Molecular Mechanisms of Desensitization and Drug Modulation in Ligand-Gated Ion C

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DESCRIPTION (provided by applicant): The long-term goal of this proposal is to determine the structural basis for allosteric mechanisms in pentameric ligand gated ion channels (LGIC). Upon neurotransmitter binding, LGIC rapidly switch from closed to open to desensitized channel conformations. Despite a substantial understanding of the physiological and pathophysiological role of desensitization, there is very little structural information available on this state. The fcus of this study is to use bacterial LGIC homologues, GLIC and ELIC, to identify the mechanism of desensitization and to determine how drugs modulate transitions between the closed, open and desensitized states. We propose to apply spectroscopic techniques to functionally defined states of GLIC and ELIC in a membrane environment. Our studies will involve a multidisciplinary approach that includes spectroscopic techniques (EPR and fluorescence), patch-clamp measurements in reconstituted proteoliposomes, and thermodynamic measurements using isothermal titration calorimetry. We will 1) Measure the conformational transitions underlying the desensitized conformation. 2) Determine the role of lipid-water-protein interface in gating transitions. 3) Identify the structural basis for allosteric modulation by general anesthetics and alcohols. These experiments will test the central hypothesis that membrane lipids play a key role in maintaining the conformational integrity, in driving gating transitions, and in governing drug modulation. The proposed studies are expected to be significant in that they will provide a dynamic view of LGIC gating in a physiologically- relevant membrane environment. These findings will establish a structural framework to enhance our understanding of LGIC function at the molecular level and thereby improve therapeutic strategies and drug design.