The nicotinic acetylcholine receptor (nAChR) represents the prototype for the large supergene family of ligand-gated channels which now includes the neuronal subtypes of nAChRs, as well as the glycine, GABA, 5-HT, and glutamate receptors. The long term goals in this project are: a) to determine the structures of the agonist and antagonist binding sited of the nAChR through analysis of the structures of the N-terminal extracellular domains of the receptor subunits, b) to characterize local, ligand-induced changes in structure and to relate these to the transduction of agonist binding to channel opening, c) to elucidate structural determinants underlying the assembly of receptor subunits, and d) to determine the significance of specific neuronal forms of the nAChR whose detailed role in behavior and nervous system function is largely unknown. Understanding the molecular mechanisms subserving nAChR function will have an important impact on studies of the other members of this structurally related family. A better understanding of the structure and functions of the extracellular domain of the skeletal muscle-type nAChR may shed light on the reasons for the great diversity of neuronal nAChR subunits. Structural information is of special value to the study of receptors such as the nAChR in that any such information can significantly contribute to the design of drugs targeted for specific receptor subtypes. Such drug design efforts could eventually lead to more selective neuromuscular blockers, to drugs that are selective for specific subtypes of neuronal nAChRs, and possibly, to improved anesthetics as well.
The aim of this proposal is, through the concomitant and coordinated use of both site-directed mutagenesis and NMR-based structural analyses, to identify in detail the structural determinants on the nAChR that form the ligand binding domain of the receptor. The investigators aim to determine the NMR solution structure of a 62-amino acid, recombinantly expressed, soluble receptor fragment that comprises 30 percent of the entire N-terminal, extracellular domain of the alpha-subunit. This fragment binds the curaremimetic antagonist, alpha-bungarotoxin (BGTX), with an apparent affinity very near to that of the native, intact receptor. They will use site-directed mutagenesis of heterologously expressed nAChR to test the predictions of the previously described NMR solution structure of a BGTX/receptor peptide complex. They also plan to construct recombinantly expressed soluble receptor fragments of the alpha, gamma, and delta subunits that will be useful in identifying inter-subunit contact regions contributing to subunit assembly. Inter- subunit contacts may be important in regulating the subunit composition of nAChRs and may ultimately determine the apparent subunit diversity of neuronal nAChRs. A combined structural and mutational approach to these issues is in line with the current objectives of structural biology which aims to elucidate the underlying relationship between structure and function. Working together the two approaches can provide insights into molecular mechanisms and the underlying basis for molecular recognition in the nervous system.
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