RESUMO
BACKGROUND: The purpose of this study was to determine whether pairs of compounds, including general anesthetics, could simultaneously modulate receptor function in a synergistic manner, thus demonstrating the existence of multiple intraprotein anesthetic binding sites. METHODS: Using standard electrophysiologic methods, we measured the effects of at least one combination of benzene, isoflurane (ISO), halothane (HAL), chloroform, flunitrazepam, zinc, and pentobarbital on at least one of the following ligand gated ion channels: N-methyl-D-aspartate receptors, glycine receptors and gamma-aminobutyric acid type A receptors. RESULTS: All drug-drug-receptor combinations were found to exhibit additive, not synergistic modulation. ISO with benzene additively depressed N-methyl-D-aspartate receptors function. ISO with HAL additively enhanced glycine receptors function, as did ISO with zinc. ISO with HAL additively enhanced gamma-aminobutyric acid type A receptors function as did all of the following: HAL with chloroform, pentobarbital with ISO, and flunitrazepam with ISO. CONCLUSION: The simultaneous allosteric modulation of ligand gated ion channels by general anesthetics is entirely additive. Where pairs of general anesthetic drugs interact synergistically to produce general anesthesia, they must do so on systems more complex than a single receptor.
Assuntos
Anestésicos Gerais/farmacologia , Canais Iônicos/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Receptores de Glicina/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Animais , Clorofórmio/administração & dosagem , Clorofórmio/farmacologia , Eletrofisiologia , Halotano/administração & dosagem , Halotano/farmacologia , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/metabolismo , Isoflurano/administração & dosagem , Isoflurano/farmacologia , Oócitos , Técnicas de Patch-Clamp , Pentobarbital/administração & dosagem , Pentobarbital/farmacologia , Receptores de GABA-A/metabolismo , Receptores de Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Xenopus laevisRESUMO
There is substantial evidence that GABAergic neurotransmission is important for many behavioral actions of ethanol and there are reports spanning more than 30 years of literature showing that low to moderate (3-30 mM) concentrations of ethanol enhance GABAergic neurotransmission. A key question is which GABA receptor subunits are sensitive to low concentrations of ethanol in vivo and in vitro. Recent evidence points to a role for extrasynaptic receptors. Another question is which behavioral actions of alcohol result from enhancement of GABAergic neurotransmission. Some clues are beginning to emerge from studies of knock-out and knock-in mice and from genetic analysis of human alcoholics. These approaches are converging on a role for GABAergic actions in regulating alcohol consumption and, perhaps, the development of alcoholism.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Alcoolismo/genética , Animais , Sítios de Ligação/efeitos dos fármacos , Ligação Genética , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptores de GABA-A/genéticaRESUMO
BACKGROUND: Acamprosate (calcium acetylhomotaurinate) has proven to be a moderately effective pharmacological adjunct for the treatment of alcoholism. However, the central nervous system mechanism by which acamprosate reduces alcohol relapse remains unclear. Here we survey a number of metabotropic receptors, ligand-gated ion channels, and voltage-gated ion channels, to determine if acamprosate has actions at these sites in the central nervous system. METHODS: Xenopus oocytes were injected with cDNAs or cRNAs encoding metabotropic glutamate receptors 1 and 5, M1 muscarinic receptors, glycine alpha1 homomeric and alpha1beta1 heteromeric receptors, gamma-aminobutyric acid A (GABA(A)alpha4beta3delta, alpha4beta3gamma2s, and alpha1beta2gamma2s) receptors, vanilloid receptor 1, and various combinations of alpha and beta subunits of voltage-gated Na+ channels. Electrophysiological responses were measured using two-electrode voltage clamp parameters after activation with agonists or voltage steps (for the voltage-gated channels). Acamprosate (0.1 to 100 microM) was pre-applied for 1 minute, followed by co-application with agonist. Acamprosate was also applied with ethanol to determine if it altered ethanol responses at some of these receptors and channels. RESULTS: None of the receptors or ion channels responded to acamprosate alone. Acamprosate also failed to alter the activation of receptors or channels by agonists or after activation of voltage-gated channels. There was no effect of acamprosate on ethanol responses at GABA(A)alpha1beta2gamma2s receptors or Na+ channels. CONCLUSIONS: Acamprosate does not significantly modulate the function of these receptors and ion channels at clinically relevant concentrations. Thus, the clinical effectiveness of acamprosate in the treatment of alcoholism is not likely due to direct effects on these receptors or ion channels.
Assuntos
Dissuasores de Álcool/farmacologia , Canais Iônicos/efeitos dos fármacos , Receptores de Neurotransmissores/efeitos dos fármacos , Taurina/análogos & derivados , Acamprosato , Animais , Relação Dose-Resposta a Droga , Etanol/farmacologia , Oócitos/efeitos dos fármacos , Receptor de Glutamato Metabotrópico 5 , Receptor Muscarínico M1/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Receptores de Glicina/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacos , Canais de Cátion TRPV/efeitos dos fármacos , Taurina/farmacologia , Xenopus laevisRESUMO
The glycine receptor is a member of the Cys-loop, ligand-gated ion channel family and is responsible for inhibition in the CNS. We examined the orientation of amino acids I229 in transmembrane 1 (TM1) and A288 in TM3, which are both critical for alcohol and volatile anesthetic action. We mutated these two amino acids to cysteines either singly or in double mutants and expressed the receptors in Xenopus laevis oocytes. We tested whether disulfide bonds could form between A288C in TM3 paired with M227C, Y228C, I229C, or S231C in TM1. Application of cross-linking (mercuric chloride) or oxidizing (iodine) agents had no significant effect on the glycine response of wild-type receptors or the single mutants. In contrast, the glycine response of the I229C/A288C double mutant was diminished after application of either mercuric chloride or iodine only in the presence of glycine, indicating that channel gating causes I229C and A288C to fluctuate to be within 6 A apart and form a disulfide bond. Molecular modeling was used to thread the glycine receptor sequence onto a nicotinic acetylcholine receptor template, further demonstrating that I229 and A288 are near-neighbors that can cross-link and providing evidence that these residues contribute to a single binding cavity.
