SITES OF ANESTHETIC ACTION ON GABA A RECEPTORS

该项目旨在确定一般性的行动地点， 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，该项目 涉及抑制性GABA/A受体-氯离子通道（GABAR）， 全身麻醉药的主要候选分子靶点。如同 GABAR超家族的所有成员都是杂五聚体，包括 从18个子单元中选出三种不同的子单元。这 导致大约24种不同的同种型， 药理学特性GABAR对麻醉药的不同敏感性 允许使用基因工程的 定点嵌合和诱变以鉴定氨基酸残基 参与麻醉调节。其他人最近已经确定了几个 跨膜结构域中的残基;我们将进行一系列 N-末端胞外区的定点突变研究 对麻醉剂调节的不同敏感性的GABAR亚基在 以确定哪些残基是负责的。更直接 麻醉剂结合位点的鉴定可以通过 用放射性麻醉剂进行光亲和标记和测定 通过微测序共价结合的氨基酸残基。这 研究将使用在昆虫Sf 9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 麻醉类固醇光亲和标记的GABAR工作。的重要性 标记残基的突变将通过诱变和分析进行验证， 麻醉敏感性使用绑定和电生理学。的 这些方法的组合，以及与相互作用位点的比较 在受体超家族的其他成员中，尤其是烟碱受体， 乙酰胆碱受体，将导致更好地了解 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.