using single-channel recordings in planar lipid bilayers. The recombinant Ca2+ release channel exhibited a K+ conductance of 780 pS when symmetrical 250 mM KCl was used as the conducting ion and a Ca2+ conductance of 116 pS in 50 mM luminal Ca2+. Opening events of the recombinant channels were brief, with an open time constant of approximately 0.22 ms. The recombinant Ca2+ release channel was more permeable to Ca2+ than to K+, with a pCa2+/pK+ ratio of 6.8. The response of the recombinant Ca2+ release channel to various concentrations of Ca2+ was biphasic, with the channel being activated by micromolar Ca2+ and inhibited by millimolar Ca2+. The recombinant channels were activated by ATP and caffeine, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Most recombinant channels were asymmetrically blocked, conducting current unidirectionally from the luminal to the cytoplasmic side of the channel. These data demonstrate that the properties of recombinant Ca2+ release channel expressed in HEK-293 cells are very similar, if not identical, to those of the native channel. Images FIGURE 1 PMID:9336186

  13. N-glycosylation sites on the nicotinic ACh receptor subunits regulate receptor channel desensitization and conductance.

    PubMed

    Nishizaki, Tomoyuki

    2003-06-10

    The present study investigated the effects of N-glycosylation sites on Torpedo acetylcholine (ACh) receptors expressed in Xenopus oocytes by monitoring whole-cell membrane currents and single-channel currents from excised patches. Receptors with the mutant subunit at the asparagine residue on the conserved N-glycosylation site (mbetaN141D, mgammaN141D, or mdeltaN143D) or the serine/threonine residue (mbetaT143A, mgammaS143A, or mdeltaS145A) delayed the rate of current decay as compared with wild-type receptors, and the most striking effect was found with receptors with mbetaT143A or mgammaS143A. For wild-type receptors, the lectin concanavalin A, that binds to glycosylated membrane proteins with high affinity, mimicked this effect. Receptors with mbetaN141D or mdeltaN143D exhibited lower single-channel conductance, but those with mbetaT143A, mgammaS143A, or mdeltaS145A otherwise revealed higher conductance than wild-type receptors. Mean opening time of single-channel currents was little affected by the mutation. N-glycosylation sites, thus, appear to play a role in the regulation of ACh receptor desensitization and ion permeability. PMID:12829329

  14. TRP Channels in Insect Stretch Receptors as Insecticide Targets.

    PubMed

    Nesterov, Alexandre; Spalthoff, Christian; Kandasamy, Ramani; Katana, Radoslav; Rankl, Nancy B; Andrés, Marta; Jähde, Philipp; Dorsch, John A; Stam, Lynn F; Braun, Franz-Josef; Warren, Ben; Salgado, Vincent L; Göpfert, Martin C

    2015-05-01

    Defining the molecular targets of insecticides is crucial for assessing their selectivity and potential impact on environment and health. Two commercial insecticides are now shown to target a transient receptor potential (TRP) ion channel complex that is unique to insect stretch receptor cells. Pymetrozine and pyrifluquinazon disturbed Drosophila coordination and hearing by acting on chordotonal stretch receptor neurons. This action required the two TRPs Nanchung (Nan) and Inactive (Iav), which co-occur exclusively within these cells. Nan and Iav together sufficed to confer cellular insecticide responses in vivo and in vitro, and the two insecticides were identified as specific agonists of Nan-Iav complexes that, by promoting cellular calcium influx, silence the stretch receptor cells. This establishes TRPs as insecticide targets and defines specific agonists of insect TRPs. It also shows that TRPs can render insecticides cell-type selective and puts forward TRP targets to reduce side effects on non-target species. PMID:25950634

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

    PubMed

    Krishtal, Oleg

    2015-07-01

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

  16. Mechanisms of Barbiturate Inhibition of Acetylcholine Receptor Channels

    PubMed Central

    Dilger, James P.; Boguslavsky, Rebecca; Barann, Martin; Katz, Tamir; Vidal, Ana Maria

    1997-01-01

    We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts is 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 μM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 μM, n = 1.09; for barbital, Ki = 1900 μM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. <0.1 ms at the Ki). Pentobarbital binds ≥10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at >100 μM pentobarbital. PMID:9089445

  17. Mechanisms of barbiturate inhibition of acetylcholine receptor channels.

    PubMed

    Dilger, J P; Boguslavsky, R; Barann, M; Katz, T; Vidal, A M

    1997-03-01

    We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts in 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 microM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 microM, n = 1.09; for barbital, Ki = 1900 microM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. < 0.1 ms at the Ki). Pentobarbital binds > or = 10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at > 100 microM pentobarbital. PMID:9089445

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

    PubMed

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

    2009-10-01

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

  19. Transient Receptor Potential Channels as Targets for Phytochemicals

    PubMed Central

    2015-01-01

    To date, 28 mammalian transient receptor potential (TRP) channels have been cloned and characterized. They are grouped into six subfamilies on the basis of their amino acid sequence homology: TRP Ankyrin (TRPA), TRP Canonical (TRPC), TRP Melastatin (TRPM), TRP Mucolipin (TRPML), TRP Polycystin (TRPP), and TRP Vanilloid (TRPV). Most of the TRP channels are nonselective cation channels expressed on the cell membrane and exhibit variable permeability ratios for Ca2+ versus Na+. They mediate sensory functions (such as vision, nociception, taste transduction, temperature sensation, and pheromone signaling) and homeostatic functions (such as divalent cation flux, hormone release, and osmoregulation). Significant progress has been made in our understanding of the specific roles of these TRP channels and their activation mechanisms. In this Review, the emphasis will be on the activation of TRP channels by phytochemicals that are claimed to exert health benefits. Recent findings complement the anecdotal evidence that some of these phytochemicals have specific receptors and the activation of which is responsible for the physiological effects. Now, the targets for these phytochemicals are being unveiled; a specific hypothesis can be proposed and tested experimentally to infer a scientific validity of the claims of the health benefits. The broader and pressing issues that have to be addressed are related to the quantities of the active ingredients in a given preparation, their bioavailability, metabolism, adverse effects, excretion, and systemic versus local effects. PMID:24926802

  20. Transient receptor potential (TRP) channels: a clinical perspective

    PubMed Central

    Kaneko, Yosuke; Szallasi, Arpad

    2014-01-01

    Transient receptor potential (TRP) channels are important mediators of sensory signals with marked effects on cellular functions and signalling pathways. Indeed, mutations in genes encoding TRP channels are the cause of several inherited diseases in humans (the so-called ‘TRP channelopathies’) that affect the cardiovascular, renal, skeletal and nervous systems. TRP channels are also promising targets for drug discovery. The initial focus of research was on TRP channels that are expressed on nociceptive neurons. Indeed, a number of potent, small-molecule TRPV1, TRPV3 and TRPA1 antagonists have already entered clinical trials as novel analgesic agents. There has been a recent upsurge in the amount of work that expands TRP channel drug discovery efforts into new disease areas such as asthma, cancer, anxiety, cardiac hypertrophy, as well as obesity and metabolic disorders. A better understanding of TRP channel functions in health and disease should lead to the discovery of first-in-class drugs for these intractable diseases. With this review, we hope to capture the current state of this rapidly expanding and changing field. LINKED ARTICLES This article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-10 PMID:24102319

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

    PubMed

    Gunthorpe, M J; Lummis, S C

    2001-06-15

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

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

    PubMed

    Gunthorpe, M J; Lummis, S C

    2001-04-01

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

  3. Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic beta cells.

    PubMed

    Wagner, Thomas F J; Loch, Sabine; Lambert, Sachar; Straub, Isabelle; Mannebach, Stefanie; Mathar, Ilka; Düfer, Martina; Lis, Annette; Flockerzi, Veit; Philipp, Stephan E; Oberwinkler, Johannes

    2008-12-01

    Transient receptor potential (TRP) cation channels are renowned for their ability to sense diverse chemical stimuli. Still, for many members of this large and heterogeneous protein family it is unclear how their activity is regulated and whether they are influenced by endogenous substances. On the other hand, steroidal compounds are increasingly recognized to have rapid effects on membrane surface receptors that often have not been identified at the molecular level. We show here that TRPM3, a divalent-permeable cation channel, is rapidly and reversibly activated by extracellular pregnenolone sulphate, a neuroactive steroid. We show that pregnenolone sulphate activates endogenous TRPM3 channels in insulin-producing beta cells. Application of pregnenolone sulphate led to a rapid calcium influx and enhanced insulin secretion from pancreatic islets. Our results establish that TRPM3 is an essential component of an ionotropic steroid receptor enabling unanticipated crosstalk between steroidal and insulin-signalling endocrine systems. PMID:18978782

  4. Transient receptor potential channel 1 (TRPC1) reduces calcium permeability in heteromeric channel complexes.

    PubMed

    Storch, Ursula; Forst, Anna-Lena; Philipp, Maximilian; Gudermann, Thomas; Mederos y Schnitzler, Michael

    2012-01-27

    Specific biological roles of the classical transient receptor potential channel 1 (TRPC1) are still largely elusive. To investigate the function of TRPC1 proteins in cell physiology, we studied heterologously expressed TRPC1 channels and found that recombinant TRPC1 subunits do not form functional homomeric channels. Instead, by electrophysiological analysis TRPC1 was shown to form functional heteromeric, receptor-operated channel complexes with TRPC3, -4, -5, -6, and -7 indicating that TRPC1 proteins can co-assemble with all members of the TRPC subfamily. In all TRPC1-containing heteromers, TRPC1 subunits significantly decreased calcium permeation. The exchange of select amino acids in the putative pore-forming region of TRPC1 further reduced calcium permeability, suggesting that TRPC1 subunits contribute to the channel pore. In immortalized immature gonadotropin-releasing hormone neurons endogenously expressing TRPC1, -2, -5, and -6, down-regulation of TRPC1 resulted in increased calcium permeability and elevated basal cytosolic calcium concentrations. We did not observe any involvement of TRPC1 in store-operated cation influx. Notably, TRPC1 suppressed the migration of gonadotropin-releasing hormone neurons without affecting cell proliferation. Conversely, in TRPC1 knockdown neurons, specific migratory properties like distance covered, locomotion speed, and directionality were increased. These findings suggest a novel regulatory mechanism relying on the expression of TRPC1 and the subsequent formation of heteromeric TRPC channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. PMID:22157757

  5. Transient Receptor Potential Channel 1 (TRPC1) Reduces Calcium Permeability in Heteromeric Channel Complexes

    PubMed Central

    Storch, Ursula; Forst, Anna-Lena; Philipp, Maximilian; Gudermann, Thomas; Mederos y Schnitzler, Michael

    2012-01-01

    Specific biological roles of the classical transient receptor potential channel 1 (TRPC1) are still largely elusive. To investigate the function of TRPC1 proteins in cell physiology, we studied heterologously expressed TRPC1 channels and found that recombinant TRPC1 subunits do not form functional homomeric channels. Instead, by electrophysiological analysis TRPC1 was shown to form functional heteromeric, receptor-operated channel complexes with TRPC3, -4, -5, -6, and -7 indicating that TRPC1 proteins can co-assemble with all members of the TRPC subfamily. In all TRPC1-containing heteromers, TRPC1 subunits significantly decreased calcium permeation. The exchange of select amino acids in the putative pore-forming region of TRPC1 further reduced calcium permeability, suggesting that TRPC1 subunits contribute to the channel pore. In immortalized immature gonadotropin-releasing hormone neurons endogenously expressing TRPC1, -2, -5, and -6, down-regulation of TRPC1 resulted in increased calcium permeability and elevated basal cytosolic calcium concentrations. We did not observe any involvement of TRPC1 in store-operated cation influx. Notably, TRPC1 suppressed the migration of gonadotropin-releasing hormone neurons without affecting cell proliferation. Conversely, in TRPC1 knockdown neurons, specific migratory properties like distance covered, locomotion speed, and directionality were increased. These findings suggest a novel regulatory mechanism relying on the expression of TRPC1 and the subsequent formation of heteromeric TRPC channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. PMID:22157757

