Neuronal nicotinic acetylcholine receptors (nAChRs) are of great interest because they are critical sites at which acetylcholine must act to excite the forebrain. These receptors are of particular interest because they are the sites at which nicotine exerts its psychoactive and addictive effects. Pharmacologic intervention at neuronal nAChRs holds promise for treating the effects of diseases of the central nervous system and for understanding and treating addictive processes. A particularly important therapeutic target is the site at which nAChRs bind acetylcholine. Unfortunately, our understanding of the structural basis for this interaction is limited. Also, for therapeutic agents to be effective, they must be capable of distinguishing among the multiple functional subtypes of nAChRs that are present in the nervous system. The research project that I describe in this application will identify the specific amino acid residues that determine the pharmacological properties of the neurotransmitter binding sites of neuronal nAChRs. The experimental strategy is to use the pharmacological differences between members of this receptor family as probes of receptor structure. This will be done using the techniques of molecular biology. Chimeric receptor subunits will be constructed by replacing specific sequence segments of one subunit with the analogous sequence segment of a pharmacologically distinct subunit. By determining the pharmacological properties of receptors formed by chimeric subunits, the sequence segments that determine pharmacological specificity will be identified. Drawing on the results of these experiments, individual amino acid changes will be made between pharmacologically distinct receptors. These experiments will identify the specific amino acids that determine pharmacological specificity. The results of these experiments will aid in the design of therapeutic agents selective for individual receptor subtypes. Specific drugs will be of great use in treating, as well as probing the mechanism of, nicotine addiction.
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