Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.

Abstract

Synthetic peptides and their respective antibodies were used in an attempt to localize and identify the ligand-binding site of the nicotinic acetylcholine receptor. Two peptides of the receptor .alpha. subunit were synthesized, the 1st corresponding to the NH2-terminal domain (positions 1-20) and the other, to a segment (residues 126-143) that contains the 1st 2 cysteine residues. Specific antipeptide antibodies were elicited in rabbits after immunization with the peptides conjugated to bovine serum albumin. The antipeptide antibodies obtained cross-reacted with the receptor and bound specifically to its .alpha. subunit. The antipeptide antibodies were used to test whether the peptide sequences corresponded to the .alpha.-bungarotoxin (.alpha.-BTX)-binding site. Staphylococcus aureus V8-protease digestion of the isolated receptor .alpha. subunit generated several fragments. Antipeptide(1-20) and antipeptide(126-143) both bound a 26-kDa fragment, whereas only antipeptide(126-143) bound a 17-kDa fragment. None of these fragments were found to bind .alpha.-BTX. On the other hand, .alpha.-BTX bound to an 18-kDa fragment that did not react with either of the antipeptide antibodies. Moreover, the 26-kDa and 17-kDa fragments were also found to contain the endoglycosidase H-susceptible oligosaccharide chain. The toxin-binding site apparently lies beyond the first possible V8 protease cleavage site after residues 126-143; i.e., Asp-152. This location is in agreement with the possibility that cysteine residues 192 and/or 193 are in close proximity to or contiguous with the ligand-binding site.