ALCOHOL ACTION ON A CLONED POTASSIUM CHANNEL

克隆钾通道上的酒精作用

• 批准号: 2855779
• 负责人: MANUEL L COVARRUBIAS
• 金额: $ 18.03万
• 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 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2855779
• 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.

申请人的摘要：乙醇中毒的生物学基础和 人们认为全身麻醉部分涉及离子通道的影响 调节神经兴奋性。 但是，尚不知道乙醇和 一般麻醉剂改变离子通道在分子水平和至 他们有多大的共同机制。 这个长期目标 建议是了解A的分子机制 克隆的钾通道和其他脂肪醇 （N-酒精）。 脂肪醇的表现为全身麻醉药。 这 研究的渠道由Shaw2编码，Shaw2是从果蝇克隆的基因。 该实验室的先前研究表明Shaw2钾通道 通过临床上浓度的乙醇有选择地抑制 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中的表达以及斑块钳 记录将用于解决以下方面：1） Shaw2钾的电生理特性上的N-醇 频道。 2）鉴定Shaw2蛋白质结构域和氨基酸残基 这可能与N-醇相互作用。 3）过表达，纯化和 重组Shaw2钾通道的重组。 前两个 各个方面将帮助我们了解生物物理和分子基础 通道抑制。 第三方面将允许净化足够 通道蛋白的数量允许使用生化和 生物物理方法可以更直接研究 N-酒精和通道。 分子的全面研究 N-酒精对钾通道抑制的基础机制是一种 了解乙醇如何影响离子渠道的重要步骤 通常，功能，引起全身麻醉和其他急性改变 影响大脑和其他器官。