  6. Pharmacology of transient receptor potential melastatin channels in the vasculature

    PubMed Central

    Zholos, Alexander

    2010-01-01

    Mammalian transient receptor potential melastatin (TRPM) non-selective cation channels, the largest TRP subfamily, are widely expressed in excitable and non-excitable cells where they perform diverse functions ranging from detection of cold, taste, osmolarity, redox state and pH to control of Mg2+ homeostasis and cell proliferation or death. Recently, TRPM gene expression has been identified in vascular smooth muscles with dominance of the TRPM8 channel. There has been in parallel considerable progress in decoding the functional roles of several TRPMs in the vasculature. This research on native cells is aided by the knowledge of the activation mechanisms and pharmacological properties of heterologously expressed TRPM subtypes. This paper summarizes the present state of knowledge of vascular TRPM channels and outlines several anticipated directions of future research in this area. PMID:20233227

  7. Transient receptor potential channel C5 in cancer chemoresistance

    PubMed Central

    He, Dong-xu; Ma, Xin

    2016-01-01

    The transient receptor potential (TRP) superfamily contains at least 28 homologs in mammalian. These proteins form TRP channels are permeable to monovalent and divalent cations and participate in a variety of physiological functions. Dysregulation of TRP channels is responsible for numerous diseases. This review provides a brief short overview of mammalian TRP channels with a focus on TRPC5 and its role in cancers. Dysregulation of TRPC5 interrupts Ca2+ homeostasis in cancer cells, which activates signaling pathways that are highly associated with cancer progression, especially cancer chemoresistance. Based on the important role of TRPC5, we also discuss the potential of TRPC5 as a target for therapeutic intervention. Either direct targeting of TRPC5 or indirect interruption of TRPC5-related signaling pathways may effectively overcome cancer chemoresistance. PMID:26657058

  8. TRP Channel Cannabinoid Receptors in Skin Sensation, Homeostasis, and Inflammation

    PubMed Central

    2015-01-01

    In the skin, cannabinoid lipids, whether of endogenous or exogenous origin, are capable of regulating numerous sensory, homeostatic, and inflammatory events. Although many of these effects are mediated by metabotropic cannabinoid receptors, a growing body of evidence has revealed that multiple members of the transient receptor potential (TRP) ion channel family can act as “ionotropic cannabinoid receptors”. Furthermore, many of these same TRP channels are intimately involved in cutaneous processes that include the initiation of pain, temperature, and itch perception, the maintenance of epidermal homeostasis, the regulation of hair follicles and sebaceous glands, and the modulation of dermatitis. Ionotropic cannabinoid receptors therefore represent potentially attractive targets for the therapeutic use of cannabinoids to treat sensory and dermatological diseases. Furthermore, the interactions between neurons and other cell types that are mediated by cutaneous ionotropic cannabinoid receptors are likely to be recapitulated during physiological and pathophysiological processes in the central nervous system and elsewhere, making the skin an ideal setting in which to dissect general complexities of cannabinoid signaling. PMID:24915599

  9. The transient receptor potential family of ion channels

    PubMed Central

    2011-01-01

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

  10. The transient receptor potential family of ion channels.

    PubMed

    Nilius, Bernd; Owsianik, Grzegorz

    2011-01-01

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

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

    PubMed

    Kawai, Fusao; Miyachi, Ei-ichi

    2007-11-01

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

  12. Function and regulation of endothelin type A receptor-operated transient receptor potential canonical channels.

    PubMed

    Horinouchi, Takahiro; Terada, Koji; Higa, Tsunaki; Aoyagi, Hiroyuki; Nishiya, Tadashi; Suzuki, Hiroyuki; Miwa, Soichi

    2011-01-01

    The purpose of this study is to identify transient receptor potential canonical (TRPC) channels responsible for receptor-operated Ca(2+) entry (ROCE) triggered by activation of endothelin type A receptor (ET(A)R) and to clarify the importance of calmodulin (CaM) / inositol 1,4,5-trisphosphate (IP(3)) receptor binding (CIRB) domain at the C terminus of TRPC channels in ET(A)R-activated channel regulation. In HEK293 cells coexpressing ET(A)R and one of seven TRPC isoforms, ET(A)R stimulation induced ROCE through TRPC3, TRPC5, TRPC6, and TRPC7. The TRPC3- and TRPC6-mediated ROCE was inhibited by selective inhibitors of G(q) protein, phospholipase C (PLC), and CaM. The CIRB domain deletion mutants of TRPC3 and TRPC6 failed to induce ET(A)R-mediated ROCE. Either deletion of the CIRB domain or pharmacological inhibition of CaM did not inhibit the targeting of these channels to the plasma membrane. These results suggest that 1) TRPC3, TRPC5, TRPC6, and TRPC7 can function as ET(A)R-operated Ca(2+) channels; 2) G(q) protein, PLC, and CaM are involved in TRPC3- and TRPC6-mediated ROCE; 3) ET(A)R-mediated activation of TRPC3 and TRPC6 requires the CIRB domain; and 4) abolition of ET(A)R-induced ROCE by CIRB domain deletion and CaM inhibition is due to loss of CaM binding to the channels but not loss of cell surface TRPC3 and TRPC6. PMID:22129540

  13. Ca(2+)-permeable AMPA and NMDA receptor channels in basket cells of rat hippocampal dentate gyrus.

    PubMed Central

    Koh, D S; Geiger, J R; Jonas, P; Sakmann, B

    1995-01-01

    1. Glutamate receptor (GluR) channels were studied in basket cells in the dentate gyrus of rat hippocampal slices. Basket cells were identified by their location, dendritic morphology and high frequency of action potentials generated during sustained current injection. 2. Dual-component currents were activated by fast application of glutamate to outside-out membrane patches isolated from basket cell somata (10 microM glycine, no external Mg2+). The fast component was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the slow component by D-2-amino-5-phosphonopentanoic acid (D-AP5). This suggests that the two components were mediated by alpha-amino-3- hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR)/kainate receptor and N-methyl-D-aspartate receptor (NMDAR) channels, respectively. The mean ratio of the peak current of the NMDAR component to that of the AMPAR/kainate receptor component was 0.22 (1 ms pulses of 10 mM glutamate). 3. The AMPAR/kainate receptor component, which was studied in isolation in the presence of D-AP5, was identified as AMPAR mediated on the basis of the preferential activation by AMPA as compared with kainate, the weak desensitization of kainate-activated currents, the cross-desensitization between AMPA and kainate, and the reduction of desensitization by cyclothiazide. 4. Deactivation of basket cell AMPARs following 1 ms pulses of glutamate occurred with a time constant (tau) of 1.2 +/- 0.1 ms (mean +/- S.E.M.). During 100 ms glutamate pulses AMPARs desensitized with a tau of 3.7 +/- 0.2ms. 5. The peak current-voltage (I-V) relation of AMPAR-mediated currents in Na(+)-rich extracellular solution showed a reversal potential of -4.0 +/- 2.6 mV and was characterized by a a doubly rectifying shape. The conductance of single AMPAR channels was estimated as 22.6 +/- 1.6 pS using non-stationary fluctuation analysis. AMPARs expressed in hippocampal basket cells were highly Ca2+ permeable (PCa/PK = 1.79). 6. NMDARs in

  14. Ethanol directly depresses AMPA and NMDA glutamate currents in spinal cord motor neurons independent of actions on GABAA or glycine receptors.

    PubMed

    Wang, M Y; Rampil, I J; Kendig, J J

    1999-07-01

    Ethanol is a general anesthetic agent as defined by abolition of movement in response to noxious stimulation. This anesthetic endpoint is due to spinal anesthetic actions. This study was designed to test the hypothesis that ethanol acts directly on motor neurons to inhibit excitatory synaptic transmission at glutamate receptors. Whole cell recordings were made in visually identified motor neurons in spinal cord slices from 14- to 23-day-old rats. Currents were evoked by stimulating a dorsal root fragment or by brief pulses of glutamate. Ethanol at general anesthetic concentrations (50-200 mM) depressed both responses. Ethanol also depressed glutamate-evoked responses in the presence of tetrodotoxin (300 nM), showing that its actions are postsynaptic. Block of inhibitory gamma-aminobutyric acidA and glycine receptors by bicuculline (50 microM) and strychnine (5 microM), respectively, did not significantly reduce the effects of ethanol on glutamate currents. Ethanol also depressed glutamate-evoked currents when the inhibitory receptors were blocked and either D, L-2-amino-5-phosphonopentanoic acid (40 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione disodium (10 microM) were applied to block N-methyl-D-aspartate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors, respectively. The results show that ethanol exerts direct depressant effects on both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate glutamate currents in motor neurons. Enhancement of gamma-aminobutyric acidA and glycine inhibition is not required for this effect. Direct depression of glutamatergic excitatory transmission by a postsynaptic action on motor neurons thus may contribute to general anesthesia as defined by immobility in response to a noxious stimulus. PMID:10381800

  15. Acid-sensing ion channels and transient-receptor potential ion channels in zebrafish taste buds.

    PubMed

    Levanti, M; Randazzo, B; Viña, E; Montalbano, G; Garcia-Suarez, O; Germanà, A; Vega, J A; Abbate, F

    2016-09-01

    Sensory information from the environment is required for life and survival, and it is detected by specialized cells which together make up the sensory system. The fish sensory system includes specialized organs that are able to detect mechanical and chemical stimuli. In particular, taste buds are small organs located on the tongue in terrestrial vertebrates that function in the perception of taste. In fish, taste buds occur on the lips, the flanks, and the caudal (tail) fins of some species and on the barbels of others. In fish taste receptor cells, different classes of ion channels have been detected which, like in mammals, presumably participate in the detection and/or transduction of chemical gustatory signals. However, since some of these ion channels are involved in the detection of additional sensory modalities, it can be hypothesized that taste cells sense stimuli other than those specific for taste. This mini-review summarizes current knowledge on the presence of transient-receptor potential (TRP) and acid-sensing (ASIC) ion channels in the taste buds of teleosts, especially adult zebrafish. Up to now ASIC4, TRPC2, TRPA1, TRPV1 and TRPV4 ion channels have been found in the sensory cells, while ASIC2 was detected in the nerves supplying the taste buds. PMID:27513962

  16. 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

  17. Epithelial P2X purinergic receptor channel expression and function

    PubMed Central

    Taylor, Amanda L.; Schwiebert, Lisa M.; Smith, Jeffrey J.; King, Chris; Jones, Julie R.; Sorscher, Eric J.; Schwiebert, Erik M.

