GATING MECHANISMS OF CHEMICALLY ACTIVATED CHANNELS

化学激活通道的门控机制

• 批准号: 2264866
• 负责人: MEYER B. JACKSON
• 金额: $ 12.26万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2264866
• 关键词: Drosophilidae; GABA receptor; Xenopus oocyte; allosteric site; chemical binding; chemical kinetics; gamma aminobutyrate; gene mutation; molecular cloning; molecular site; neural inhibition; neuropharmacology; picrotoxin; protein structure function; site directed mutagenesis; synapses; transfection; voltage /patch clamp

Patch clamp and molecular genetic techniques will be combined to study structure-function relationships in the Drosophila GABA-A receptor. The recent cloning of the GABA-A receptor of Drosophila provides an opportunity to carry out molecular genetic modifications of the receptor. The functional consequences of these structural alterations will be examined in expression systems such as Xenopus oocytes and transfected cells, and in a native cellular environment provided by cell culture of the Drosophila nervous system. In addition, Drosophila provides a powerful method of isolating mutants based on selection in terms of function, rather than site-directed mutagenesis. These advantages will be exploited to investigate the following basic problems relevant to the function of ligand-gated channels. We will use site-directed mutagenesis to determine the residues responsible for the anion selectivity of this channel. This information will then be exploited to elucidate the number of subunits in the native receptor and the number of copies of each cloned subunit in the native receptor. Ultimately, this work will lead to an understanding of the subunit stoichiometry of the GABA-A receptor of Drosophila. There is already some evidence to suggest that this receptor is a homomultimer, and if this is actually the case it would have considerable evolutionary and mechanistic significance. Proposed experiments will provide a clear test for this hypothesis. Along a different line, experiments will exploit mutants with reduced sensitivity to the classical GA BAA receptor antagonist picrotoxin. These mutants were isolated in fields sprayed with cyclodiene insecticides. Experiments proposed with these and other mutants will determine whether picrotoxin blocks by an allosteric mechanism, or by blockade of the open channel, or by both of these mechanisms. These experiments will also identify the residues of the GABA-A receptor that form the picrotoxin binding site. The information about the GABA-A receptor obtained in these studies will help researchers understand the nature of inhibition throughout the nervous system. Researchers will be able to develop better drugs in the treatment of epilepsy and many other neural disorders. The insight that will result from this study will be of general value in the development of drugs that act at other receptors in addition to the GABA-A receptor.

斑块夹和分子遗传技术将合并以研究 果蝇GABA-A受体中的结构功能关系。这 果蝇的GABA-A受体的最新克隆提供了 进行受体的分子遗传修饰的机会。 这些结构改变的功能后果将是 在诸如爪蟾卵母细胞等表达系统中检查并转染 细胞，以及在由细胞培养的天然细胞环境中 果蝇神经系统。此外，果蝇提供了强大的 基于选择的方法隔离突变体的方法， 而不是定向诱变。这些优势将被利用 调查与功能有关的以下基本问题 配体门控通道。我们将使用定向诱变来确定 负责该通道阴离子选择性的残基。这 然后，将利用信息来阐明 本地受体和每个克隆亚基的副本数量 天然受体。最终，这项工作将导致对 果蝇的GABA-A受体的亚基化学计量法。有 已经有一些证据表明该受体是同源物，并且 如果实际上是这种情况，它将具有相当大的进化和 机械意义。拟议的实验将提供明确的测试 为此假设。沿着不同的线，实验将利用 对经典GA BAA受体敏感性降低的突变体 拮抗剂野毒素。这些突变体在喷涂的田野中分离 环肽杀虫剂。这些突变体提出的实验 将通过变构机制或通过 开放通道的封锁或通过这两种机制的封锁。这些 实验还将确定GABA-A受体的残基 形成野毒素结合位点。有关GABA-A的信息 在这些研究中获得的受体将帮助研究人员了解 整个神经系统抑制的性质。研究人员会 能够在治疗癫痫和许多其他许多方面开发更好的药物 神经疾病。这项研究将产生的见解将是 在对其他受体作用的药物开发中的一般价值 加上GABA-A受体。