Alcohol Actions--Molecular Targets on Brain Proteins

描述（由申请人提供）：我们认为乙醇对神经元功能的急性影响已从非特异性膜作用演变为对特定蛋白质上特定位点的扰动。一些新的技术和方法使人们有可能提出关于酒精与大脑蛋白质相互作用的关键问题，并将蛋白质功能的变化与酒精的特定行为联系起来。在过去的资助期内，我们开发了烷醇类试剂作为醇类似物，可用于共价标记感兴趣的蛋白质中假定的醇结合位点。我们建议使用这种方法来测试的假设，乙醇改变甘氨酸和GABAa受体的功能，通过结合在充满水的蛋白质腔，这些腔的占领改变通道门控。确定对酒精作用于目标蛋白质至关重要的氨基酸应该有助于我们回答关键问题：乙醇的哪些行为效应是由于对哪些特定蛋白质的作用？最近构建的小鼠敲除模型说明了从关键氨基酸到行为研究的路径，其中单个GABAa受体亚基中的单个氨基酸突变会改变苯二氮卓类药物的特定行为作用。这些类型的突变小鼠提供了一种强大的新方法，将单一蛋白质与特定行为联系起来。目前的建议的一个主要目标是使用这种方法来定义甘氨酸和GABA α受体在体内酒精作用的重要性。除了敲入方法，更传统的敲除或无效突变小鼠也将用于提供有关特定蛋白质在药物作用中的作用的有价值的信息，并将用于本项目。这项工作的长期目标是确定关键蛋白质位点，这些位点可以作为酒精强化，依赖和复发的新疗法的靶点。

DESCRIPTION (provided by applicant): Our view of the acute effects of ethanol on neuronal function has evolved from nonspecific membrane actions to perturbation of specific sites on specific proteins. Several new techniques and approaches make it possible to ask key questions about the interaction of alcohol with brain proteins and to link changes in protein function with specific behavioral actions of alcohol. During the past grant period we developed alkanethiol reagents as alcohol analogs that could be used to covalently label putative alcohol binding sites in proteins of interest. We propose to use this approach to test the hypotheses that ethanol alters the function of glycine and GABAa receptors by binding within water-filled protein cavities, and that occupation of these cavities alters channel gating. Identification of amino acids critical for alcohol action on target proteins should help us to answer the key question: Which behavioral effects of ethanol are due to actions on which specific proteins? The path from critical amino acids to behavioral studies is illustrated by the recent construction of knock in mice in which mutation of a single amino acid in a single GABAa receptor subunit alters a specific behavioral action of benzodiazepines. These types of mutant mice provide a powerful new approach to link a single protein with a specific behavior. A major goal of the current proposal is to use this approach to define the importance of glycine and GABAa receptors in alcohol actions in vivo. In addition to the knock in approach, the more conventional knock out or null mutant mice will also be used to provide valuable information about the role of specific proteins in drug action and will be employed in this project. The long-range goal of this work is to define key protein sites that can serve as targets for new therapies for alcohol reinforcement, dependence and relapse.