    1999-01-01

    P2X purinergic receptor (P2XR) channels bind ATP and mediate Ca2+ influx — 2 signals that stimulate secretory Cl– transport across epithelia. We tested the hypotheses that P2XR channels are expressed by epithelia and that P2XRs transduce extracellular ATP signals into stimulation of Cl– transport across epithelia. Electrophysiological data and mRNA analysis of human and mouse pulmonary epithelia and other epithelial cells indicate that multiple P2XRs are broadly expressed in these tissues and that they are active on both apical and basolateral surfaces. Because P2X-selective agonists bind multiple P2XR subtypes, and because P2X agonists stimulate Cl– transport across nasal mucosa of cystic fibrosis (CF) patients as well as across non-CF nasal mucosa, P2XRs may provide novel targets for extracellular nucleotide therapy of CF. PMID:10510328

  18. The role of transient receptor potential channels in metabolic syndrome.

    PubMed

    Liu, Daoyan; Zhu, Zhiming; Tepel, Martin

    2008-11-01

    Metabolic syndrome is correlated with increased cardiovascular risk and characterized by several factors, including visceral obesity, hypertension, insulin resistance, and dyslipidemia. Several members of a large family of nonselective cation entry channels, e.g., transient receptor potential (TRP) canonical (TRPC), vanilloid (TRPV), and melastatin (TRPM) channels, have been associated with the development of cardiovascular diseases. Thus, disruption of TRP channel expression or function may account for the observed increased cardiovascular risk in metabolic syndrome patients. TRPV1 regulates adipogenesis and inflammation in adipose tissues, whereas TRPC3, TRPC5, TRPC6, TRPV1, and TRPM7 are involved in vasoconstriction and regulation of blood pressure. Other members of the TRP family are involved in regulation of insulin secretion, lipid composition, and atherosclerosis. Although there is no evidence that a single TRP channelopathy may be the cause of all metabolic syndrome characteristics, further studies will help to clarify the role of specific TRP channels involved in the metabolic syndrome. (Hypertens Res 2008; 31: 1989-1995). PMID:19098369

  19. Classical Transient Receptor Potential 1 (TRPC1): Channel or Channel Regulator?

    PubMed Central

    Dietrich, Alexander; Fahlbusch, Meike; Gudermann, Thomas

    2014-01-01

    In contrast to other Classical Transient Receptor Potential TRPC channels the function of TRPC1 as an ion channel is a matter of debate, because it is often difficult to obtain substantial functional signals over background in response to over-expression of TRPC1 alone. Along these lines, heterologously expressed TRPC1 is poorly translocated to the plasma membrane as a homotetramer and may not function on its own physiologically, but may rather be an important linker and regulator protein in heteromeric TRPC channel tetramers. However, due to the lack of specific TRPC1 antibodies able to detect native TRPC1 channels in primary cells, identification of functional TRPC1 containing heteromeric TRPC channel complexes in the plasma membrane is still challenging. Moreover, an extended TRPC1 cDNA, which was recently discovered, may seriously question results obtained in heterologous expression systems transfected with shortened cDNA versions. Therefore, this review will focus on the current status of research on TRPC1 function obtained in primary cells and a TRPC1-deficient mouse model. PMID:25268281

  20. Transient receptor potential cation channels in visceral sensory pathways

    PubMed Central

    Blackshaw, L Ashley

    2014-01-01

    The extensive literature on this subject is in direct contrast to the limited range of clinical uses for ligands of the transient receptor potential cation channels (TRPs) in diseases of the viscera. TRPV1 is the most spectacular example of this imbalance, as it is in other systems, but it is nonetheless the only TRP target that is currently targeted clinically in bladder sensory dysfunction. It is not clear why this discrepancy exists, but a likely answer is in the promiscuity of TRPs as sensors and transducers for environmental mechanical and chemical stimuli. This review first describes the different sensory pathways from the viscera, and on which nociceptive and non-nociceptive neurones within these pathways TRPs are expressed. They not only fulfil roles as both mechano-and chemo-sensors on visceral afferents, but also form an effector mechanism for cell activation after activation of GPCR and cytokine receptors. Their role may be markedly changed in diseased states, including chronic pain and inflammation. Pain presents the most obvious potential for further development of therapeutic interventions targeted at TRPs, but forms of inflammation are emerging as likely to benefit also. However, despite much basic research, we are still at the beginning of exploring such potential in visceral sensory pathways. LINKED ARTICLES This article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-10 PMID:24641218

  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. Melatonin receptor and KATP channel modulation in experimental vascular dementia.

    PubMed

    Singh, Prabhat; Gupta, Surbhi; Sharma, Bhupesh

    2015-04-01

    Cerebrovascular and cardiovascular diseases are stated as important risk factors of vascular dementia (VaD) and other cognitive disorders. In the central nervous system, melatonin (MT1/MT2) as well as serotonin subtype 2C (5-HT2C) receptors is pharmacologically associated with various neurological disorders. Brain mitochondrial potassium channels have been reported for their role in neuroprotection. This study has been structured to investigate the role of agomelatine, a melatonergic MT1/MT2 agonist and nicorandil, a selective ATP sensitive potassium (KATP) channel opener in renal artery ligation (two-kidney-one-clip: 2K1C) hypertension induced endothelial dysfunction, brain damage and VaD. 2K1C-renovascular hypertension has increased mean arterial blood pressure (MABP), impaired memory (elevated plus maze and Morris water maze), endothelial function, reduced serum nitrite/nitrate and increased brain damage (TTC staining of brain sections). Furthermore, 2K1C animals have shown high levels of oxidative stress in serum (increased thiobarbituric acid reactive species-TBARS with decreased levels of glutathione-GSH, superoxide dismutase-SOD and catalase-CAT), in the aorta (increased aortic superoxide anion) and in the brain (increased TBARS with decreased GSH, SOD and CAT). 2K1C has also induced a significant increase in brain inflammation (myeloperoxidase-MPO levels), acetylcholinesterase activity (AChE) and calcium levels. Impairment in mitochondrial complexes like NADH dehydrogenase (complex-I), succinate dehydrogenase (complex-II) and cytochrome oxidase (complex-IV) was also noted in 2K1C animals. Administration of agomelatine, nicorandil and donepezil significantly attenuated 2K1C-hypertension induced impairments in memory, endothelial function, nitrosative stress, mitochondrial dysfunction, inflammation and brain damage. Therefore, modulators of MT1/MT2 receptors and KATP channels may be considered as potential agents for the management of renovascular

  3. Transient Receptor Potential Channels and Corneal Stromal Inflammation.

    PubMed

    Okada, Yuka; Reinach, Peter S; Shirai, Kumi; Kitano-Izutani, Ai; Miyajima, Masayasu; Yamanaka, Osamu; Sumioka, Takayoshi; Saika, Shizuya

    2015-11-01

    Corneal transparency is dependent on the maintenance of the structural integrity and functional activity of its epithelial and endothelial limiting layers and the stroma. Different transient receptor potential (TRP) channel subtypes are expressed in cells and on corneal sensory nerve endings. They serve as sensors and transducers of environmental stimuli that can reduce tissue transparency. These nonselective cation channels are members of a superfamily sharing TRP box protein sequence homology having 6 membrane spanning domains with a pore between the fifth and sixth segments. TRP channels are composed of 4 monomeric subunits that oligomerize in homomeric or heteromeric configurations derived from different TRP subtypes belonging to the same or any of 6 different subfamilies. TRP subfamily members identified in the cornea include those belonging to the canonical, vanilloid, ankyrin, or melastatin subfamilies. In this review, we specifically focus on the functional roles of TRPV1 and TRPA1 expression in the cornea as their activation provides adaptive nociceptive and immune responses to noxious environmental stresses such as irritating ligands, temperature fluctuations, rises in ambient osmolarity, mechanical stretch, decline in pH, and tissue injury. Our previous studies have indicated that TRPV1 and TRPA1 subtypes are potential drug targets for improving corneal wound healing after alkali burns, because injury-induced fibrosis, neovascularization, and inflammation in either TRPV1 or TRPA1 gene-silenced mice were all significantly reduced. PMID:26448171

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-01-01

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

  6. Glycine311, a determinant of paxilline block in BK channels: a novel bend in the BK S6 helix

    PubMed Central

    Zhou, Yu; Tang, Qiong-Yao; Xia, Xiao-Ming

    2010-01-01

    The tremorogenic fungal metabolite, paxilline, is widely used as a potent and relatively specific blocker of Ca2+- and voltage-activated Slo1 (or BK) K+ channels. The pH-regulated Slo3 K+ channel, a Slo1 homologue, is resistant to blockade by paxilline. Taking advantage of the marked differences in paxilline sensitivity and the homology between subunits, we have examined the paxilline sensitivity of a set of chimeric Slo1/Slo3 subunits. Paxilline sensitivity is associated with elements of the S5–P loop–S6 module of the Slo1 channel. Replacement of the Slo1 S5 segment or the second half of the P loop results in modest changes in paxilline sensitivity. Replacing the Slo1 S6 segment with the Slo3 sequence abolishes paxilline sensitivity. An increase in paxilline affinity and changes in block kinetics also result from replacing the first part of the Slo1 P loop, the so-called turret, with Slo3 sequence. The Slo1 and Slo3 S6 segments differ at 10 residues. Slo1-G311S was found to markedly reduce paxilline block. In constructs with a Slo3 S6 segment, S300G restored paxilline block, but most effectively when paired with a Slo1 P loop. Other S6 residues differing between Slo1 and Slo3 had little influence on paxilline block. The involvement of Slo1 G311 in paxilline sensitivity suggests that paxilline may occupy a position within the central cavity or access its blocking position through the central cavity. To explain the differences in paxilline sensitivity between Slo1 and Slo3, we propose that the G311/S300 position in Slo1 and Slo3 underlies a structural difference between subunits in the bend of S6, which influences the occupancy by paxilline. PMID:20421373

  7. Extrasynaptic localization of glycine receptors in the rat supraoptic nucleus: further evidence for their involvement in glia-to-neuron communication.

    PubMed

    Deleuze, C; Alonso, G; Lefevre, I A; Duvoid-Guillou, A; Hussy, N

    2005-01-01

    Neurons of the rat supraoptic nucleus (SON) express glycine receptors (GlyRs), which are implicated in the osmoregulation of neuronal activity. The endogenous agonist of the receptors has been postulated to be taurine, shown to be released from astrocytes. We here provide additional pieces of evidence supporting the absence of functional glycinergic synapses in the SON. First, we show that blockade of GlyRs with strychnine has no effect on either the amplitude or frequency of miniature inhibitory postsynaptic currents recorded in SON neurons, whereas they were all suppressed by the GABA(A) antagonist gabazine. Then, double immunostaining of sections with presynaptic markers and either GlyR or GABA(A) receptor (GABA(A)R) antibodies indicates that, in contrast with GABA(A)Rs, most GlyR membrane clusters are not localized facing presynaptic terminals, indicative of their extrasynaptic localization. Moreover, we found a striking anatomical association between SON GlyR clusters and glial fibrillary acidic protein (GFAP)-positive astroglial processes, which contain high levels of taurine. This type of correlation is specific to GlyRs, since GABA(A)R clusters show no association with GFAP-positive structures. These results substantiate and strengthen the concept of extrasynaptic GlyRs mediating a paracrine communication between astrocytes and neurons in the SON. PMID:15893641

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

    PubMed Central

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

    2012-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1987-12-01

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

  10. AMPA receptors undergo channel arrest in the anoxic turtle cortex.

    PubMed

    Pamenter, Matthew Edward; Shin, Damian Seung-Ho; Buck, Leslie Thomas

    2008-02-01

    Without oxygen, all mammals suffer neuronal injury and excitotoxic cell death mediated by overactivation of the glutamatergic N-methyl-D-aspartate receptor (NMDAR). The western painted turtle can survive anoxia for months, and downregulation of NMDAR activity is thought to be neuroprotective during anoxia. NMDAR activity is related to the activity of another glutamate receptor, the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR). AMPAR blockade is neuroprotective against anoxic insult in mammals, but the role of AMPARs in the turtle's anoxia tolerance has not been investigated. To determine whether AMPAR activity changes during hypoxia or anoxia in the turtle cortex, whole cell AMPAR currents, AMPAR-mediated excitatory postsynaptic potentials (EPSPs), and excitatory postsynaptic currents (EPSCs) were measured. The effect of AMPAR blockade on normoxic and anoxic NMDAR currents was also examined. During 60 min of normoxia, evoked peak AMPAR currents and the frequencies and amplitudes of EPSPs and EPSCs did not change. During anoxic perfusion, evoked AMPAR peak currents decreased 59.2 +/- 5.5 and 60.2 +/- 3.5% at 20 and 40 min, respectively. EPSP frequency (EPSP(f)) and amplitude decreased 28.7 +/- 6.4% and 13.2 +/- 1.7%, respectively, and EPSC(f) and amplitude decreased 50.7 +/- 5.1% and 51.3 +/- 4.7%, respectively. In contrast, hypoxic (Po(2) = 5%) AMPAR peak currents were potentiated 56.6 +/- 20.5 and 54.6 +/- 15.8% at 20 and 40 min, respectively. All changes were reversed by reoxygenation. AMPAR currents and EPSPs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In neurons pretreated with CNQX, anoxic NMDAR currents were reversibly depressed by 49.8 +/- 7.9%. These data suggest that AMPARs may undergo channel arrest in the anoxic turtle cortex. PMID:18056983

  11. Deactivation of the Arabidopsis BRI1 receptor kinase by autophosphorylation within the glycine-rich loop involved in ATP binding

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The activity of the dual-specificity brassinosteroid receptor kinase, BRI1, reflects the balance between phosphorylation-dependent activation and several potential mechanisms for deactivation of the receptor. In the present report, we identify regions of the juxtamembrane domain that are essential f...

