The combined effects of zinc and ethanol at the glycine receptor

描述(申请人提供)：乙醇是最广泛使用的药物之一，但它与产生作用的分子靶标结合还不完全清楚。在乙醇强烈支持的蛋白质靶标中，甘氨酸受体除了受到酒精和麻醉剂的调节外，还受到内源性锌的调节。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尚未见报道。我们的初步数据表明，锌在醉人浓度的乙醇对甘氨酸受体(GlyR)功能的作用中起关键作用，因此强调了解锌/乙醇的相互作用对于确定乙醇在GlyR中的作用机制是必要的。随后，了解锌/乙醇的相互作用可能会导致更准确地近似乙醇在体内的作用。这项拟议的项目将使用体外和活体技术来研究酒精对甘氨酸受体上锌作用的影响。目的1检测非洲爪哇卵母细胞表达的3个甘氨酸受体亚基(11、12、13)中锌与乙醇的相互作用。目的2将通过定点突变消除甘氨酸受体上可能的锌结合部位，确定目标1中锌-乙醇相互作用的机制。突变的甘氨酸受体将基于已知对锌结合很重要的氨基酸残基来构建，并将允许确定甘氨酸受体上的锌/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。突变最初将在1 1亚基中产生，但如果获得显著结果，则将在1 2和1 3亚基中的同源位置产生额外的突变。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)小鼠在可能的1 1甘氨酸受体锌结合位点处存在突变，将为甘氨酸受体锌/乙醇相互作用的行为学研究提供一个动物模型，并可能成为第一个测试酒精消费和其他行为指标的甘氨酸受体纯合子敲除小鼠。与其他尚未存活的甘氨酸受体KI纯合子小鼠不同，Glra1(D80A)纯合子成年后仍可存活。这个项目的发现将提供关于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。) 与公共健康相关：乙醇是最广泛使用的药物之一，但它与产生作用的分子靶标结合在一起并不完全清楚。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。

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.