ALCOHOL ACTION ON A CLONED POTASSIUM CHANNEL

克隆钾通道上的酒精作用

• 批准号: 2000495
• 负责人: MANUEL L COVARRUBIAS
• 金额: $ 17.33万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2000495
• 关键词: alcohols; ethanol; general anesthesia; molecular cloning; neural inhibition; neural transmission; neurophysiology; neurotoxicology; potassium channel; protein purification; toad

APPLICANT'S ABSTRACT: The biological basis of ethanol intoxication and general anesthesia are believed to involve, in part, effects on ion channels that regulate nerve excitability. However, it is not known how ethanol and general anesthetics alter ion channel function at the molecular level and to what extent they share a common mechanism. The long-term goal of this proposal is to understand the molecular mechanism underlying inhibition of a cloned potassium channel by ethanol and other aliphatic alcohols (n-alcohols). Aliphatic alcohols behave as general anesthetics. The channel under study is encoded by Shaw2, a gene cloned from Drosophila. Previous studies in this laboratory have shown that Shaw2 potassium channels are selectively inhibited by clinically-relevant concentrations of ethanol in a manner consistent with a direct drug-channel interaction. Recombinant DNA technology, expression in frog oocytes or insect cells and patch-clamp recording will be used to address the following aspects: 1) The action of n-alcohols on the electrophysiological properties of Shaw2 potassium channels. 2)Identification of Shaw2 protein domains and amino acid residues that may interact with n-alcohols. 3)Overexpression, purification and reconstitution of recombinant Shaw2 potassium channels. The first two aspects will help us to understand the biophysical and molecular basis of channel inhibition. The third aspect will allow purification of sufficient quantities of the channel protein to permit the use of biochemical and biophysical methods to study more directly the interaction between n-alcohols and the channel. A comprehensive study of the molecular mechanism underlying inhibition of a potassium channel by n-alcohols is an important step towards understanding how ethanol can affect ion channel function in general, causing general anesthesia and other acute alterations affecting the brain and other organs.

申请人摘要：乙醇中毒的生物学基础和 据信全身麻醉部分涉及对离子通道的影响 调节神经兴奋性。 然而，目前尚不清楚乙醇和 全身麻醉剂在分子水平上改变离子通道功能并 他们在多大程度上拥有共同的机制。 本次活动的长远目标 提议是了解抑制a的分子机制 通过乙醇和其他脂肪醇克隆钾通道 （n-醇）。 脂肪醇起到全身麻醉剂的作用。 这 正在研究的通道由 Shaw2 编码，Shaw2 是从果蝇中克隆的基因。 该实验室之前的研究表明 Shaw2 钾通道 被临床相关浓度的乙醇选择性抑制 以与直接药物通道相互作用一致的方式。 重组 DNA技术、青蛙卵母细胞或昆虫细胞表达和膜片钳 录音将用于解决以下几个方面： 1） 正醇对 Shaw2 钾电生理特性的影响 渠道。 2)Shaw2蛋白结构域和氨基酸残基的鉴定 可能与正醇发生相互作用。 3)过表达、纯化和 重组Shaw2钾通道的重建。 前两个 方面将帮助我们了解生物物理和分子基础 通道抑制。 第三个方面将允许纯化足够的 通道蛋白的数量以允许使用生化和 生物物理学方法更直接地研究之间的相互作用 n-醇和通道。 分子的综合研究 正醇抑制钾通道的机制是 理解乙醇如何影响离子通道的重要一步 一般功能，导致全身麻醉和其他急性改变 影响大脑和其他器官。