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

    PubMed Central

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

    1983-01-01

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

  13. A Glycine soja ABA-responsive receptor-like cytoplasmic kinase, GsRLCK, positively controls plant tolerance to salt and drought stresses.

    PubMed

    Sun, XiaoLi; Sun, Mingzhe; Luo, Xiao; Ding, XiaoDong; Ji, Wei; Cai, Hua; Bai, Xi; Liu, XiaoFei; Zhu, YanMing

    2013-06-01

    Receptor such as protein kinases are proposed to work as sensors to initiate signaling cascades in higher plants. However, little is known about the precise functions of receptor such as protein kinases in abiotic stress response in plants, especially in wild soybean. Here, we focused on characterization of the biological functions of a receptor-like cytoplasmic serine/threonine protein kinase gene, GsRLCK, which was previously identified as a putative salt-alkali stress-related gene from the transcriptome profiles of Glycine soja. Bioinformatic analysis showed that GsRLCK protein contained a conserved kinase catalytic domain and two transmembrane domains at the N-terminus, but no typical extracellular domain. Consistently, GsRLCK-eGFP fusion protein was observed on the plasma membrane, but eGFP alone was distributing throughout the cytoplasm in onion epidermal cells. Quantitative real-time PCR analysis revealed the induced expression of GsRLCK by ABA, salt, alkali, and drought stresses. However, the expression levels of GsRLCK seemed to be similar in different tissues, except soybean pod. Phenotypic assays demonstrated that GsRLCK overexpression decreased ABA sensitivity and altered expression levels of ABA-responsive genes. Furthermore, we also found that GsRLCK conferred increased tolerance to salt and drought stresses and increased expression levels of a handful of stress-responsive genes, when overexpressing in Arabidopsis. In a word, we gave exact evidence that GsRLCK was a novel receptor-like cytoplasmic protein kinase and played a crucial role in plant responses to ABA, salt, and drought stresses. PMID:23494614

  14. Altered mnemonic functions and resistance to N-METHYL-d-Aspartate receptor antagonism by forebrain conditional knockout of glycine transporter 1.

    PubMed

    Singer, P; Yee, B K; Feldon, J; Iwasato, T; Itohara, S; Grampp, T; Prenosil, G; Benke, D; Möhler, H; Boison, D

    2009-06-30

    Converging evidence from pharmacological and molecular studies has led to the suggestion that inhibition of glycine transporter 1 (GlyT1) constitutes an effective means to boost N-methyl-d-aspartate receptor (NMDAR) activity by increasing the extra-cellular concentration of glycine in the vicinity of glutamatergic synapses. However, the precise extent and limitation of this approach to alter cognitive function, and therefore its potential as a treatment strategy against psychiatric conditions marked by cognitive impairments, remain to be fully examined. Here, we generated mutant mice lacking GlyT1 in the entire forebrain including neurons and glia. This conditional knockout system allows a more precise examination of GlyT1 downregulation in the brain on behavior and cognition. The mutation was highly effective in attenuating the motor-stimulating effect of acute NMDAR blockade by phencyclidine, although no appreciable elevation in NMDAR-mediated excitatory postsynaptic currents (EPSC) was observed in the hippocampus. Enhanced cognitive performance was observed in spatial working memory and object recognition memory while spatial reference memory and associative learning remained unaltered. These findings provide further credence for the potential cognitive enhancing effects of brain GlyT1 inhibition. At the same time, they indicated potential phenotypic differences when compared with other constitutive and conditional GlyT1 knockout lines, and highlighted the possibility of a functional divergence between the neuronal and glia subpopulations of GlyT1 in the regulation of learning and memory processes. The relevance of this distinction to the design of future GlyT1 blockers as therapeutic tools in the treatment of cognitive disorders remains to be further investigated. PMID:19332109

  15. Transient Receptor Potential Canonical 1 (TRPC1) Channels as Regulators of Sphingolipid and VEGF Receptor Expression

    PubMed Central

    Asghar, Muhammad Yasir; Magnusson, Melissa; Kemppainen, Kati; Sukumaran, Pramod; Löf, Christoffer; Pulli, Ilari; Kalhori, Veronica; Törnquist, Kid

    2015-01-01

    The identity of calcium channels in the thyroid is unclear. In human follicular thyroid ML-1 cancer cells, sphingolipid sphingosine 1-phosphate (S1P), through S1P receptors 1 and 3 (S1P1/S1P3), and VEGF receptor 2 (VEGFR2) stimulates migration. We show that human thyroid cells express several forms of transient receptor potential canonical (TRPC) channels, including TRPC1. In TRPC1 knockdown (TRPC1-KD) ML-1 cells, the basal and S1P-evoked invasion and migration was attenuated. Furthermore, the expression of S1P3 and VEGFR2 was significantly down-regulated. Transfecting wild-type ML-1 cells with a nonconducting TRPC1 mutant decreased S1P3 and VEGFR2 expression. In TRPC1-KD cells, receptor-operated calcium entry was decreased. To investigate whether the decreased receptor expression was due to attenuated calcium entry, cells were incubated with the calcium chelator BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). In these cells, and in cells where calmodulin and calmodulin-dependent kinase were blocked pharmacologically, S1P3 and VEGFR2 expression was decreased. In TRPC1-KD cells, both hypoxia-inducible factor 1α expression and the secretion and activity of MMP2 and MMP9 were attenuated, and proliferation was decreased in TRPC1-KD cells. This was due to a prolonged G1 phase of the cell cycle, a significant increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27, and a decrease in the expression of cyclin D2, cyclin D3, and CDK6. Transfecting TRPC1 to TRPC1-KD cells rescued receptor expression, migration, and proliferation. Thus, the expression of S1P3 and VEGFR2 is mediated by a calcium-dependent mechanism. TRPC1 has a crucial role in this process. This regulation is important for the invasion, migration, and proliferation of thyroid cancer cells. PMID:25971967

  16. A case of autism with an interstitial deletion on 4q leading to hemizygosity for genes encoding for glutamine and glycine neurotransmitter receptor sub-units (AMPA 2, GLRA3, GLRB) and neuropeptide receptors NPY1R, NPY5R

    PubMed Central

    Ramanathan, Subhadra; Woodroffe, Abigail; Flodman, Pamela L; Mays, Lee Z; Hanouni, Mona; Modahl, Charlotte B; Steinberg-Epstein, Robin; Bocian, Maureen E; Spence, M Anne; Smith, Moyra

    2004-01-01

    Background Autism is a pervasive developmental disorder characterized by a triad of deficits: qualitative impairments in social interactions, communication deficits, and repetitive and stereotyped patterns of behavior. Although autism is etiologically heterogeneous, family and twin studies have established a definite genetic basis. The inheritance of idiopathic autism is presumed to be complex, with many genes involved; environmental factors are also possibly contributory. The analysis of chromosome abnormalities associated with autism contributes greatly to the identification of autism candidate genes. Case presentation We describe a child with autistic disorder and an interstitial deletion on chromosome 4q. This child first presented at 12 months of age with developmental delay and minor dysmorphic features. At 4 years of age a diagnosis of Pervasive Developmental Disorder was made. At 11 years of age he met diagnostic criteria for autism. Cytogenetic studies revealed a chromosome 4q deletion. The karyotype was 46, XY del 4 (q31.3-q33). Here we report the clinical phenotype of the child and the molecular characterization of the deletion using molecular cytogenetic techniques and analysis of polymorphic markers. These studies revealed a 19 megabase deletion spanning 4q32 to 4q34. Analysis of existing polymorphic markers and new markers developed in this study revealed that the deletion arose on a paternally derived chromosome. To date 33 genes of known or inferred function are deleted as a consequence of the deletion. Among these are the AMPA 2 gene that encodes the glutamate receptor GluR2 sub-unit, GLRA3 and GLRB genes that encode glycine receptor subunits and neuropeptide Y receptor genes NPY1R and NPY5R. Conclusions The deletion in this autistic subject serves to highlight specific autism candidate genes. He is hemizygous for AMPA 2, GLRA3, GLRB, NPY1R and NPY5R. GluR2 is the major determinant of AMPA receptor structure. Glutamate receptors maintain structural

  17. Intracellular calcium channels: inositol-1,4,5-trisphosphate receptors

    PubMed Central

    Fedorenko, Olena A.; Popugaeva, Elena; Enomoto, Masahiro; Stathopulos, Peter B.; Ikura, Mitsuhiko; Bezprozvanny, Ilya

    2014-01-01

    The inositol-1,4,5-trisphosphate receptors (InsP3Rs) are the major intracellular Ca2+-release channels in cells. Activity of InsP3Rs is essential for elementary and global Ca2+ events in the cell. There are three InsP3Rs isoforms that are present in mammalian cells. In this review review we will focus primarily on InsP3R type 1. The InsP3R1 is a predominant isoform in neurons and it is most extensively studied isoform. Combination of biophysical and structural methods revealed key mechanisms of InsP3R function and modulation. Cell biological and biochemical studies lead to identification of a large number of InsP3R-binding proteins. InsP3Rs are involved in the regulation of numerous physiological processes, including learning and memory, proliferation, differentiation, development and cell death. Malfunction of InsP3R1 play a role in a number of neurodegenerative disorders and other disease states. InsP3Rs represent a potentially valuable drug target for treatment of these disorders and for modulating activity of neurons and other cells. Future studies will provide better understanding of physiological functions of InsP3Rs in health and disease. PMID:24300389

  18. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na/sup +/ channel interaction

    SciTech Connect

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-12

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na/sup +/ channels is a coupled event mediated by guanine nucleotide binding protein(s) (G-protein(s)). These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of (/sup 3/H) acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of (/sup 3/H)batrachotoxin to Na/sup +/ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced /sup 22/Na/sup +/ uptake in the presence and absence of tetrodotoxin, which blocks Na/sup +/ channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na/sup +/channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na/sup +/ channel-is such that at resting potential the muscarinic receptor induces opening of Na/sup +/ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.

  19. The N-terminal domain of GluR6-subtype glutamate receptor ion channels

    SciTech Connect

    Kumar, Janesh; Schuck, Peter; Jin, Rongsheng; Mayer, Mark L.

    2009-09-25

    The amino-terminal domain (ATD) of glutamate receptor ion channels, which controls their selective assembly into AMPA, kainate and NMDA receptor subtypes, is also the site of action of NMDA receptor allosteric modulators. Here we report the crystal structure of the ATD from the kainate receptor GluR6. The ATD forms dimers in solution at micromolar protein concentrations and crystallizes as a dimer. Unexpectedly, each subunit adopts an intermediate extent of domain closure compared to the apo and ligand-bound complexes of LIVBP and G protein-coupled glutamate receptors (mGluRs), and the dimer assembly has a markedly different conformation from that found in mGluRs. This conformation is stabilized by contacts between large hydrophobic patches in the R2 domain that are absent in NMDA receptors, suggesting that the ATDs of individual glutamate receptor ion channels have evolved into functionally distinct families.

