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：酒精中毒的生物学基础和 全身麻醉被认为部分涉及对离子通道的影响。 调节神经兴奋性的物质。然而，目前尚不清楚乙醇和 全麻药在分子水平上改变离子通道功能 它们在多大程度上共享一个共同的机制。这样做的长期目标是 建议理解抑制一种基因的分子机制 乙醇等脂肪醇克隆的钾通道 (n-醇)。脂肪醇的作用类似于全身麻醉剂。这个 正在研究的通道是由从果蝇中克隆的基因Shaw2编码的。 本实验室以前的研究表明，Shaw2钾通道 被临床上相关浓度的乙醇选择性地抑制 以与药物-通道直接相互作用相一致的方式。重组 DNA技术、在青蛙卵母细胞或昆虫细胞中的表达及膜片钳 录音将用于解决以下方面：1)行动 正醇对Shaw2钾的电生理性质的影响 频道。2)Shaw2蛋白结构域和氨基酸残基的鉴定 可能与正构醇相互作用的物质。3)过表达、纯化和 重组Shaw2钾通道的重组。前两个 Aspects将帮助我们理解生物物理和分子基础 通道抑制。第三方面将允许提纯足够的 允许使用生化和蛋白质的通道蛋白的量 生物物理方法更直接地研究人与人的相互作用 N-醇和通道。分子的综合研究 正构醇抑制钾通道的机制是 了解乙醇如何影响离子通道的重要一步 全身功能，引起全身麻醉和其他急性改变 影响大脑和其他器官。