FUNCTIONAL MAPPING OF NICOTINIC RECEPTOR BINDING SITES

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DESCRIPTION (adapted from applicant's description): The aim of this proposal is to develop a dynamic structural model of the nicotinic acetylcholine receptor (AChR) agonist binding site by combining mutagenesis of the receptor with functional and biochemical analysis. The application of molecular biological techniques to studies of the nicotinic receptor has resulted in significant advances in the understanding of the relationship between receptor structure and function. For example, the regions of the receptor responsible for acetylcholine binding and the region which forms the ion channel proper have been identified. The task now is to understand how these regions interact to generate the functional response. Where are the specific contact points between acetylcholine and the binding domain? What are the physical changes in the protein that occur upon binding which ultimately lead to the opening of the ion channel? By using a panel of genetically modified receptors in which sites for chemical modification have been introduced throughout the binding domain, the roles of individual residues will be examined. Biochemical analysis will yield information on the accessibility of residues and secondary structure of the binding site. Protein conformational changes will also be monitored by examining acetylcholine-dependent differences in chemical modification. Overall the goal of this work is to enhance understanding of the specific mechanisms of acetylcholine action and to provide information applicable to studies of other ligand-activated ion channels.