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.
