The Binding Site of the Nicotinic Acetylcholine Receptor in Animal Species Resistant to β-Bungarotoxin

The ligand binding site of the nicotinic acetylcholine receptor (AChR) is located in the α-subunit, within a small fragment containing the tandem cysteines at positions 192 and 193. We have been analyzing the binding site domain of AChRs from several animal species exhibiting various degrees of resistance to α-bungarotoxin (α-BTX). Our earlier work on the snake and mongoose AChR, both of which do not bind α-BTX, suggested that amino acid substitutions at positions 187, 189, and 194 of the AChR α-subunit are important in determining the resistance of these AChRs to α-BTX. In the present study, we have examined the correlation between a-BTX binding and the structure of the binding site domain of AChR from the hedgehog, shrew, cat, and human. Fragments of the AChR α-subunit corresponding to residues 122-205 from these species were cloned, sequenced, and expressed in Escherichia coli. The hedgehog fragment does not bind α-BTX, in common with the snake and mongoose AChR, and the human fragment is a partial binder. The shrew and cat fragments bind α-BTX to a similar extent as the mouse fragment. The hedgehog and human AChRs have nonaromatic amino acid residues at positions 187 and 189 of the α-subunit, as is seen with the “toxin resistant” snake and mongoose, and in contrast with the “toxin binders”, which have aromatic residues at these two positions. Thus, it appears that aromatic amino acid residues at positions 187 and 189 of the AChR α-subunit are required for proper α-BTX binding, and that changes at these positions to nonaromatic residues are important in determining resistance to α-BTX.