The long-term goal of this project is the elucidation of the high-resolution structure of the extracellular domain of the skeletal muscle-type nicotinic acetylcholine receptor (nAChR). Near term, a major focus is placed on elucidating the structure of the ligand binding site with particular emphasis on the binding of the classic, nicotinic, high affinity ligand, a- bungarotoxin (Bgtx). A clear delineation of the contact zone involved in Bgtx recognition would contribute not only towards the elucidation of the molecular basis for receptor-toxin interaction but would also further our understanding of the basic principles that underlie molecular recognition in other important protein-protein interactions. In the first aim we will use multi-dimensional NMR techniques to determine the solution structures of 15N-enriched receptor- derived peptides (an a1 18mer and an a7 19mer) each bound in a stoichiometric complex with a-bungarotoxin. These two peptides, which correspond to the sequences on their respective native receptors forming the major binding determinants for Bgtx, bind Bgtx with nM affinity.
Aim 2 will use a site-directed Cysteine substitution approach to map the solvent accessible surface of the nAChR and to identify positions in proximity to the Bgtx binding site.
In aim 3, we will study chimeric neuronal/muscle a-subunits expressed in Xenopus oocytes to further delineate the residues contributing to Bgtx binding. New chimeric constructs will be prepared using a4 and a2 subunits to determine whether sensitivity to Bgtx can be conferred with minimal sequence replacements. Recent technological advances in the expression and analysis of recombinant proteins and in NMR-based structure determinations make this an opportune time to pursue these goals. The proposed studies, directed towards the ultimate understanding of how ligand-gated receptors operate at the molecular and mechanistic level, will also be of considerable value in the design of better and more specific neuromuscular blocking agents. Information gained from the proposed studies will also be relevant to the entire family of neuronal nAChRs whose structure and function are less well characterized than those of the muscle receptor. Neuronal nAChRs are involved in cognitive function and also appear to play a role in nicotine dependency and in neurological disorders such as schizophrenia and Alzheimer's disease.
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