BINDING SITES OF THE ACETYLCHOLINE ACCEPTOR

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The nicotine acetylcholine receptor contains a ligand-gated ion channel which mediates the chemo-electrical transduction at the neuromuscular junction by binding acetylcholine released from the nerve terminal. The broad long-term objectives of this proposal is to understand how the agonist-binding induces the channel opening and conformational changes of the receptor. Specifically, the role of charge-charge interactions in acetylcholine receptor function will be systematically studied. The specific aims are: to determine electrostatic interactions receptor function will be systematically studied. The specific aims are: to determine electrostatic interactions between agonist. The specific aims are: to determine electrostatic interactions between agonist and its binding sites, and whether the charged groups exist at the sites and how they distribute; to determine the effect of electrostatic interactions on the conformation equilibrium of the acetylcholine receptor, and whether electrostatic interactions are responsible for conformational conversions; and to determine the role of electrostatic interactions between non- competitive blocker (NCB) and the channel site, and whether the changes in the ion channel distribute differently for different conformations. The equilibrium, association and dissociation constants of agonist and NCB will be determined by conventional fluorescence binding measurements and stopped flow fluorescence spectroscopy at various ionic strength. These measurements will also characterize the populations of the native conformations and rate of conformational transitions. The acetylcholine receptor is the target of auto-immune antibodies in the disease myasthenia gravis and local anesthetics. The proposed research will help define the structure of those domains relative to ligand binding and thus will contribute to rational drug design.