Assuntos
Álcoois/metabolismo , Glicina/metabolismo , Receptores de Glicina/química , Receptores de Glicina/metabolismo , Animais , Sítios de Ligação , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismo , Cisteína/metabolismo , Dissulfetos/metabolismo , Relação Dose-Resposta a Droga , Feminino , Glicina/farmacologia , Humanos , Modelos Moleculares , Mutagênese , Oócitos/fisiologia , Oxirredução , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Glicina/genética , Xenopus laevisRESUMO
Glycine receptors (GlyRs) are members of the ligand-gated ion channel superfamily. Each subunit has four transmembrane segments (TM1-TM4). Several studies suggest that amino acids in all four TMs face into a water-filled, alcohol and anesthetic binding cavity in the extracellular portion of the transmembrane domain. TM4 should contribute a "wall" to this cavity, but the residues involved are unknown. Here, we determined the ability of an alcohol analog, propyl methanethiosulfonate (propyl MTS), to covalently react with twelve GlyR TM4 positions (I401-I412) after mutating the original amino acids to cysteines. Reactivity of a cysteine with propyl MTS implies that the cysteine is exposed to water. W407C, I409C, Y410C, and K411C showed altered receptor function following reaction with propyl MTS in the presence or absence of glycine. The cysteine mutations alone eliminated the effects of ethanol for I409C, Y410C, and K411C, and reduced the effects of octanol for I409C and isoflurane for K411C. The ability of propyl MTS to reduce isoflurane and chloroform potentiation was examined in the reactive mutants. Potentiation by isoflurane was significantly reduced for I409C after reaction. These data demonstrate water-accessibility of specific TM4 positions in the GlyR and suggest involvement of these residues with alcohol and anesthetic action.
Assuntos
Receptores de Glicina/efeitos dos fármacos , Substituição de Aminoácidos , Anestésicos Inalatórios , Depressores do Sistema Nervoso Central/farmacologia , Clorofórmio/farmacologia , Cisteína/fisiologia , DNA Complementar/biossíntese , DNA Complementar/genética , Interpretação Estatística de Dados , Relação Dose-Resposta a Droga , Eletrofisiologia , Etanol/farmacologia , Glicina/farmacologia , Humanos , Isoflurano/farmacologia , Mutação/genética , Mutação/fisiologia , Octanóis/farmacologiaRESUMO
Contact points between transmembrane segments (TMs) two and three of the glycine receptor are undefined and may play an important role in channel gating. We tested whether two amino acids in TM2 (S267) and TM3 (A288), known to be critical for alcohol and volatile anesthetic action, could cross-link by mutating both to cysteines and expressing the receptors in Xenopus laevis oocytes. In contrast with the wild-type receptor and single cysteine mutants, the S267C/A288C double mutant displayed unusual responses, including a tonic leak activity that was closed by strychnine and a run-down of the response upon repeated applications of glycine. We hypothesized that these characteristics were due to cross-linking of the two cysteines on opposing faces of these adjacent, alpha helical TMs. This would alter the movement of these two regions required for normal gating. To test this hypothesis, we used dithiothreitol to reduce the putative S267C-A288C disulfide bond. Reduction abolished the leak current and provided normal responses to glycine. Subsequent application of the cross-linking agent mercuric chloride caused the initial characteristics to return. These data demonstrate that S267 and A288 are near-neighbors and provide insight towards the location and role of the TM2-TM3 interface in ligand-gated ion channels.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Glicina/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Receptores de Glicina/efeitos dos fármacos , Receptores de Glicina/metabolismo , Álcoois/farmacologia , Substituição de Aminoácidos/genética , Anestésicos Inalatórios/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Humanos , Ativação do Canal Iônico/genética , Ligantes , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Receptores de Glicina/genética , Relação Estrutura-Atividade , Xenopus laevisRESUMO
The glycine receptor is a target for both alcohols and anesthetics, and certain amino acids in the alpha1 subunit transmembrane segments (TM) are critical for drug effects. Introducing larger amino acids at these positions increases the potency of glycine, suggesting that introducing larger residues, or drug molecules, into the drug-binding cavity facilitates channel opening. A possible mechanism for these actions is that the volume of the cavity expands and contracts during channel opening and closing. To investigate this hypothesis, mutations for amino acids in TM1 (I229C) and TM2 (G256C, T259C, V260C, M263C, T264C, S267C, S270C) and TM3 (A288C) were individually expressed in Xenopus laevis oocytes. The ability of sulfhydryl-specific alkyl methanethiosulfonate (MTS) compounds of different lengths to covalently react with introduced cysteines in both the closed and open states of the receptor was determined. S267C was accessible to short chain (C3-C8) MTS in both open and closed states, but was only accessible to longer chain (C10-C16) MTS compounds in the open state. Reaction with S267C was faster in the open state. I229C and A288C showed state-dependent reaction with MTS only in the presence of agonist. M263C and S270C were also accessible to MTS labeling. Mutated residues more intracellular than M263C did not react, indicating a floor of the cavity. These data demonstrate that the conformational changes accompanying channel gating increase accessibility to amino acids critical for drug action in TM1, TM2, and TM3, which may provide a mechanism by which alcohols and anesthetics can act on glycine (and likely other) receptors.