Molecular dissection of subunit interfaces in the acetylcholine receptor - Identification of residues that determine agonist selectivity

Agonists and antagonists select between the alpha gamma and alpha delta binding sites of the fetal muscle acetylcholine receptor owing to different contributions by the gamma and delta subunits, To identify determinants of selectivity for agonists, we constructed a panel of gamma-delta subunit chimeras, co-expressed them with the alpha subunit in 293 HEK cells, and measured carbamylcholine binding affinity of intracellular complexes, Wild-type alpha delta complexes bind carbamyl choline about 30-fold more tightly than alpha gamma complexes. This degree of selectivity is similar to that of the resting state of the receptor determined by kinetic analysis of single-channel events, We identify a primary set of determinants of selectivity, Lys(gamma 34)/Ser(delta 36) and phe(gamma 172)/Ile(delta 178), and a secondary set, Glu(gamma 57)/Asp(delta 59) and Cys(gamma 115)/Tyr(delta 117). The contributions of all four determinants are subunit-dependent and are modified by interaction with one another, Coexpression of point mutant subunits with complementary wild-type subunits to form cell surface pentamers shows that Lys(gamma 34)/Ser(delta 36) and phe(gamma 172)/Ile(delta 178) contribute in a manner consistent with affecting selectivity of the resting state of the receptor, while Glu(gamma 57) appears to contribute to the affinity of the desensitized state. The four determinants either coincide with or are close to residues known to contribute to the acetylcholine binding site. These results suggest that a minimum of four loops in the gamma and delta subunits contribute to the agonist binding site.