EFFECTS OF MUTATIONS OF TORPEDO ACETYLCHOLINE-RECEPTOR ALPHA-1 SUBUNIT RESIDUES 184-200 ON ALPHA-BUNGAROTOXIN BINDING IN A RECOMBINANT FUSION PROTEIN

Residues between positions 184 and 200 of the Torpedo acetylcholine re ceptor alpha1 subunit were changed by oligonucleotide-directed mutagen esis in a recombinant fusion protein containing residues 166-211. Amin o acids were substituted with residues present in the snake alpha subu nit, with an alanine, or with a functionally dissimilar residue. The c ompetitive antagonist alpha-bungarotoxin bound to the fusion protein w ith high apparent affinity (IC50 = 3.2 x 10(-8) M), and binding was co mpeted by agonists and antagonists. Mutation of His-186, Tyr-189, Tyr- 190, Cys-192, Cys-193, Pro-194, and Asp-195 greatly reduced or abolish ed alpha-bungarotoxin binding, while mutation of Tyr-198 reduced bindi ng, indicating these residues play an important role in binding either through functional interaction with neurotoxin residues or by stabili zing the conformation of the binding site. Molecular modeling of acety lcholine receptor residues 184-200 and knowledge of both neurotoxin an d receptor residues essential for binding allow analysis of possible s tructure-function relationships of the interaction of alpha-bungarotox in with this region of the receptor.