The combined effects of zinc and ethanol at the glycine receptor

锌和乙醇对甘氨酸受体的联合作用

• 批准号: 8155327
• 负责人: Lindsay M. McCracken
• 金额: $ 1.9万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8155327
• 关键词: Acoustics; Acute; Adult; Affinity; Alcohol abuse; Alcohol consumption; Alcohol withdrawal syndrome; Alcoholism; Alcohols; Amino Acids; Anesthetics; Animal Model; Animals; Behavior; Behavioral; Binding; Binding Sites; Buffers; Convulsions; Data; Drug Kinetics; Drug usage; Ethanol; Ethanol Metabolism; Glycine; Glycine Receptors; Homozygote; In Vitro; Individual; Investigation; Ion Channel; Knock-in Mouse; Lead; Measures; Molecular Target; Mus; Mutation; Neuraxis; Pharmacology; Physiological; Positioning Attribute; Proteins; Publishing; Recombinants; Role; Site; Site-Directed Mutagenesis; Solutions; Strychnine; Techniques; Testing; Wild Type Mouse; Xenopus oocyte; Zinc; alcohol behavior; alcohol effect; base; behavior measurement; behavior test; drinking; effective therapy; in vivo; insight; mutant; novel; public health relevance; receptor; receptor function; receptor sensitivity; research study; response

DESCRIPTION (provided by applicant): Ethanol is among the most widely used drugs, yet the molecular targets to which it binds to produce its effects are not completely understood. Among the strongly-supported protein targets of ethanol are glycine receptors, which in addition to being modulated by alcohol and anesthetics, are also modulated by endogenous zinc. Although the individual effects of zinc and ethanol at the glycine receptor have been studied, the role of endogenous zinc in ethanol enhancement glycine receptor function has not. Our preliminary data indicate that zinc is critical for the action of intoxicating concentrations of ethanol on glycine receptor (GlyR) function, and therefore highlight that understanding zinc/ethanol interactions is necessary in defining the mechanism of action of ethanol at the GlyR. Subsequently, understanding zinc/ethanol interactions could lead to more accurate approximations of ethanol's effects in vivo. The proposed project will investigate the effects of zinc on alcohol action at the glycine receptor using in vitro and vivo techniques. Aim 1 will test and characterize a zinc/ethanol interaction in three glycine receptor subunits (11, 1 2, and 13) expressed in Xenopus oocytes. Aim 2 will determine a mechanism for the zinc-ethanol interaction characterized in Aim 1 using site-directed mutagenesis to eliminate putative zinc binding sites on the glycine receptor. Mutant glycine receptors will be constructed based on amino acid residues known to be important for zinc binding and will allow for it to be determined if zinc/ethanol interactions at the glycine receptor are due to zinc binding at known sites or via action at alternative sites. The mutations will be initially created in the 1 1 subunit, but if significant results are obtained, then additional mutations in the homologous positions in the 1 2 and 1 3 subunits will be made. Aim 3 will evaluate the effects of a zinc-insensitive glycine receptor mutation on alcohol consumption and other behavioral tests in mice. Homozygous Glra1(D80A) mice, which contain a mutation at a putative 1 1 glycine receptor zinc binding site, will provide an animal mode for behavioral studies of zinc/ethanol interactions at the glycine receptor, and will be potentially the first glycine receptor homozygous knock-in mice in which alcohol consumption and other behavioral measures of ethanol action will be tested. Unlike other homozygous glycine receptor KI mice, which have not been viable, Glra1(D80A) homozygotes are viable as adults. Findings from this project will provide insight about the effects of zinc on alcohol's action in the central nervous system. By better understanding the sites and mechanisms of action of alcohol, more effective treatments for alcohol abuse and alcoholism can be developed. ) PUBLIC HEALTH RELEVANCE: Ethanol is among the most widely used drugs, yet the molecular targets to which it binds to produce its effects are not completely understood. By better understanding the sites and mechanisms of action of alcohol, more effective treatments for alcohol abuse and alcoholism can be developed.

描述（由申请人提供）：乙醇是最广泛使用的药物之一，但其结合产生其作用的分子靶标尚未完全了解。在乙醇的强支持蛋白靶点中有甘氨酸受体，它除了被酒精和麻醉剂调节外，还被内源性锌调节。虽然已经研究了锌和乙醇对甘氨酸受体的单独作用，但内源性锌在乙醇增强甘氨酸受体功能中的作用还没有研究。我们的初步数据表明，锌对于乙醇中毒浓度对甘氨酸受体（GlyR）功能的作用至关重要，因此，了解锌/乙醇的相互作用对于确定乙醇对GlyR的作用机制是必要的。随后，了解锌/乙醇的相互作用可以更准确地估计乙醇在体内的作用。拟建项目将利用体外和体内技术研究锌对乙醇作用于甘氨酸受体的影响。目的1将测试和表征锌/乙醇在爪蟾卵母细胞中表达的三个甘氨酸受体亚基（11、12和13）中的相互作用。Aim 2将确定Aim 1中描述的锌-乙醇相互作用的机制，使用位点定向诱变消除甘氨酸受体上假定的锌结合位点。突变型甘氨酸受体将基于已知对锌结合很重要的氨基酸残基构建，并将允许确定锌/乙醇在甘氨酸受体上的相互作用是由于锌在已知位点结合还是通过在替代位点的作用。突变将首先在11亚基上产生，但如果获得显著的结果，那么将在12和13亚基的同源位置上产生额外的突变。目的3将评估锌不敏感甘氨酸受体突变对小鼠饮酒和其他行为测试的影响。纯合子Glra1（D80A）小鼠，在假定的11甘氨酸受体锌结合位点上含有突变，将为甘氨酸受体上锌/乙醇相互作用的行为研究提供动物模型，并且可能是第一个甘氨酸受体纯合子敲入小鼠，用于测试酒精消耗和乙醇作用的其他行为测量。与其他纯合子甘氨酸受体KI小鼠不同，Glra1（D80A）纯合子在成年后是存活的。这个项目的发现将为锌对酒精在中枢神经系统中的作用的影响提供见解。通过更好地了解酒精的作用部位和机制，可以开发出更有效的治疗酒精滥用和酒精中毒的方法。