This project is aimed at understanding the structure of the neurotransmitter binding sites of neuronal nicotinic acetylcholine receptors (nAChRs). Neuronal nAChRs are of interest for several reasons. Neuronal nAChRs are the sites at which nicotine exerts its psychoactive and addictive effects Nicotinic ligands are also potentially useful as anxiolytics and analgesics, and in the treatment of neurological disorders such as schizophrenia, Parkinson's disease, and Alzheimer's disease. Thus pharmacological intervention at neuronal nAChRs holds promise for treating the effects of diseases of the central nervous system, and for understanding and treating addictive processes. Critical to the realization of this potential is the development of subtype selective nAChR ligands. Pursuit of this goal requires an understanding of the molecular structure of the ligand binding sites of neuronal nAChRs. Specifically, the features of the nicotinic binding sites that are responsible for nAChR subtype selectivity must be identified. The neuronal nAChR family consists of a large number of related subunits, which can associate in a variety of combinations to form pharmacologically distinct receptors. The ligand binding sites on these receptors are complex, each being formed by several segments of amino acid sequence from two different subunits. We will identify the amino acid residues on neuronal nAChR subunits that confer specificity for agonists and competitive antagonists using a combination of molecular biological, electrophysiological and pharmacological techniques. We will identify critical amino acid residues on peptide antagonists of neuronal nAChRs, such as Neuronal Bungarotoxin, by studying a series of recombinant mutant toxin preparations. With this information, we will then use mutant cycle analysis to identify interacting pairs of residues on toxin and receptor, ultimately leading to an experimentally-based, three-dimensional model of amino acid residues of neuronal nAChR that determine subtype specificity.
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