  20. Transient neuromotor phenotype in transgenic spastic mice expressing low levels of glycine receptor β-subunit: an animal model of startle disease

    PubMed Central

    Becker, Lore; Hartenstein, Bettina; Schenkel, Johannes; Kuhse, Jochen; Betz, Heinrich; Weiher, Hans

    2000-01-01

    Startle disease or hereditary hyperekplexia has been shown to result from mutations in the α1-subunit gene of the inhibitory glycine receptor (GlyR). In hyperekplexia patients, neuromotor symptoms generally become apparent at birth, improve with age, and often disappear in adulthood. Loss-of-function mutations of GlyR α or β-subunits in mice show rather severe neuromotor phenotypes. Here, we generated mutant mice with a transient neuromotor deficiency by introducing a GlyR β transgene into the spastic mouse (spa/spa), a recessive mutant carrying a transposon insertion within the GlyR β-subunit gene. In spa/spa TG456 mice, one of three strains generated with this construct, which expressed very low levels of GlyR β transgene-dependent mRNA and protein, the spastic phenotype was found to depend upon the transgene copy number. Notably, mice carrying two copies of the transgene showed an age-dependent sensitivity to tremor induction, which peaked at ∼ 3–4 weeks postnatally. This closely resembles the development of symptoms in human hyperekplexia patients, where motor coordination significantly improves after adolescence. The spa/spa TG456 line thus may serve as an animal model of human startle disease. PMID:10651857

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

    NASA Astrophysics Data System (ADS)

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

    2001-06-01

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

  2. Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

    PubMed Central

    Tank, D W; Huganir, R L; Greengard, P; Webb, W W

    1983-01-01

    Small unilamellar vesicles containing purified and reconstituted nicotinic acetylcholine receptors from Torpedo electroplax have been fused by a simple freeze-thaw procedure to form large liposomes. Giga-seal patch-recording techniques were used to form isolated patches of liposome-membrane and to measure single-channel properties of the reconstituted receptor-ion channel complex. The observed properties are quantitatively similar to those reported for vertebrate muscle nicotinic acetylcholine receptor species recorded in situ. The results demonstrate that the pentameric complex consisting of the alpha 2 beta gamma delta subunits is fully functional. The methods used in these experiments should be useful in studying the effects of chemical alterations on the properties of acetylcholine receptor channels as well as other types of purified and reconstituted ion channels. PMID:6308673

  3. 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

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

    PubMed

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

    2016-09-01

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

  5. Inhibition of mechanosensitivity in visceral primary afferents by GABAB receptors involves calcium and potassium channels.

    PubMed

    Page, A J; O'Donnell, T A; Blackshaw, L A

    2006-01-01

    GABA(B) receptors inhibit mechanosensitivity of visceral afferents. This is associated with reduced triggering of events that lead to gastro-esophageal reflux, with important therapeutic consequences. In other neuronal systems, GABA(B) receptor activation may be linked via G-proteins to reduced N-type Ca(2+) channel opening, increased inward rectifier K(+) channel opening, plus effects on a number of intracellular messengers. Here we aimed to determine the role of Ca(2+) and K(+) channels in the inhibition of vagal afferent mechanoreceptor function by the GABA(B) receptor agonist baclofen. The responses of three types of ferret gastro-esophageal vagal afferents (mucosal, tension and tension mucosal receptors) to graded mechanical stimuli were investigated in vitro. The effects of baclofen (200 microM) alone on these responses were quantified, and the effects of baclofen in the presence of the G-protein-coupled inward rectifier potassium channel blocker Rb(+) (4.7 mM) and/or the N-type calcium channel blocker omega-conotoxin GVIA (0.1 microM). Baclofen inhibition of mucosal receptor mechanosensitivity was abolished by both blockers. Its inhibitory effect on tension mucosal receptors was partly reduced by both. The inhibitory effect of baclofen on tension receptors was unaffected. The data indicate that the inhibitory action of GABA(B) receptors is mediated via different pathways in mucosal, tension and tension mucosal receptors via mechanisms involving both N-type Ca(2+) channels and inwardly rectifying K(+) channels and others. PMID:16289839

  6. Alteration of glycine receptor immunoreactivity in the auditory brainstem of mice following three months of exposure to radiofrequency radiation at SAR 4.0 W/kg

    PubMed Central

    MASKEY, DHIRAJ; KIM, HYUNG GUN; SUH, MYUNG-WHAN; ROH, GU SEOB; KIM, MYEUNG JU

    2014-01-01

    The increasing use of mobile communication has triggered an interest in its possible effects on the regulation of neurotransmitter signals. Due to the close proximity of mobile phones to hearing-related brain regions during usage, its use may lead to a decrease in the ability to segregate sounds, leading to serious auditory dysfunction caused by the prolonged exposure to radiofrequency (RF) radiation. The interplay among auditory processing, excitation and inhibitory molecule interactions plays a major role in auditory function. In particular, inhibitory molecules, such a glycine, are predominantly localized in the auditory brainstem. However, the effects of exposure to RF radiation on auditory function have not been reported to date. Thus, the aim of the present study was to investigate the effects of exposure to RF radiation on glycine receptor (GlyR) immunoreactivity (IR) in the auditory brainstem region at 835 MHz with a specific absorption rate of 4.0 W/kg for three months using free-floating immunohistochemistry. Compared with the sham control (SC) group, a significant loss of staining intensity of neuropils and cells in the different subdivisions of the auditory brainstem regions was observed in the mice exposed to RF radiation (E4 group). A decrease in the number of GlyR immunoreactive cells was also noted in the cochlear nuclear complex [anteroventral cochlear nucleus (AVCN), 31.09%; dorsal cochlear nucleus (DCN), 14.08%; posteroventral cochlear nucleus (PVCN), 32.79%] and the superior olivary complex (SOC) [lateral superior olivary nucleus (LSO), 36.85%; superior paraolivary nucleus (SPN), 24.33%, medial superior olivary nucleus (MSO), 23.23%; medial nucleus of the trapezoid body (MNTB), 10.15%] of the mice in the E4 group. Auditory brainstem response (ABR) analysis also revealed a significant threshold elevation of in the exposed (E4) group, which may be associated with auditory dysfunction. The present study suggests that the auditory brainstem region

  7. Pharmacology of the capsaicin receptor, transient receptor potential vanilloid type-1 ion channel.

    PubMed

    Nagy, Istvan; Friston, Dominic; Valente, Jojo Sousa; Torres Perez, Jose Vicente; Andreou, Anna P

    2014-01-01

    The capsaicin receptor, transient receptor potential vanilloid type 1 ion channel (TRPV1), has been identified as a polymodal transducer molecule on a sub-set of primary sensory neurons which responds to various stimuli including noxious heat (> -42 degrees C), protons and vanilloids such as capsaicin, the hot ingredient of chilli peppers. Subsequently, TRPV1 has been found indispensable for the development of burning pain and reflex hyperactivity associated with inflammation of peripheral tissues and viscera, respectively. Therefore, TRPV1 is regarded as a major target for the development of novel agents for the control of pain and visceral hyperreflexia in inflammatory conditions. Initial efforts to introduce agents acting on TRPV1 into clinics have been hampered by unexpected side-effects due to wider than expected expression in various tissues, as well as by the complex pharmacology, of TRPV1. However, it is believed that better understanding of the pharmacological properties of TRPV1 and specific targeting of tissues may eventually lead to the development of clinically useful agents. In order to assist better understanding of TRPV1 pharmacology, here we are giving a comprehensive account on the activation and inactivation mechanisms and the structure-function relationship of TRPV1. PMID:24941664

  8. 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.

  9. Block of NMDA receptor channels by endogenous neurosteroids: implications for the agonist induced conformational states of the channel vestibule.

    PubMed

    Vyklicky, Vojtech; Krausova, Barbora; Cerny, Jiri; Balik, Ales; Zapotocky, Martin; Novotny, Marian; Lichnerova, Katarina; Smejkalova, Tereza; Kaniakova, Martina; Korinek, Miloslav; Petrovic, Milos; Kacer, Petr; Horak, Martin; Chodounska, Hana; Vyklicky, Ladislav

    2015-01-01

    N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor's ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system. PMID:26086919

  10. N-methyl-D-aspartate receptor channel blocker-like discriminative stimulus effects of nitrous oxide gas.

    PubMed

    Richardson, Kellianne J; Shelton, Keith L

    2015-01-01

    Nitrous oxide (N2O) gas is a widely used anesthetic adjunct in dentistry and medicine that is also commonly abused. Studies have shown that N2O alters the function of the N-methyl-d-aspartate (NMDA), GABAA, opioid, and serotonin receptors among others. However, the receptors systems underlying the abuse-related central nervous system effects of N2O are unclear. The present study explores the receptor systems responsible for producing the discriminative stimulus effects of N2O. B6SJLF1/J male mice trained to discriminate 10 minutes of exposure to 60% N2O + 40% oxygen versus 100% oxygen served as subjects. Both the high-affinity NMDA receptor channel blocker (+)-MK-801 maleate [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate] and the low-affinity blocker memantine partially mimicked the stimulus effects of N2O. Neither the competitive NMDA antagonist, CGS-19755 (cis-4-[phosphomethyl]-piperidine-2-carboxylic acid), nor the NMDA glycine-site antagonist, L701-324 [7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1H)-quinolinone], produced N2O-like stimulus effects. A range of GABAA agonists and positive modulators, including midazolam, pentobarbital, muscimol, and gaboxadol (4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol), all failed to produce N2O-like stimulus effects. The μ-, κ-, and δ-opioid agonists, as well as 5-hydroxytryptamine (serotonin) 1B/2C (5-HT1B/2C) and 5-HT1A agonists, also failed to produce N2O-like stimulus effects. Ethanol partially substituted for N2O. Both (+)-MK-801 and ethanol but not midazolam pretreatment also significantly enhanced the discriminative stimulus effects of N2O. Our results support the hypothesis that the discriminative stimulus effects of N2O are at least partially mediated by NMDA antagonist effects similar to those produced by channel blockers. However, as none of the drugs tested fully mimicked the stimulus effects of N2O, other mechanisms may also be involved. PMID:25368340

  11. The Role of Canonical Transient Receptor Potential Channels in Seizure and Excitotoxicity

    PubMed Central

    Zheng, Fang; Phelan, Kevin D.

    2014-01-01

    Canonical transient receptor potential (TRPC) channels are a family of polymodal cation channels with some degree of Ca2+ permeability. Although initially thought to be channels mediating store-operated Ca2+ influx, TRPC channels can be activated by stimulation of Gq-coupled G-protein coupled receptors, or by an increase in intracellular free Ca2+ concentration. Thus, activation of TRPC channels could be a common downstream event of many signaling pathways that contribute to seizure and excitotoxicity, such as N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ influx, or metabotropic glutamate receptor activation. Recent studies with genetic ablation of various TRPC family members have demonstrated that TRPC channels, in particular heteromeric TRPC1/4 channels and homomeric TRPC5 channels, play a critical role in both pilocarpine-induced acute seizures and neuronal cell death. However, exact underlying mechanisms remain to be fully elucidated, and selective TRPC modulators and antibodies with better specificity are urgently needed for future research. PMID:24722470

  12. Channel catfish, Ictalurus punctatus, chemokine receptor CXCR4 cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chemokine receptor CXCR4, a member of the G protein-coupled receptor superfamily, binds selectively CXCL12. This protein plays many important roles in immunological as well as pathophysiological functions. In this communication, we identified and characterized the channel catfish CXCR4 transcript....

  13. Mapping the energy and diffusion landscapes of membrane proteins at the cell surface using high-density single-molecule imaging and Bayesian inference: application to the multiscale dynamics of glycine receptors in the neuronal membrane.

