Receptor-Ligand Interactions in the Muscle Type Nicotinic ACh Receptor

Washington University School Of Medicine, Saint Louis MO

Investigators

Abstract

SUMMARY: The muscle type nicotinic acetylcholine receptor (AChR) is located in the vertebrate neuromuscular junction and in the electric organ of some species of fish. Its function is to promote the depolarization of the endplate. As a member of the ligand-gated ion channel (LGIC) superfamily, its structural and functional properties are closely related to those of other members of the family such as the neuronal AChR, the GABAA receptor, the glycine receptor and others. The experiments in this proposal have been designed to study the very basics of the receptor-ligand interaction and channel gating, such as which residues on the receptor interact with which functional groups on the agonist, and the contributions made by the individual agonist binding sites to channel gating. The long-term goal of this project is to facilitate the understanding of the properties of the AChR and LGIC, in general. The proposed experiments will utilize single-channel patch clamp coupled to the kinetic analysis techniques which will allow detailed studies of the effects of changes in the structure of the receptor or the ligand, and to correlate them with changes in the functional response, i.e. affinity, gating or desensitization, of the receptor. The analysis of the interactions between the receptor and the ligand will be examined using thermodynamic mutant cycle analysis. This method is based on the determination of whether mutations are additive or not, and allows to establish whether amino acid residues or functional groups interact with each other. The AChR is used as a biophysical model system as it possesses relatively simple kinetic properties and a high single-channel conductance (or signal-to-noise ratio), necessary for studies involving kinetic analysis. The results of these studies can be applied in studies of other LGIC due to a high degree of homology between the members of the LGIC superfamily.