The long-term aim of this research is to understand the regulation of ligand-gated ion channels by the binding of neurotransmitters. The particular goal of this application is to understand the importance of charge-charge interactions in the regulation of the nicotinic acetylcholine receptor by the binding of acetylcholine. Specifically, the structure activity relationship of d-tubocurarine to the nicotinic acetylcholine receptor will be examined by analyzing the binding of d-tubocurarine analogs to native and mutated acetylcholine receptors. The role of electrostatic attraction in binding will be examined to determine whether charge- charge attraction governs the rapid rate of acetylcholine binding and contributes to the stabilization of the agonist cation. The channel movements associated with the conformational transition of the acetylcholine receptor will be measured by mutagenesis of residues near the narrow pore of the channel, and by direct measurements of electrostatic potential using fluorescence lifetime spectroscopic methods. The experiments will define functionally relevant components of the receptor structure that contribute to ligand binding, to ion channel structure, and to function. This will improve our understanding of synaptic transmission, a process that underlies the complex phenomena of learning, memory and thought. The skeletal muscle nicotinic acetylcholine receptor (and particularly the extracellular domain) is the target of autoimmune antibodies in the disease Myasthenia Gravis. The neuronal homologues of this protein are involved in nicotine addiction and possibly in Alzheimer's disease. A better understanding of the structure of this protein and especially the acetylcholine binding sites will be important for a full understanding of these ailments and for developing treatments. A fundamental understanding of the binding site stricture will improve rational drug design for this protein while the methodology proposed here may constitute a new paradigm for rational drug design for receptor targets in general.
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