Structural analysis of alcohol-dependent activation of GIRKs

描述(申请人提供)：酒精摄入通过调节不同类型的离子通道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实现的。酒精在大脑中的靶点之一是G蛋白门控的内向整流钾(GIRK)通道，该通道被乙醇激活。与野生型小鼠相比，缺乏GIRK2通道的小鼠表现出酒精诱导的疼痛耐受性降低，自我给予更多的乙醇。此外，一个对镇静停药倾向有很大影响的数量性状基因座，如来自乙醇的，被缩小到小鼠1号染色体上包含GIRK3基因的区域。GIRK3基因敲除小鼠表现出不那么严重的镇静-催眠戒断。尽管GIRK2和GIRK2/3通道与乙醇相关的行为有关，但这种反应的分子机制尚不清楚。最近，我们用高分辨率结构研究表明，醇直接与内向整流钾通道的疏水口袋结合。GIRK2通道酒精结合口袋的突变显著改变了乙醇的激活。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，该变化传递到通道的门并打开通道。在这个研究方案中，我们计划使用高分辨率结晶学研究、基于结构的突变和先进的电生理记录的创新方法来研究这一假说。具体地说，我们将进行GIRK2/3通道中乙醇结合口袋的结构-功能分析(1)，解决与乙醇复杂的GIRK通道的高分辨率结构，以揭示乙醇依赖门控时通道蛋白的构象变化(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的离子通道的机制提供深入的认识。了解乙醇调节离子通道的分子机制可以开发治疗酒精依赖的新型药物，直接造福人类健康。 与公共健康相关：乙醇是美国吸毒和滥用的主要药物，它直接调节大脑离子通道，控制脑神经元的兴奋性。这项资助的目的是研究酒精依赖激活神经元钾通道的分子和结构机制。这些研究的结果可能会导致开发出专门调节钾通道或拮抗乙醇作用的新型药物。

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.