Alcohol Actions--Molecular Targets on Brain Proteins

描述(由申请人提供)：我们对乙醇对神经元功能的急性影响的观点已经从非特定的膜作用演变为对特定蛋白质上的特定位置的扰动。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行为联系起来。在过去的资助期间，我们开发了烷硫醇试剂作为酒精类似物，可以用来共价标记感兴趣蛋白质中的假定酒精结合部位。我们建议使用这种方法来检验这样的假设，即乙醇通过与充满水的蛋白质腔内结合而改变甘氨酸和GABAA受体的功能，并且这些腔的占据改变了通道门控。识别酒精对靶蛋白作用的关键氨基酸应该有助于我们回答这个关键问题：乙醇的哪些行为影响是由于对哪些特定蛋白质的作用？从关键氨基酸到行为学研究的途径被最近在小鼠中构建的敲打结构所阐明，在该结构中，单个GABAA受体亚基中的单个氨基酸的突变改变了苯二氮类药物的特定行为行为。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。当前提案的一个主要目标是使用这种方法来确定甘氨酸和GABAA受体在体内酒精作用中的重要性。除了敲入方法，更传统的敲除或零突变小鼠也将被用来提供关于特定蛋白质在药物作用中的作用的有价值的信息，并将在这个项目中使用。这项工作的长期目标是确定关键的蛋白质位点，这些蛋白质位点可以作为酒精强化、依赖和复发新疗法的靶点。

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.