Mapping the alcohol site of a neuronal potassium channel

描述（由申请人提供）：本项目的长期目标是确定酒精对脑离子通道功能调节的结构基础。本研究的主题是神经元Shaw 2钾通道，它是直接抑制乙醇和其他短链成员的同源系列的1-烷醇的相关浓度。在本研究先前结果的指导下，该项目的中心假设指出，Shaw 2通道的激活门贡献了一个两亲性蛋白质-蛋白质界面，该界面构成了醇结合位点。该项目的重点是研究激活门作为控制酒精诱导的Shaw 2通道反应的相互作用的位点。本课题的具体目标是：1）研究Shaw 2钾通道S4-S5环中酒精结合的特定结构决定因素。2)研究酒精诱导的Shaw 2钾通道S6节段C端部分的结构特征。3)以原核细胞KvAP通道为模型，探讨Shaw 2钾通道中乙醇结合位点的结构。前两个目标系统地将联合收割机重组DNA技术、电生理学和生物物理学分析相结合，以绘制Shaw 2通道中的酒精位点。最后一个目标是根据最近解决的KvAP通道的晶体结构，寻求结构生物学的潜在应用，以确定工程醇位点的结构。在离子通道中结合酒精的生理相关两亲界面的详细地图是在原子水平上了解急性酒精中毒并针对这些位点进行治疗应用的第一步。

DESCRIPTION (provided by applicant): The long-term goal of this project is to determine the structural basis of the functional modulation of brain ion channels by alcohol. The subject of this study is the neuronal Shaw2 potassium channel, which is directly inhibited by pharmacologically relevant concentrations of ethanol and other short-chain members of the homologous series of 1-alkanols. Guided by previous results from this study, the central hypothesis of the project states that the activation gate of the Shaw2 channel contributes an amphipathic protein-protein interface that constitutes the alcohol binding site. This project focuses on investigating the activation gate as the locus of the interactions that control the alcohol-induced responses of the Shaw2 channel. The specific aims of the project are: 1) To investigate the specific structural determinants of alcohol binding in the S4-S5 loop of the Shaw2 potassium channel. 2) To investigate the structural features that govern the alcohol-induced responses of the Shaw2 potassium channel in the C-terminal section of the S6 segment. 3) To develop the prokaryotic KvAP channel as a model to explore the achitecture of the alcohol binding site in the Shaw2 potassium channel. The first two aims systematically combine recombinant DNA technology, electrophysiology and biophysical analyses to map the alcohol site in the Shaw2 channel. In light of the recently solved crystal structure of the KvAP channel, the last aim seeks the potential application of structural biology to determine the achitecture of an engineered alcohol site. A detailed map of the physiologically relevant amphipathic interfaces that bind alcohol in ion channels is the first step toward understanding acute alcohol intoxication at the atomic level and targeting these sites for therapeutic applications.