    PubMed

    Masson, Jean-Baptiste; Dionne, Patrice; Salvatico, Charlotte; Renner, Marianne; Specht, Christian G; Triller, Antoine; Dahan, Maxime

    2014-01-01

    Protein mobility is conventionally analyzed in terms of an effective diffusion. Yet, this description often fails to properly distinguish and evaluate the physical parameters (such as the membrane friction) and the biochemical interactions governing the motion. Here, we present a method combining high-density single-molecule imaging and statistical inference to separately map the diffusion and energy landscapes of membrane proteins across the cell surface at ~100 nm resolution (with acquisition of a few minutes). Upon applying these analytical tools to glycine neurotransmitter receptors at inhibitory synapses, we find that gephyrin scaffolds act as shallow energy traps (~3 kBT) for glycine neurotransmitter receptors, with a depth modulated by the biochemical properties of the receptor-gephyrin interaction loop. In turn, the inferred maps can be used to simulate the dynamics of proteins in the membrane, from the level of individual receptors to that of the population, and thereby, to model the stochastic fluctuations of physiological parameters (such as the number of receptors at synapses). Overall, our approach provides a powerful and comprehensive framework with which to analyze biochemical interactions in living cells and to decipher the multiscale dynamics of biomolecules in complex cellular environments. PMID:24411239

  14. Functional analysis of duplicated Symbiosis Receptor Kinase (SymRK) genes during nodulation and mycorrhizal infection in soybean (Glycine max).

    PubMed

    Indrasumunar, Arief; Wilde, Julia; Hayashi, Satomi; Li, Dongxue; Gresshoff, Peter M

    2015-03-15

    Association between legumes and rhizobia results in the formation of root nodules, where symbiotic nitrogen fixation occurs. The early stages of this association involve a complex of signalling events between the host and microsymbiont. Several genes dealing with early signal transduction have been cloned, and one of them encodes the leucine-rich repeat (LRR) receptor kinase (SymRK; also termed NORK). The Symbiosis Receptor Kinase gene is required by legumes to establish a root endosymbiosis with Rhizobium bacteria as well as mycorrhizal fungi. Using degenerate primer and BAC sequencing, we cloned duplicated SymRK homeologues in soybean called GmSymRKα and GmSymRKβ. These duplicated genes have high similarity of nucleotide (96%) and amino acid sequence (95%). Sequence analysis predicted a malectin-like domain within the extracellular domain of both genes. Several putative cis-acting elements were found in promoter regions of GmSymRKα and GmSymRKβ, suggesting a participation in lateral root development, cell division and peribacteroid membrane formation. The mutant of SymRK genes is not available in soybean; therefore, to know the functions of these genes, RNA interference (RNAi) of these duplicated genes was performed. For this purpose, RNAi construct of each gene was generated and introduced into the soybean genome by Agrobacterium rhizogenes-mediated hairy root transformation. RNAi of GmSymRKβ gene resulted in an increased reduction of nodulation and mycorrhizal infection than RNAi of GmSymRKα, suggesting it has the major activity of the duplicated gene pair. The results from the important crop legume soybean confirm the joint phenotypic action of GmSymRK genes in both mycorrhizal and rhizobial infection seen in model legumes. PMID:25617765

  15. Differential contribution of the NR1- and NR2A-subunits to the selectivity filter of recombinant NMDA receptor channels.

    PubMed Central

    Wollmuth, L P; Kuner, T; Seeburg, P H; Sakmann, B

    1996-01-01

    1. The molecular determinants for the narrow constriction of recombinant N-methyl-D-aspartate (NMDA) receptor channels composed of wild-type and mutant NR1- and NR2A-subunits were studied in Xenopus oocytes. 2. The relative permeability of differently sized organic cations was used as an indicator of the size of the narrow constriction. From measured reversal potentials under bi-ionic conditions with K+ as the reference solution, permeability ratios were calculated with the Lewis equation. 3. For wild-type NMDA receptor channels, five organic cations showed clear reversal potentials, with permeability ratios (PX/PK): ammonium, 1.28; methylammonium, 0.48; dimethylammonium (DMA), 0.20; diethylammonium, 0.07; and dimethylethanol-ammonium, 0.02. 4. Mutation of the N-site asparagine (N) to glutamine (Q) at homologous positions in either NR1 (position 598) or NR2A (position 595) increased the permeability of DMA relative to wild-type channels about equally. However, for larger sized organic cations, the NR1(N598Q) mutation had stronger effects on increasing their permeability whereas the NR2A(N595Q) mutation was without effect. These changes in organic cation permeability suggest that the NR1(N598Q) mutation increases the pore size while the NR2A(N595Q) mutation does not. 5. Channels in which the NR1 N-site asparagine was replaced by the smaller glycine (G), NR1(N598G)-NR2A, showed the largest increase in pore size of all sites examined in either subunit. In contrast, in the NR2A-subunit the same N-site substitution to glycine produced only small effects on pore size. 6. For the NR2A-subunit, an asparagine residue (position 596) on the C-terminal side of the N-site, when mutated to larger or smaller sized amino acids, produced large, volume-specific effects on pore size. The mutant channel NR1-NR2A(N596G) had the largest increase in pore size of all sites examined in the NR2A-subunit. In contrast, mutation of the homologous position in the NR1-subunit had no effect on

  16. Mitochondrial Ryanodine Receptors and Other Mitochondrial Ca2+ Permeable Channels

    PubMed Central

    Ryu, Shin-Young; Beutner, Gisela; Dirksen, Robert T.; Kinnally, Kathleen W.; Sheu, Shey-Shing

    2010-01-01

    Ca2+ channels that underlie mitochondrial Ca2+ transport first reported decades ago have now just recently been precisely characterized electrophysiologically. Numerous data indicate that mitochondrial Ca2+ uptake via these channels regulates multiple intracellular processes by shaping cytosolic and mitochondrial Ca2+ transients, as well as altering the cellular metabolic and redox state. On the other hand, mitochondrial Ca2+ overload also initiates a cascade of events that leads to cell death. Thus, characterization of mitochondrial Ca2+ channels is central to a comprehensive understanding of cell signaling. Here, we discuss recent progresses in the biophysical and electrophysiological characterization of several distinct mitochondrial Ca2+ channels. PMID:20096690

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

    PubMed Central

    Corradi, Jeremías; Gumilar, Fernanda; Bouzat, Cecilia

    2009-01-01

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

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

    PubMed Central

    Wolstenholme, Adrian J.

    2011-01-01

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

  19. The opioid peptide dynorphin directly blocks NMDA receptor channels in the rat.

    PubMed Central

    Chen, L; Gu, Y; Huang, L Y

    1995-01-01

    1. The actions of dynorphin on N-methyl-D-aspartate (NMDA) responses were examined in acutely dissociated trigeminal neurons in rat. Whole-cell and single-channel currents were recorded using the patch clamp technique. 2. Dynorphins reduced NMDA-activated currents (INMDA). The IC50 was 0.25 microM for dynorphin (1-32), 1.65 microM for dynorphin (1-17) and 1.8 microM for dynorphin (1-13). 3. The blocking action of dynorphin is voltage independent. 4. The inhibitory action of dynorphin cannot be blocked by high concentration of the non-selective opioid receptor antagonist naloxone, nor by the specific kappa-opioid receptor antagonist nor-Binaltorphimine (nor-BNI). 5. Single-channel analyses indicate that dynorphin reduces the fraction of time the channel is open without altering the channel conductance. 6. We propose that dynorphin acts directly on NMDA receptors. PMID:7537820

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

    PubMed Central

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

    2004-01-01

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

  1. Suppression of spreading depolarization and stabilization of dendritic spines by GLYX-13, an NMDA receptor glycine-site functional partial agonist.

    PubMed

    Zhang, Xiao-Lei; Shuttleworth, C William; Moskal, Joseph R; Stanton, Patric K

    2015-11-01

    Cortical spreading depolarization (SD) is a slow self-propagating wave of mass cellular depolarization in brain tissue, thought to be the underlying cause of migraine scintillating scotoma and aura, and associated with stroke, traumatic brain injury, and termination of status epilepticus. The N-methyl-d-aspartate subtype of glutamate receptor (NMDAR), which gates influx of calcium and is an important trigger of long-term synaptic plasticity, is also a contributor to the initiation and propagation of SD. The current study tested the potential of pharmacological modulation of NMDAR activity through the obligatory co-agonist binding site, to suppress the initiation of SD, and modulate the effects of SD on dendritic spine morphology, in in vitro hippocampal slices. A novel NMDAR functional glycine site partial agonist, GLYX-13, sometimes completely prevented the induction of SD and consistently slowed its rate of propagation. The passage of SD through the hippocampal CA1 region produced a rapid retraction of dendritic spines which reversed after neuronal depolarization had recovered. GLYX-13 improved the rate and extent of return of dendritic spines to their original sizes and locations following SD, suggesting that NMDAR modulators can protect synaptic connections in the brain from structural alterations elicited by SD. These data indicate that NMDAR modulation to renormalize activity may be an effective new treatment strategy for suppression or amelioration of the contribution of SD to short and long-term symptoms of migraine attacks, as well as the effects of SD on tissue damaged by stroke or traumatic brain injury. PMID:26244282

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

    PubMed

    Ivanova, T T; Caprio, J

    1993-12-01

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

  3. Transient receptor potential (TRP) channels, vascular tone and autoregulation of cerebral blood flow.

    PubMed

    Brayden, Joseph E; Earley, Scott; Nelson, Mark T; Reading, Stacey

    2008-09-01

    Members of the transient receptor potential (TRP) channel superfamily are present in vascular smooth muscle cells and play important roles in the regulation of vascular contractility. The TRPC3 and TRPC6 channels are activated by stimulation of several excitatory receptors in vascular smooth muscle cells. Activation of these channels leads to myocyte depolarization, which stimulates Ca2+ entry via voltage-dependent Ca2+ channels (VDCC), leading to vasoconstriction. The TRPV4 channels in arterial myocytes are activated by epoxyeicosatrienoic acids, and activation of the channels enhances Ca2+ spark and transient Ca2+-sensitive K+ channel activity, thereby hyperpolarizing and relaxing vascular smooth muscle cells. The TRPC6 and TRPM4 channels are activated by mechanical stimulation of cerebral artery myocytes. Subsequent depolarization and activation of VDCC Ca2+ entry is directly linked to the development of myogenic tone in vitro and to autoregulation of cerebral blood flow in vivo. These findings imply a fundamental importance of TRP channels in the regulation of vascular smooth muscle tone and suggest that TRP channels could be important targets for drug therapy under conditions in which vascular contractility is disturbed (e.g. hypertension, stroke, vasospasm). PMID:18215190

  4. Ion channel profile of TRPM8 cold receptors reveals a role of TASK-3 potassium channels in thermosensation.

    PubMed

    Morenilla-Palao, Cruz; Luis, Enoch; Fernández-Peña, Carlos; Quintero, Eva; Weaver, Janelle L; Bayliss, Douglas A; Viana, Félix

    2014-09-11

    Animals sense cold ambient temperatures through the activation of peripheral thermoreceptors that express TRPM8, a cold- and menthol-activated ion channel. These receptors can discriminate a very wide range of temperatures from innocuous to noxious. The molecular mechanism responsible for the variable sensitivity of individual cold receptors to temperature is unclear. To address this question, we performed a detailed ion channel expression analysis of cold-sensitive neurons, combining bacterial artificial chromosome (BAC) transgenesis with a molecular-profiling approach in fluorescence-activated cell sorting (FACS)-purified TRPM8 neurons. We found that TASK-3 leak potassium channels are highly enriched in a subpopulation of these sensory neurons. The thermal threshold of TRPM8 cold neurons is decreased during TASK-3 blockade and in mice lacking TASK-3, and, most importantly, these mice display hypersensitivity to cold. Our results demonstrate a role of TASK-3 channels in thermosensation, showing that a channel-based combinatorial strategy in TRPM8 cold thermoreceptors leads to molecular specialization and functional diversity. PMID:25199828

