Molecular Mechanism Underlying Alcohol Modulation of Inward Rectifying K+ Channel

项目描述（由申请人提供）：本项目主要目的是研究醇类调节离子通道的分子机制。g蛋白门控内整流钾通道（Kir3或GIRK）是控制神经元电活动的重要通道，可被乙醇激活。活化的机制尚不清楚。最近，在一个相关的内向整流器Kir2.1的三维结构中发现了一个二醇的结合位点。由于二醇和乙醇在结构上是相关的，这个疏水口袋被认为是醇调节的位点。拟议的研究将评估这种疏水结合袋是否介导二醇和醇在Kir3和Kir2通道上的作用。具体而言，研究的目的是：1)利用膜片钳技术确定二醇和醇对相关Kir2通道电流调制的定量构效关系；2)表征二醇诱导的Kir3电流激活，并比较乙醇对这些通道的调节作用；3)通过结构域特异性和位点定向诱变确定二醇结合疏水袋是否是乙醇对Kir3和Kir2的作用位点。全细胞膜片钳记录将用于研究二醇和醇对哺乳动物细胞中异种表达的Kir通道的影响。这些实验将有助于更好地理解醇调节离子通道的机理。在美国和世界范围内，酒精是一种主要的成瘾和滥用药物。饮酒会导致中毒，这被描述为中枢神经系统抑制的全面增加。这是由酒精对离子通道的影响介导的，离子通道是决定神经元如何放电的关键蛋白质。人们对醇类如何直接影响离子通道知之甚少。提出的实验将使我们更好地了解醇对一类受醇影响的离子通道的作用。这项研究还可能导致药物制剂的开发，旨在预防或治疗与饮酒有关的成瘾和滥用。

DESCRIPTION (provided by applicant): The major goal of this project is to investigate the molecular mechanisms underlying modulation of ion channels by alcohols. G-protein gated inwardly rectifying potassium (Kir3 or GIRK) channels, which are important for controlling electrical activity of neurons, are activated by ethanol. The mechanism of activation is not well understood. Recently, a binding site for a diol was discovered in a 3D structure of a related inward rectifier, Kir2.1. Since both diol and ethanol are structurally related, this hydrophobic pocket is postulated to be the site of alcohol modulation. The proposed research will assess whether this hydrophobic binding pocket mediates the actions of diols and alcohols on both Kir3 and Kir2 channels. Specifically the aim of the research is to: 1) determine the quantitative structure-activity relationship for diols and alcohols on modulation of related Kir2 channel currents by using patch-clamp techniques; 2) Characterize diol induced activation of Kir3 currents and compare with effects of ethanol modulation of these channel; and 3) Determine whether the diol binding hydrophobic pocket is the site of action of ethanol for Kir3 and Kir2 by domain specific, and site directed mutagenesis. Whole-cell patch-clamp recordings will be used to study the effect of diols and alcohols on Kir channels expressed heterologously in mammalian cells. These experiments will lead to a better understanding of the mechanism of alcohol modulation of ion channels. Alcohol is a major drug of addiction and abuse in the U.S. and worldwide. Consumption of alcohol leads to intoxication which is described as an overall increase in inhibition of the central nervous system. This is mediated by alcohol's effect on ion channels, proteins which are critical in determining how neurons fire. How alcohols directly affect ion channels is poorly understood. The proposed experiments will lead to a better understanding of where alcohols act on a class of ion channels affected by alcohol. This study could also lead to development of pharmaceutical agents designed to prevent or treat addiction and abuse associated with consumption of alcohol.