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: 8002749
• 负责人: Lindsay M. McCracken
• 金额: $ 3.15万
• 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 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8002749
• 关键词: Adult; Alcohol abuse; Alcohol consumption; Alcoholism; Alcohols; Amino Acids; Anesthetics; Animals; Behavioral; Binding; Binding Sites; Data; Drug usage; Ethanol; Glycine Receptors; Homozygote; In Vitro; Individual; Knock-in Mouse; Lead; Molecular Target; Mus; Mutation; Neuraxis; Positioning Attribute; Proteins; Role; Site; Site-Directed Mutagenesis; Techniques; Testing; Xenopus oocyte; Zinc; alcohol effect; base; behavior measurement; behavior test; effective therapy; in vivo; insight; mutant; public health relevance; receptor function

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）：乙醇是使用最广泛的药物之一，但是它与之结合以产生其作用的分子靶标尚未完全了解。乙醇的强有力蛋白质靶标是甘氨酸受体，除了通过酒精和麻醉调节外，还由内源性锌调节。尽管已经研究了锌和乙醇对甘氨酸受体的个体作用，但内源性锌在乙醇增强甘氨酸受体功能中的作用尚未。我们的初步数据表明，锌对于乙醇对甘氨酸受体（GLYR）功能的作用至关重要，因此强调，理解锌/乙醇相互作用对于确定乙醇在Glyr的作用机理是必要的。随后，理解锌/乙醇相互作用可能会导致对乙醇在体内作用的近似值。拟议的项目将使用体外和体内技术研究锌对甘氨酸受体的酒精作用的影响。 AIM 1将测试并表征三个甘氨酸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。 AIM 2将确定使用位置定向的诱变在AIM 1中特征的锌 - 乙醇相互作用的机制，以消除甘氨酸受体上推定的锌结合位点。突变甘氨酸受体将基于已知对于锌结合很重要的氨基酸残基构建，并可以确定甘氨酸受体处的锌/乙醇相互作用是由于已知位点的锌结合还是通过替代位点的作用引起的。该突变最初将在1 1个亚基中产生，但是如果获得了显着的结果，则将在1 2和1 3亚基中的同源位置中的其他突变。 AIM 3将评估锌不敏感的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1 1甘氨酸受体锌结合位点中包含突变，将为甘氨酸受体的锌/乙醇相互作用的行为研究提供动物模式，并且可能是第一个纯化的乙醇敲击型乙醇和其他行动素的乙醇量和其他行动量的乙醇受体受体识别小鼠。与其他不可行的纯合甘氨酸受体Ki小鼠不同，GLRA1（D80A）纯合子作为成年人是可行的。该项目的发现将提供有关锌对中枢神经系统中酒精作用的影响的见解。通过更好地了解酒精的作用的地点和机制，可以开发出对酒精滥用和酒精中毒的更有效治疗方法。 ） 公共卫生相关性：乙醇是使用最广泛的药物之一，但是它与之结合以产生其作用的分子靶标尚未完全了解。通过更好地了解酒精的作用的地点和机制，可以开发出对酒精滥用和酒精中毒的更有效治疗方法。