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

Agonists and antagonists select between the αγ and αδ binding sites of the fetal muscle acetylcholine receptor owing to different contributions by the γ and δ subunits. To identify determinants of selectivity for agonists, we constructed a panel of γ-δ subunit chimeras, co-expressed them with the α subunit in 293 HEK cells, and measured carbamylcholine binding affinity of intracellular complexes. Wild-type αδ complexes bind carbamylcholine about 30-fold more tightly than αγ 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(γ34)/Ser(δ36) and Phe(γ172)/Ile(δ178), and a secondary set, Glu(γ57)/Asp(δ59) and Cys(γ115)/Tyr(δ117). 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(γ34)/Ser(δ36) and Phe(γ172)/Ile(δ178) contribute in a manner consistent with affecting selectivity of the resting state of the receptor, while Glu(γ57) 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 γ and δ subunits contribute to the agonist binding site.