Analysis of Acetylcholine Receptor Subunit

Universidad Central Del Caribe, Bayamon PR

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Abstract

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