  5. Cyclic nucleotide-activated channels in carp olfactory receptor cells.

    PubMed

    Kolesnikov, S S; Kosolapov, A V

    1993-07-25

    When applied from the cytoplasmic side, cyclic 3',5'-adenosine and guanosine monophosphates reversibly increased the ion permeability of inside-out patches of carp olfactory neuron plasma membrane. The cAMP (cGMP)-induced permeability via cAMP (cGMP) concentration was fitted by Hill's equation with the exponents of 1.07 +/- 0.15 (1.12 +/- 0.05) and EC50 = 1.3 +/- 0.6 microM (0.9 +/- 0.3 microM). Substitution of NaCl in the bathing solution by chlorides of other alkali metals resulted in a slight shift of reversal potential of the cyclic nucleotide-dependent (CN) current, which indicates a weak selectivity of the channels. Permeability coefficients calculated by Goldman-Hodgkin-Katz's equation corresponded to the following relation: PNa/PK/PLi/PRb/PCs = 1:0.98:0.94:0.70:0.61. Ca2+ and Mg2+ in physiological concentrations blocked the channels activated by cyclic nucleotides (CN-channels). In the absence of divalent cations the conductance of single CN-channels was equal to 51 +/- 9 pS in 100 mM NaCl solution. Channel density did not exceed 1 micron-2. The maximal open state probability of the channel (Po) tended towards 1.0 at a high concentration of cAMP or cGMP. Dichlorobenzamil decreased Po without changing the single CN-channel' conductance. CN-channels exhibited burst activity. Mean open and closed times as well as the burst duration depended on agonist concentration. A kinetic model with four states (an inactivated, a closed and two open ones) is suggested to explain the regularities of CN-channel gating and dose-response relations. PMID:8334139

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

    PubMed

    Uchida, Kunitoshi; Tominaga, Makoto

    2011-01-01

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

  7. Role of transient receptor potential and acid-sensing ion channels in peripheral inflammatory pain.

    PubMed

    White, John P M; Cibelli, Mario; Rei Fidalgo, Antonio; Paule, Cleoper C; Noormohamed, Faruq; Urban, Laszlo; Maze, Mervyn; Nagy, Istvan

    2010-03-01

    Pain originating in inflammation is the most common pathologic pain condition encountered by the anesthesiologist whether in the context of surgery, its aftermath, or in the practice of pain medicine. Inflammatory agents, released as components of the body's response to peripheral tissue damage or disease, are now known to be collectively capable of activating transient receptor potential vanilloid type 1, transient receptor potential vanilloid type 4, transient receptor potential ankyrin type 1, and acid-sensing ion channels, whereas individual agents may activate only certain of these ion channels. These ionotropic receptors serve many physiologic functions-as, indeed, do many of the inflammagens released in the inflammatory process. Here, we introduce the reader to the role of these ionotropic receptors in mediating peripheral pain in response to inflammation. PMID:20179512

  8. The anticonvulsant and behavioural profile of L-687,414, a partial agonist acting at the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex.

    PubMed Central

    Tricklebank, M D; Bristow, L J; Hutson, P H; Leeson, P D; Rowley, M; Saywell, K; Singh, L; Tattersall, F D; Thorn, L; Williams, B J

    1994-01-01

    1. The anticonvulsant and behavioural effects of the glycine/NMDA receptor partial agonist, L-687,414 (R(+)-cis-beta-methyl-3-amino-1-hydroxypyrrolid-2-one) have been investigated in rodents. 2. L-687,414 dose-dependently antagonized seizures induced by N-methyl-D,L- aspartic acid (NMDLA, ED50 = 19.7 mg kg-1), pentylenetetrazol (PTZ, ED50 = 13.0 mg kg-1) and electroshock (ED50 = 26.1 mg kg-1) when given intravenously 15 min before test, in male Swiss Webster mice but was most potent against audiogenic seizures induced by a 120 dB bell in DBA/2 mice (ED50 = 5.1 mg kg-1, i.p., 30 min before test). 3. L-687,414 also induced impairments of performance in a rotarod test in both Swiss Webster and DBA/2 mice and the ratio [rotarod MED:anticonvulsant ED50] varied between 0.9 and 5, depending on the convulsant used. 4. Similar behaviours to those seen after administration of the non-competitive NMDA receptor antagonist, MK-801 (head weaving, body rolling, hyperlocomotion) were seen in the mouse after giving L-687,414, although the peak effect occurred at a dose (100 mg kg-1) which was 5-20 times the anticonvulsant ED50S, depending on the convulsant used. Unlike MK-801, however, doses of L-687,414 that were behaviourally stimulant did not increase dopamine turnover in the nucleus accumbens. 5. Consistent with the interaction of L-687,414 with the glycine/NMDA receptor, the anticonvulsant, ataxic and motor stimulant effects of the compound were significantly attenuated by the glycine/NMDA receptor agonist, D-serine (10-100 micrograms per mouse, i.c.v.).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7858861

  9. The Role of Transient Receptor Potential Cation Channels in Ca2+ Signaling

    PubMed Central

    Gees, Maarten; Colsoul, Barbara; Nilius, Bernd

    2010-01-01

    The 28 mammalian members of the super-family of transient receptor potential (TRP) channels are cation channels, mostly permeable to both monovalent and divalent cations, and can be subdivided into six main subfamilies: the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and the TRPA (ankyrin) groups. TRP channels are widely expressed in a large number of different tissues and cell types, and their biological roles appear to be equally diverse. In general, considered as polymodal cell sensors, they play a much more diverse role than anticipated. Functionally, TRP channels, when activated, cause cell depolarization, which may trigger a plethora of voltage-dependent ion channels. Upon stimulation, Ca2+ permeable TRP channels generate changes in the intracellular Ca2+ concentration, [Ca2+]i, by Ca2+ entry via the plasma membrane. However, more and more evidence is arising that TRP channels are also located in intracellular organelles and serve as intracellular Ca2+ release channels. This review focuses on three major tasks of TRP channels: (1) the function of TRP channels as Ca2+ entry channels; (2) the electrogenic actions of TRPs; and (3) TRPs as Ca2+ release channels in intracellular organelles. PMID:20861159

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

    PubMed Central

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

    2013-01-01

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

  11. LE135, a retinoid acid receptor antagonist, produces pain through direct activation of TRP channels

    PubMed Central

    Yin, Shijin; Luo, Jialie; Qian, Aihua; Yu, Weihua; Hu, Hongzhen

    2014-01-01

    Background and PurposeRetinoids, through their activation of retinoic acid receptors (RARs) and retinoid X receptors, regulate diverse cellular processes, and pharmacological intervention in their actions has been successful in the treatment of skin disorders and cancers. Despite the many beneficial effects, administration of retinoids causes irritating side effects with unknown mechanisms. Here, we demonstrate that LE135 [4-(7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid], a selective antagonist of RARβ, is a potent activator of the capsaicin (TRPV1) and wasabi (TRPA1) receptors, two critical pain-initiating cation channels. Experimental ApproachWe performed to investigate the excitatory effects of LE135 on TRPV1 and TRPA1 channels expressed in HEK293T cells and in dorsal root ganglia neurons with calcium imaging and patch-clamp recordings. We also used site-directed mutagenesis of the channels to determine the structural basis of LE135-induced activation of TRPV1 and TRPA1 channels and behavioural testing to examine if pharmacological inhibition and genetic deletion of the channels affected LE135-evoked pain-related behaviours. Key ResultsLE135 activated both the capsaicin receptor (TRPV1) and the allyl isothiocyanate receptor (TRPA1) heterologously expressed in HEK293T cells and endogenously expressed by sensory nociceptors. Mutations disrupting the capsaicin-binding site attenuated LE135 activation of TRPV1 channels and a single mutation (K170R) eliminated TRPA1 activity evoked by LE135. Intraplantar injection of LE135 evoked pain-related behaviours. Both TRPV1 and TRPA1 channels were involved in LE135-elicited pain-related responses, as shown by pharmacological and genetic ablation studies. Conclusions and ImplicationsThis blocker of retinoid acid signalling also exerted non-genomic effects through activating the pain-initiating TRPV1 and TRPA1 channels. PMID:24308840

  12. Synthesis of 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides and their evaluation as ligands for NMDA receptor glycine binding site.

    PubMed

    Bluke, Zanda; Paass, Einars; Sladek, Meik; Abel, Ulrich; Kauss, Valerjans

    2016-08-01

    A series of 2-substituted 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides were synthesized and evaluated for their affinity to the glycine binding site of the N-methyl-d-aspartate (NMDA) receptor. The binding affinity was determined by the displacement of radioligand [(3)H]MDL-105,519 from rat cortical membrane preparations. The most attractive structures in the search for prospective NMDA receptor ligands were identified to be 2-arylcarbonylmethyl substituted 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides. It has been demonstrated for the first time that the replacement of NH group in the ligand by sp(3) CH2 is tolerated. This finding may pave the way for previously unexplored approaches for designing new ligands of the NMDA receptor. PMID:26114309

  13. Vector-averaged gravity does not alter acetylcholine receptor single channel properties

    NASA Technical Reports Server (NTRS)

    Reitstetter, R.; Gruener, R.

    1994-01-01

    To examine the physiological sensitivity of membrane receptors to altered gravity, we examined the single channel properties of the acetylcholine receptor (AChR), in co-cultures of Xenopus myocytes and neurons, to vector-averaged gravity in the clinostat. This experimental paradigm produces an environment in which, from the cell's perspective, the gravitational vector is "nulled" by continuous averaging. In that respect, the clinostat simulates one aspect of space microgravity where the gravity force is greatly reduced. After clinorotation, the AChR channel mean open-time and conductance were statistically not different from control values but showed a rotation-dependent trend that suggests a process of cellular adaptation to clinorotation. These findings therefore suggest that the ACHR channel function may not be affected in the microgravity of space despite changes in the receptor's cellular organization.

  14. Regulation of canonical transient receptor potential (TRPC) channel function by diacylglycerol and protein kinase C.

    PubMed

    Venkatachalam, Kartik; Zheng, Fei; Gill, Donald L

    2003-08-01

    The mechanism of receptor-induced activation of the ubiquitously expressed family of mammalian canonical transient receptor potential (TRPC) channels has been the focus of intense study. Primarily responding to phospholipase C (PLC)-coupled receptors, the channels are reported to receive modulatory input from diacylglycerol, endoplasmic reticulum inositol 1,4,5-trisphosphate receptors and Ca2+ stores. Analysis of TRPC5 channels transfected within DT40 B cells and deletion mutants thereof revealed efficient activation in response to PLC-beta or PLC-gamma activation, which was independent of inositol 1,4,5-trisphoshate receptors or the content of stores. In both HEK293 cells and DT40 cells, TRPC5 and TRPC3 channel responses to PLC activation were highly analogous, but only TRPC3 and not TRPC5 channels responded to the addition of the permeant diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG). However, OAG application or elevated endogenous DAG, resulting from either DAG lipase or DAG kinase inhibition, completely prevented TRPC5 or TRPC4 activation. This inhibitory action of DAG on TRPC5 and TRPC4 channels was clearly mediated by protein kinase C (PKC), in distinction to the stimulatory action of DAG on TRPC3, which is established to be PKC-independent. PKC activation totally blocked TRPC3 channel activation in response to OAG, and the activation was restored by PKC-blockade. PKC inhibition resulted in decreased TRPC3 channel deactivation. Store-operated Ca2+ entry in response to PLC-coupled receptor activation was substantially reduced by OAG or DAG-lipase inhibition in a PKC-dependent manner. However, store-operated Ca2+ entry in response to the pump blocker, thapsigargin, was unaffected by PKC. The results reveal that each TRPC subtype is strongly inhibited by DAG-induced PKC activation, reflecting a likely universal feedback control on TRPCs, and that DAG-mediated PKC-independent activation of TRPC channels is highly subtype-specific. The

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

    PubMed Central

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

    2011-01-01

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

  16. Transient Receptor Potential Channel Polymorphisms Are Associated with the Somatosensory Function in Neuropathic Pain Patients

    PubMed Central

    Baron, Ralf; Maier, Christoph; Tölle, Thomas R.; Treede, Rolf-Detlef; Berthele, Achim; Faltraco, Frank; Flor, Herta; Gierthmühlen, Janne; Haenisch, Sierk; Huge, Volker; Magerl, Walter; Maihöfner, Christian; Richter, Helmut; Rolke, Roman; Scherens, Andrea; Üçeyler, Nurcan; Ufer, Mike; Wasner, Gunnar; Zhu, Jihong; Cascorbi, Ingolf

