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）：乙醇是最广泛使用的药物之一，但其结合产生其作用的分子靶点尚未完全了解。乙醇的强有力的蛋白质靶点是甘氨酸受体，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。我们的初步数据表明，锌是至关重要的酒精中毒浓度的甘氨酸受体（GlyR）功能的行动，因此强调，了解锌/乙醇的相互作用是必要的，在定义乙醇的作用机制在GlyR。随后，了解锌/乙醇的相互作用可能会导致更准确的近似乙醇的影响在体内。拟议的项目将研究锌对乙醇作用于甘氨酸受体的影响，采用体外和体内技术。目的1将测试和表征锌/乙醇相互作用的三个甘氨酸受体亚基（11，12和13）在非洲爪蟾卵母细胞中表达。目的2将确定锌-乙醇相互作用的机制，其特征在于在目的1中使用定点诱变来消除甘氨酸受体上的假定锌结合位点。突变甘氨酸受体将基于已知对锌结合重要的氨基酸残基构建，并将允许确定甘氨酸受体处的锌/乙醇相互作用是否是由于锌在已知位点的结合或通过在替代位点的作用。突变最初将在11亚基中产生，但如果获得显著结果，则将在12和13亚基中的同源位置中产生另外的突变。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Glra 1（D80 A）小鼠，其在推定的11甘氨酸受体锌结合位点处含有突变，将为甘氨酸受体处的锌/乙醇相互作用的行为研究提供动物模式，并且将潜在地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。与其他不能存活的纯合甘氨酸受体KI小鼠不同，Glra 1（D80 A）纯合子成年后仍能存活。该项目的发现将提供有关锌对酒精在中枢神经系统中作用的影响的见解。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酒精滥用和酒精中毒治疗方法。) 公共卫生相关性：乙醇是使用最广泛的药物之一，但它与产生其作用的分子靶点尚未完全了解。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酒精滥用和酒精中毒治疗方法。