IDENTIFICATION OF EQUIVALENT RESIDUES IN THE GAMMA-SUBUNIT, DELTA-SUBUNIT, AND EPSILON-SUBUNIT OF THE NICOTINIC RECEPTOR THAT CONTRIBUTE TOALPHA-BUNGAROTOXIN BINDING

Cysteine was introduced from residues 116 to 121 of the gamma subunit of the fetal mouse acetylcholine receptor, and the mutant receptors we re treated with methanethiosulfonate reagents and examined for changes in ligand binding properties, Of the 18 combinations of mutant and re agent, only receptors harboring gamma L119C treated with the quaternar y 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 lig ands dimethyl-d-tubocurarine (DMT) and alpha-conotoxin M1 (CTX) confir m specificity of modification for the site harboring gamma L119C, Cyst eines placed at equivalent positions of the delta and epsilon subunits also lead to selective loss of alpha-btx binding following MTSET trea tment, gamma L119C receptors treated with the primary amine reagent MT SEA (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) an d Gln(beta 119) is without effect, demonstrating subunit and residue s pecificity of MTSEA modification, MTSET modification of nearby gamma Y 117C does not block alpha-btx binding but markedly diminishes affinity for DMT and CTX, The overall findings indicate a localized point of i nteraction between alpha-btx and the modified gamma L119C, delta L121C , and epsilon L119C.