SITES OF ANESTHETIC ACTION ON GABA A RECEPTORS

该计划项目旨在确定将军的行动地点 麻醉剂作用于大脑中的蛋白质靶点，集中在配体上- 门控离子通道神经递质受体超家族，本项目 涉及抑制性GABA/A受体-氯离子通道(GABAR)， 是全麻药的主要候选分子靶点。和以前一样 GABAR超级家族的所有成员都是异戊异构体，包括 从18个曲目中选择三种不同类型的亚基。 结果产生了大约24种不同的亚型， 药理特性。GABAR对麻醉药的不同敏感性 不同的亚基组成允许使用基因工程的 定点嵌合体和突变鉴定氨基酸残基 参与了麻醉调制。其他人最近发现了几个 膜跨结构域中的残基；我们将进行一系列 N端胞外区的定点突变研究 对麻醉药调节具有不同敏感性的GABAR亚基 以确定哪些残留物应对此负责。更直接 麻醉剂结合部位的鉴定可通过以下方式获得 放射性麻醉剂的光亲和标记及其测定 通过微测序共价结合的氨基酸残基。这 研究将使用在昆虫Sf9细胞系中表达的重组GABAR 使用杆状病毒。特别是，我们将开发一种新的放射性物质 麻醉剂类固醇光亲和标签用于GABAR的工作。它的重要性 的标记残留物将通过诱变和分析 使用捆绑和电生理学的麻醉敏感性。这个 这些方法的组合，以及与交互站点的比较 在受体超家族的其他成员中，特别是尼古丁 乙酰胆碱受体，将使我们更好地了解 这些临床重要药物的作用机制。

This program project seeks to identify the sites of action of general anesthetics on protein targets in the brain, concentrating on the ligand- gated ion channel super-family of neurotransmitter receptors, this project deals with the inhibitory GABA/A receptor-chloride ion channel (GABAR), the major candidate molecular target for the general anesthetics. As with all members of the super-family, GABAR are heteropentamers, comprised of three different sorts of subunits chosen from a repertoire of 18. This results in about two dozen different isoforms which differ in pharmacological properties. Different sensitivity to anesthetics of GABAR of varying subunit composition allow the use of the genetically engineered site-directed chimerae and mutagenesis to identify amino acid residues involved in anesthetic modulation. Others recently have identified several residues in the membrane-spanning domains; we will carry out a series of site-directed mutagenesis studies on the N-terminal extracellular domain of GABAR subunits of varying sensitivity to modulation by anesthetics in order to identify which residues are responsible. More direct identification of anesthetic binding sites can be obtained by photoaffinity labeling with radioactive anesthetics and determination of the amino acid residues that are covalently bound by microsequencing. This study will employ recombinant GABAR expressed in the insect Sf9 cell line using Baculovirus. In particular we will develop a new radioactive anesthetic steroid photoaffinity label for the GABAR work. The importance of labeled residues will be verified by mutagenesis and analysis of anesthetic sensitivity using binding and electrophysiology. The combination of these approaches, and comparison with sites of interaction in other members of the receptor super-family especially nicotinic acetylcholine receptors, will lead to a greater understanding of the mechanism of action of these clinically important drugs.