The ionotropic acetylcholine receptor (AchR) from the electric organ of the marine ray Torpedo californica is a pentameric ligand-gated cation channel composed of 4 different but similar subunits with a subunit stoichiometry of alpha2-beta-gamma-delta. Each subunit presents two distinct faces, (+) and (-), to form a different interface with each of its neighboring subunits. The long-term research goals of this project are to identify the subunit residues that are essential for stabilization of these interfaces that form during receptor assembly and to identify within that set of stabilizing residues the subset of residues which determines the specificity of each subunit-subunit interaction.
SPECIFIC AIM 1 is to identify alpha and gamma subunit residues that stabilize the alpha+/-gamma interface that forms in one of the earliest steps in AchR assembly.
SPECIFIC AIM 2 is to identify alpha and beta subunit residues that stabilize the beta+/-alpha interface, which also forms early in AchR assembly.
SPECIFIC AIM 3 is to identify residues in the delta+/-beta interface that influence delta subunit assembly with beta-alpha-gamma trimers, an intermediate step in AchR assembly. In these 3 aims, site-directed mutagenesis will be used to modify specific residues that will be identified from a homology model of the interfaces constructed from the published high-resolution structure of a snail acetylcholine-binding protein. The effect of each point mutation on cell-surface AchR expression and on the assembly of intracellular oligomers will be measured.
SPECIFIC AIM 4 is to make a mutant gamma subunit (gamma') that can substitute the delta subunit in assembly of surface expressed AchR yet still present a """"""""gamma-like"""""""" (-) face to its interface with the alpha subunit.
This aim will confirm the specificity of the interactions and will produce a cell surface-expressed alpha2-beta-gamma-gamma'receptor with equivalent alpha-gamma ligand-binding domains. Such a molecule would greatly facilitate radioligand-binding studies on this receptor. The Torpedo1 AchR is an excellent model for the human muscle AchR, whose dysfunction occurs in numerous acquired, inherited and toxicological neuromuscular diseases. Understanding Torpedo AchR assembly at the molecular level will be a major step toward improving the diagnosis and treatment of such diseases.
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