ALCOHOL ACTION ON A CLONED POTASSIUM CHANNEL

申请人摘要：乙醇中毒的生物学基础， 一般认为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，不知道乙醇和 全身麻醉剂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。 长期目标是 我们的建议是要了解潜在的抑制的分子机制， 乙醇等脂肪醇克隆钾通道 （正醇）。 脂肪醇类可用作全身麻醉剂。 的 研究中的通道由Shaw 2编码，Shaw 2是从果蝇克隆的基因。 该实验室先前的研究表明，Shaw 2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中的表达和膜片钳 记录将用于解决以下几个方面：1）行动 正构醇对Shaw 2钾离子电生理特性的影响 渠道 2)Shaw 2蛋白结构域和氨基酸残基的鉴定 可以与N-醇相互作用。 3）过表达、纯化和 重组Shaw 2钾通道的重建。 前两 这些方面将帮助我们了解生物物理和分子基础， 通道抑制 第三个方面将允许足够的纯化 通道蛋白的量，以允许使用生物化学和 生物物理学方法更直接地研究 n-醇和通道。 一项关于分子生物学的综合研究 N-醇抑制钾通道的潜在机制是 了解乙醇如何影响离子通道重要一步 一般功能，引起全身麻醉和其他急性改变 影响大脑和其他器官

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.