The long term goal of the research in this application is to understand the mechanism of regulation of the gating of the nicotinic acetylcholine receptor (AChR) ion channel by acetylcholine binding. The specific goal of this project is to define the neurotransmitter binding sites on the AChR.
Three specific aims will be carried out. First, two series of homologous toxins, based on the alkaloid d-tubocurarine and on the peptide alpha- conotoxins, will be synthesized and characterized for binding to native receptors. Second, an extended set of amino acids in the AChR that interact with the toxins will be defined. Third, specific interaction energies between AChR amino acids and functional groups on the toxins will be measured to then complete a map of complementary interactions between binding site residues and the toxins. The methods for carrying out these specific aims include equilibrium binding analysis of toxins to the AChR and synthesis of new toxins, principally peptide synthesis and modification. AChR subunit chimeras will be constructed with sequentially smaller segment replacement. These chimeras will be analyzed for their toxin binding properties to identify amino acids involved in binding. Photoaffinity derivatives of toxins will also be synthesized for identification of amino acids at the binding sites by subsequent proteolytic mapping. Site directed mutations of amino acids that interact with the toxins will be constructed. Then the energy contribution of specific amino acid-toxin interactions will be measured by double mutant thermodynamic cycle analysis. The skeletal muscle AChR (and particularly the extracellular domain) is the target of autoimmune antibodies in the disease Myasthenia gravis. The neuronal homologs of this protein are likely involved in nicotine addiction and possibly Alzheimer's disease. A better understanding of the structure of the extracellular domain of the AChR and especially the neurotransmitter sites will be important for a full understanding of these ailments and may lead to insights to improve rational drug design for this class of receptors.
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