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对离子通道调节的分子机制。乙醇激活G蛋白门控内向整流钾(Kir3或GIRK)通道，该通道对控制神经元的电活动具有重要作用。激活的机制还不是很清楚。最近，在相关的内向整流子Kir2.1的3D结构中发现了二醇的结合部位。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酒精调节的部位。这项拟议的研究将评估这种疏水结合口袋是否介导了二醇和醇在Kir3和Kir2通道上的作用。具体地说，本研究的目的是：1)利用膜片钳技术确定二醇和醇对相关Kir2通道电流的调控作用的定量构效关系；2)表征二醇对Kir3电流的激活，并与乙醇对这些通道的调节作用进行比较；以及3)通过结构域特异性和定点突变来确定二醇结合的疏水口袋是否是乙醇对Kir3和Kir2的作用部位。全细胞膜片钳记录将被用来研究二醇和醇对哺乳动物细胞异源表达的KIR通道的影响。这些实验将有助于更好地理解酒精对离子通道的调节机制。在美国和全世界，酒精是一种主要的成瘾和滥用药物。饮酒会导致中毒，这被描述为对中枢神经系统的抑制全面增加。这是由酒精对离子通道的影响所介导的，离子通道是决定神经元如何放电的关键蛋白质。酒精是如何直接影响离子通道的，目前还知之甚少。拟议的实验将有助于更好地理解酒精对一类受酒精影响的离子通道的作用。这项研究还可能导致开发旨在预防或治疗与饮酒有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.