Identification of equivalent residues in the gamma, delta, and epsilon subunits of the nicotinic receptor that contribute to alpha-bungarotoxin binding

Cysteine was introduced from residues 116 to 121 of the gamma subunit of the fetal mouse acetylcholine receptor, and the mutant receptors were treated with methanethiosulfonate reagents and examined for changes in ligand binding properties, Of the 18 combinations of mutant and reagent, only receptors harboring gamma L119C treated with the quaternary ammonium reagent MTSET (trimethylammonium-ethyl methanethiosulfonate) show a decreased number of alpha-bungarotoxin (alpha-btx) sites, The decrease of 50% suggests that alpha-btx binding to the site harboring gamma L119C is blocked, Analysis of binding of the site-selective ligands dimethyl-d-tubocurarine (DMT) and alpha-conotoxin M1 (CTX) confirm specificity of modification for the site harboring gamma L119C, Cysteines placed at equivalent positions of the delta and epsilon subunits also lead to selective loss of alpha-btx binding following MTSET treatment, gamma L119C receptors treated with the primary amine reagent MTSEA (aminoethyl methanethiosulfonate) retain alpha-btx binding to both sites but show reduced affinity for DMT and CTX at the modified site, Lysine mutagenesis of Leu(gamma 119), Leu(delta 121), and Leu(epsilon 119) mimics MTSEA treatment, whereas mutagenesis of Thr(alpha 119) and Gln(beta 119) is without effect, demonstrating subunit and residue specificity of MTSEA modification, MTSET modification of nearby gamma Y117C does not block alpha-btx binding but markedly diminishes affinity for DMT and CTX, The overall findings indicate a localized point of interaction between alpha-btx and the modified gamma L119C, delta L121C, and epsilon L119C.