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，但其结合以产生其作用的分子靶标尚不完全清楚。乙醇的大力支持的蛋白质靶标之一是甘氨酸受体，其除了受到酒精和麻醉剂的调节外，还受到内源性锌的调节。尽管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尚未研究。我们的初步数据表明，锌对于醉人浓度的乙醇对甘氨酸受体 (GlyR) 功能的作用至关重要，因此强调了解锌/乙醇相互作用对于确定乙醇在 GlyR 上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。拟议的项目将利用体外和体内技术研究锌对甘氨酸受体上的酒精作用的影响。目标 1 将测试并表征非洲爪蟾卵母细胞中表达的三个甘氨酸受体亚基（11、1、2 和 13）中的锌/乙醇相互作用。目标 2 将确定目标 1 中表征的锌-乙醇相互作用的机制，使用定点诱变消除甘氨酸受体上假定的锌结合位点。突变的甘氨酸受体将基于已知对锌结合重要的氨基酸残基构建，并且将允许确定甘氨酸受体处的锌/乙醇相互作用是否是由于已知位点的锌结合或通过替代位点的作用所致。突变最初将在 1 1 亚基中产生，但如果获得显着结果，则将在 1 2 和 1 3 亚基的同源位置产生额外的突变。目标 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 Glra1(D80A) 小鼠在假定的 1 1 甘氨酸受体锌结合位点处含有突变，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式，并且可能是第一个甘氨酸受体纯合敲入小鼠，其中将测试酒精消耗和乙醇作用的其他行为测量。与其他无法存活的纯合甘氨酸受体 KI 小鼠不同，Glra1(D80A) 纯合子成年后仍可存活。该项目的研究结果将深入了解锌对酒精在中枢神经系统中的作用的影响。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酒精滥用和酒精中毒治疗方法。 ） 公共健康相关性：乙醇是使用最广泛的药物之一，但其结合产生作用的分子靶点尚不完全清楚。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酒精滥用和酒精中毒治疗方法。