SITES OF ANESTHETIC ACTION ON GABA A RECEPTORS

GABA A 受体的麻醉作用位点

• 批准号: 6410443
• 负责人: RICHARD W OLSEN
• 金额: $ 17.7万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6410443
• 关键词: Baculoviridae; GABA receptor; SDS polyacrylamide gel electrophoresis; Sf9 cell line; Xenopus oocyte; affinity labeling; alcohols; binding sites; chloride channels; drug interactions; electrophysiology; halothane; immunofluorescence technique; local anesthetics; neurotransmitter antagonist; peptide chemical synthesis; protein purification; protein sequence; receptor binding; receptor expression; scintillation counter; site directed mutagenesis; voltage /patch clamp

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.

该计划项目旨在确定一般的行动部位 大脑中蛋白质靶标麻醉药，集中于配体 神经递质受体的门控离子通道超家族，该项目 处理抑制性GABA/A受体 - 氯离子通道（GABAR）， 全身麻醉剂的主要候选分子靶标。和 超级家庭的所有成员Gabar都是异植物分子，由 从18的曲目中选择了三种不同的亚基。 导致大约二十种不同的同工型，不同 药理特性。对Gabar麻醉的敏感性不同 不同的亚基组成允许使用基因工程 定向的嵌合体和诱变，以鉴定氨基酸残基 参与麻醉调节。其他人最近确定了几个 跨膜域中的残基；我们将进行一系列 N末端细胞外结构域的位置定向诱变研究 对麻醉剂在变化的敏感性的GABAR亚基中 为了确定哪些残留物负责。更直接 可以通过识别麻醉结合位点的识别 带有放射性麻醉和确定的光性标签 通过微钉测序共价结合的氨基酸残基。这 研究将采用在昆虫SF9细胞系中表达的重组GABAR 使用杆状病毒。特别是我们将开发新的放射性 麻醉剂类固醇光附为GABAR工作的标签。重要性 标记的残基将通过诱变和分析来验证 使用结合和电生理学麻醉灵敏度。这 这些方法的组合以及与相互作用的比较 在受体的其他成员中，尤其是烟碱 乙酰胆碱受体将使对 这些临床上重要的药物的作用机理。