Structural analysis of alcohol-dependent activation of GIRKs

描述（由申请人提供）：酒精（乙醇）的摄入通过调节不同类型的离子通道来改变大脑中的神经活动。该领域的一个新兴概念是，乙醇的一些生理效应是通过直接调节大脑中的离子通道来介导的。大脑中乙醇的靶标之一是G蛋白门控的内纠偏钾（GIRK）通道，该通道由乙醇激活。缺乏GIRK2通道的小鼠表现出乙醇诱导的疼痛耐受性降低，并且比野生型小鼠自我施用更多的乙醇。此外，在小鼠1号染色体上含有Girk3基因的一个数量性状位点对镇静戒断（如乙醇戒断）的易感性有很大影响。GIRK3基因敲除小鼠表现出较轻的镇静催眠戒断反应。尽管GIRK2和GIRK2/3通道与乙醇相关行为有关，但这种反应的分子机制尚不清楚。最近，我们通过高分辨率的结构研究表明，醇直接结合到内向纠偏钾通道的疏水口袋上。GIRK2通道中酒精结合袋的突变显著改变了乙醇的活化。我们假设乙醇与GIRK2/3通道中的疏水口袋结合，并促进构象变化，从而传递到通道的门并打开通道。在本研究计划中，我们计划使用高分辨率晶体学研究，基于结构的诱变和先进的电生理记录的创新方法来研究这一假设。具体而言，我们将对GIRK2/3通道中的乙醇结合囊进行结构-功能分析(1)，求解与乙醇配合的GIRK通道的高分辨率结构，以揭示乙醇依赖性门控发生的通道蛋白构象变化(2)，并利用单通道记录和半胱氨酸取代通道的化学修饰，阐述乙醇依赖性激活GIRK通道的机制模型(3)。这些实验的完成将揭示乙醇调节GIRK通道的结构基础，这将为乙醇调节其他类型离子通道的机制提供见解。了解乙醇调控离子通道的分子机制有助于开发治疗酒精依赖的新型药物，直接造福人类健康。

DESCRIPTION (provided by applicant): Alcohol (ethanol) consumption alters neural activity in the brain by modulating different types of ion channels. An emerging concept in the field is that some of the physiological effects of ethanol are mediated by direct modulation of ion channels in the brain. One of the targets in the brain for ethanol is the G protein-gated inwardly rectifying potassium (GIRK) channel, which is activated by ethanol. Mice lacking GIRK2 channels exhibit diminished ethanol-induced tolerance to pain and self-administer more ethanol than wild-type mice. Moreover, a quantitative trait loci with a large effect on predisposition to sedative withdrawal, such as from ethanol, was narrowed to a region on chromosome 1 in mice that contains Girk3 gene. GIRK3 knockout mice exhibit less severe sedative-hypnotic withdrawal. Though GIRK2 and GIRK2/3 channels are implicated in ethanol-related behaviors, the molecular mechanism underlying this response is not well understood. Recently, we showed with high-resolution structural studies that alcohols bind directly to hydrophobic pockets of inwardly rectifying potassium channels. Mutations in the alcohol-binding pocket of GIRK2 channels significantly alter ethanol activation. We hypothesize that ethanol binds to hydrophobic pockets in GIRK2/3 channels and facilitate a conformational change that is relayed to the channel's gate and opens the channel. In this research proposal, we plan to use an innovative approach of high-resolution crystallographic studies, structure-based mutagenesis and advanced electrophysiological recordings to investigate this hypothesis. Specifically, we will conduct a structure-function analysis of the ethanol-binding pocket in GIRK2/3 channels (1), solve high-resolution structures of GIRK channels complexed with ethanol to reveal conformational changes in the channel protein that occur with ethanol-dependent gating (2), and elaborate mechanistic models for ethanol-dependent activation of GIRK channels, utilizing single-channel recordings and chemical modification of cysteine-substituted channels (3). Completion of these proposed experiments will reveal the structural basis of ethanol modulation of GIRK channels, which will provide insights into the mechanism of ethanol-modulation of other types of ion channels. Understanding the molecular mechanism underlying ethanol modulation of ion channels could lead to development of novel pharmaceutical agents for treating alcohol-dependence, directly benefiting human health. PUBLIC HEALTH RELEVANCE: Ethanol, a major drug of addiction and abuse in the U.S., directly modulates brain ion channels, which control the excitability of brain neurons. The goal of this grant is to investigate the molecular and structural mechanisms underlying ethanol-dependent activation of neuronal potassium channels. Results from these studies could lead to the development of novel pharmaceutical agents that specifically modulate potassium channels or antagonize actions of ethanol.