The nicotinic acetylcholine receptor translates the binding of acetylcholine into the flow of ions across the cell membrane and thus serves a major role in the communication between nerve and muscle (or electrocyte). In addition to acetylcholine, several types of pharmacological agents interact with the acetylcholine receptor. Of these, the noncompetitive blockers and the benzomorphan opiates bind to regulatory sites on the receptor that are distinct from the binding sites for acetylcholine. The studies proposed here will use acetylcholine receptor from Torpedo electroplaque and from rat primary myotubes to investigate the function, structure, and physiological significance of the binding sites for noncompetitive blockers and opiates. The function of the binding sites will be investigated using a multidisciplinary approach that includes radioligand binding, 19F NMR spectroscopy, stopped flow fluorescence analysis of agonist binding and ion flux, and loose and gigaseal patch clamp recording. The structure of the binding sites will be investigated using photoaffinity labeling, peptide mapping and amino acid sequence analysis. Finally, the hypothesis that an endogenous substance is present in the Torpedo electric organ which is capable of regulating the acetylcholine receptor through these binding sites will be tested and, if present, the endogenous substance will be thoroughly characterized. Preliminary results suggest that such a molecule exists and potentially can be purified using reverse phase HPLC. A detailed knowledge of the structure and regulatory properties of the acetylcholine receptor is important for a number of reasons: (1) due to the fact that it can be obtained in milligram quantities, the nicotinic acetylcholine receptor serves as a model system for the general understanding of neurotransmitter-receptor interactions; (2) the acetylcholine receptor plays an important role in a number of neuromuscular disease processes, such as myasthenia gravis and slow channel syndrome; and (3) a number of adverse drug interactions are mediated through the various ligand binding sites on the acetylcholine receptor. We hope that our studies will provide a new level of understanding of the structure and regulatory properties of the acetylcholine receptor and suggest new strategies for novel therapeutic uses of drugs that interact with the various binding sites on the receptor protein.
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