    2011-01-01

    Transient receptor potential channels are important mediators of thermal and mechanical stimuli and play an important role in neuropathic pain. The contribution of hereditary variants in the genes of transient receptor potential channels to neuropathic pain is unknown. We investigated the frequency of transient receptor potential ankyrin 1, transient receptor potential melastin 8 and transient receptor potential vanilloid 1 single nucleotide polymorphisms and their impact on somatosensory abnormalities in neuropathic pain patients. Within the German Research Network on Neuropathic Pain (Deutscher Forscbungsverbund Neuropathischer Schmerz) 371 neuropathic pain patients were phenotypically characterized using standardized quantitative sensory testing. Pyrosequencing was employed to determine a total of eleven single nucleotide polymorphisms in transient receptor potential channel genes of the neuropathic pain patients and a cohort of 253 German healthy volunteers. Associations of quantitative sensory testing parameters and single nucleotide polymorphisms between and within groups and subgroups, based on sensory phenotypes, were analyzed. Single nucleotide polymorphisms frequencies did not differ between both the cohorts. However, in neuropathic pain patients transient receptor potential ankyrin 1 710G>A (rs920829, E179K) was associated with the presence of paradoxical heat sensation (p = 0.03), and transient receptor potential vanilloid 1 1911A>G (rs8065080, I585V) with cold hypoalgesia (p = 0.0035). Two main subgroups characterized by preserved (1) and impaired (2) sensory function were identified. In subgroup 1 transient receptor potential vanilloid 1 1911A>G led to significantly less heat hyperalgesia, pinprick hyperalgesia and mechanical hypaesthesia (p = 0.006, p = 0.005 and p<0.001) and transient receptor potential vanilloid 1 1103C>G (rs222747, M315I) to cold hypaesthesia (p = 0.002), but there was absence of associations in subgroup 2. In

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2014-01-01

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

  20. Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

    PubMed Central

    Li, Mufeng; Silberberg, Shai D.; Swartz, Kenton J.

    2013-01-01

    The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg2+. Here we investigated the active forms of ATP and found that the action of MgATP2− and ATP4− differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP2− promotes opening with very low efficacy. In contrast, both free ATP and MgATP2− robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg2+ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP2− and weak regulation by Mg2+. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP2− and regulation by Mg2+, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons. PMID:23959888

  1. G-protein-coupled receptor regulation of P2X1 receptors does not involve direct channel phosphorylation

    PubMed Central

    2004-01-01

    P2X1 receptors for ATP are ligand-gated cation channels, which mediate smooth muscle contraction, contribute to blood clotting and are co-expressed with a range of GPCRs (G-protein-coupled receptors). Stimulation of Gαq-coupled mGluR1α (metabotropic glutamate receptor 1α), P2Y1 or P2Y2 receptors co-expressed with P2X1 receptors in Xenopus oocytes evoked calcium-activated chloride currents (IClCa) and potentiated subsequent P2X1-receptor-mediated currents by up to 250%. The mGluR1α-receptor-mediated effects were blocked by the phospholipase C inhibitor U-73122. Potentiation was mimicked by treatment with the phor-bol ester PMA. P2X receptors have a conserved intracellular PKC (protein kinase C) site; however, GPCR- and PMA-mediated potentiation was still observed with point mutants in which this site was disrupted. Similarly, the potentiation by GPCRs or PMA was unaffected by chelating the intracellular calcium rise with BAPTA/AM [bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis-(acetoxymethyl ester)] or the PKC inhibitors Ro-32-0432 and bisindolylmaleimide I, suggesting that the regulation does not involve a calcium-sensitive form of PKC. However, both GPCR and PMA potentiation were blocked by the kinase inhibitor staurosporine. Potentiation by phorbol esters was recorded in HEK-293 cells expressing P2X1 receptors, and radiolabelling of phosphorylated proteins in these cells demonstrated that P2X1 receptors are basally phosphorylated and that this level of phosphorylation is unaffected by phorbol ester treatment. This demonstrates that P2X1 regulation does not result directly from phosphorylation of the channel, but more likely by a staurosporine-sensitive phosphorylation of an accessory protein in the P2X1 receptor complex and suggests that in vivo fine-tuning of P2X1 receptors by GPCRs may contribute to cardiovascular control and haemostasis. PMID:15144237

  2. Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes.

    PubMed

    Toyoshima, C; Unwin, N

    1988-11-17

    The nicotinic acetylcholine receptor belongs to a class of molecules that respond transiently to chemical stimuli by opening a water-filled channel through the cell membrane for cations to diffuse. This channel lies along the central axis delineated by a ring of five homologous, membrane-spanning subunits and thus has properties, such as conductance and ion selectivity, which depend on the profile created by the encircling subunits. Insight has been gained recently about the amino-acid residues implicated directly in the ion transport, and some information about the subunit configuration around the channel has come from electron microscopy studies of postsynaptic membranes crystallized in the form of flattened tubular vesicles. The resolution along the axis of the channel has, however, been limited by the restricted range of views obtainable. Here we report the structure of the channel at 17 A resolution, determined by three-dimensional image reconstruction from tubular vesicles having receptors organized in helical arrays across their surfaces. The helical symmetry is preserved by suspending the tubes in thin films of ice, and the receptors in such tubes can be seen from all angles, allowing the channel to be revealed clearly in relation to the lipid bilayer and the peripheral protein for the first time. PMID:2461515

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

    PubMed

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

    1991-10-31

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

  4. Subunit composition of mammalian transient receptor potential channels in living cells.

    PubMed

    Hofmann, Thomas; Schaefer, Michael; Schultz, Günter; Gudermann, Thomas

    2002-05-28

    Hormones, neurotransmitters, and growth factors give rise to calcium entry via receptor-activated cation channels that are activated downstream of phospholipase C activity. Members of the transient receptor potential channel (TRPC) family have been characterized as molecular substrates mediating receptor-activated cation influx. TRPC channels are assumed to be composed of multiple TRPC proteins. However, the cellular principles governing the assembly of TRPC proteins into homo- or heteromeric ion channels still remain elusive. By pursuing four independent experimental approaches--i.e., subcellular cotrafficking of TRPC subunits, differential functional suppression by dominant-negative subunits, fluorescence resonance energy transfer between labeled TRPC subunits, and coimmunoprecipitation--we investigate the combinatorial rules of TRPC assembly. Our data show that (i) TRPC2 does not interact with any known TRPC protein and (ii) TRPC1 has the ability to form channel complexes together with TRPC4 and TRPC5. (iii) All other TRPCs exclusively assemble into homo- or heterotetramers within the confines of TRPC subfamilies--e.g., TRPC4/5 or TRPC3/6/7. The principles of TRPC channel formation offer the conceptual framework to assess the physiological role of distinct TRPC proteins in living cells. PMID:12032305

  5. 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

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

    PubMed Central

    Schindler, H; Spillecke, F; Neumann, E

    1984-01-01

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

  7. Transient receptor potential melastatin 3 is a phosphoinositide-dependent ion channel

    PubMed Central

    Badheka, Doreen; Borbiro, Istvan

    2015-01-01

    Phosphoinositides are emerging as general regulators of the functionally diverse transient receptor potential (TRP) ion channel family. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been reported to positively regulate many TRP channels, but in several cases phosphoinositide regulation is controversial. TRP melastatin 3 (TRPM3) is a heat-activated ion channel that is also stimulated by chemical agonists, such as pregnenolone sulfate. Here, we used a wide array of approaches to determine the effects of phosphoinositides on TRPM3. We found that channel activity in excised inside-out patches decreased over time (rundown), an attribute of PI(4,5)P2-dependent ion channels. Channel activity could be restored by application of either synthetic dioctanoyl (diC8) or natural arachidonyl stearyl (AASt) PI(4,5)P2. The PI(4,5)P2 precursor phosphatidylinositol 4-phosphate (PI(4)P) was less effective at restoring channel activity. TRPM3 currents were also restored by MgATP, an effect which was inhibited by two different phosphatidylinositol 4-kinase inhibitors, or by pretreatment with a phosphatidylinositol-specific phospholipase C (PI-PLC) enzyme, indicating that MgATP acted by generating phosphoinositides. In intact cells, reduction of PI(4,5)P2 levels by chemically inducible phosphoinositide phosphatases or a voltage-sensitive 5′-phosphatase inhibited channel activity. Activation of PLC via muscarinic receptors also inhibited TRPM3 channel activity. Overall, our data indicate that TRPM3 is a phosphoinositide-dependent ion channel and that decreasing PI(4,5)P2 abundance limits its activity. As all other members of the TRPM family have also been shown to require PI(4,5)P2 for activity, our data establish PI(4,5)P2 as a general positive cofactor of this ion channel subfamily. PMID:26123195

  8. Transient Receptor Potential (TRP) Channels and Cardiac Fibrosis

    PubMed Central

    Yue, Zhichao; Zhang, Yanhui; Xie, Jia; Jiang, Jianmin; Yue, Lixia

    2013-01-01

    Cardiac fibrosis is associated with most cardiac diseases. Fibrosis is an accumulation of excessive extracellular matrix proteins (ECM) synthesized by cardiac fibroblasts and myofibroblasts. Fibroblasts are the most prevalent cell type in the heart, comprising 75% of cardiac cells. Myofibroblasts are hardly present in healthy normal heart tissue, but appear abundantly in diseased hearts. Cardiac fibroblasts are activated by a variety of pathological stimuli, such as myocardial injury, oxidative stress, mechanical stretch, and elevated autocrine-paracrine mediators, thereby undergoing proliferation, differentiation to myofibroblasts, and production of various cytokines and ECM proteins. A number of signaling pathways and bioactive molecules are involved and work in concert to activate fibroblasts and myofibroblasts in the fibrogenesis cascade. Fibroblasts and myofibroblasts are not only principal ECM producers, but also play a central role in fibrogenesis and myocardial remodeling in fibrotic heart disease. Thus, understanding the biological processes of cardiac fibroblasts will provide novel insights into the underlying mechanisms of fibrosis and provide potential targets for developing anti-fibrotic drugs. Recent studies demonstrate that Ca2+ signal is essential for fibroblast proliferation, differentiation, and ECM-protein production. This review focuses on the recent advances in understanding molecular mechanisms of Ca2+ signaling in cardiac fibrogenesis, and potential role of Ca2+-permeable channels, in particular, the transient potential (TRP) channels in fibrotic heart disease. TRP channels are highly expressed in cardiac fibroblasts. TRPM7 has been shown to be essential in TGFβ1 mediated fibrogenesis, and TRPC3 has been demonstrated to play an essential role in regulating fibroblast function. Thus, the Ca2+-permeable TRP channels may serve as potential novel targets for developing anti-fibrotic drugs. PMID:23432060

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

    PubMed Central

    Horn, R

    1987-01-01

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

  10. Natural-Product-Derived Transient Receptor Potential Melastatin 8 (TRPM8) Channel Modulators.

    PubMed

    LeGay, Christina M; Gorobets, Evgueni; Iftinca, Mircea; Ramachandran, Rithwik; Altier, Christophe; Derksen, Darren J

    2016-06-01

    A library of novel structural hybrids of menthol and cubebol was tested for each derivative's ability to interact with the transient receptor potential subfamily melastatin member 8 (TRPM8) channel. This structure-activity relationship study revealed three potent modulators of the TRPM8 ion channel: a novel agonist (4) with an EC50 value of 11 ± 1 μM, an antagonist (15) with an IC50 value of 2 ± 1 μM, and an allosteric modulator (21) that minimized channel desensitization toward menthol. Each of these novel exocyclic olefin analogues of menthol is readily accessible by synthesis and was tested using Ca(2+) assays and electrophysiology. PMID:27171974