Molecular Mechanism Underlying Alcohol Modulation of Inward Rectifying K+ Channel

描述（由申请人提供）：本项目的主要目标是研究醇类调节离子通道的分子机制。G蛋白门控内向整流钾（Kir 3或GIRK）通道对于控制神经元的电活动是重要的，其被乙醇激活。激活的机制还不清楚。最近，在相关内向整流器Kir2.1的3D结构中发现了二醇的结合位点。由于二醇和乙醇在结构上相关，因此假定该疏水口袋是醇调节的位点。拟议的研究将评估这种疏水结合口袋是否介导二醇和醇对Kir 3和Kir 2通道的作用。本研究的主要目的是：1）应用膜片钳技术研究二醇和醇类化合物对相关Kir 2通道电流的调控作用的定量构效关系; 2）研究二醇对Kir 3通道电流的激活作用，并与乙醇对这些通道的调控作用进行比较;和3）通过结构域特异性和定点诱变确定二醇结合疏水口袋是否是乙醇对Kir 3和Kir 2的作用位点。将使用全细胞膜片钳记录来研究二醇和醇对哺乳动物细胞中异源表达的Kir通道的影响。这些实验将导致更好地了解酒精调节离子通道的机制。酒精是美国和世界范围内成瘾和滥用的主要药物。饮酒导致中毒，这被描述为中枢神经系统抑制的总体增加。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。酒精如何直接影响离子通道还知之甚少。拟议的实验将导致更好地了解酒精作用于一类受酒精影响的离子通道。这项研究还可能导致开发旨在预防或治疗与酒精消费相关的成瘾和滥用的药物。

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.