Mapping of a Cholinergic Binding Site by Means of Synthetic Peptides, Monoclonal Antibodies, and α-Bungarotoxin

Previous studies by several laboratories have identified a narrow sequence region of the nicotinic Acetylcholine receptor (AChR) α subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind α-bungarotoxin (α-BTX) and several α-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the α subunit, and bind to AChR in a mutually exclusive fashion with α-BTX. The binding of WF5 and W2 to Torpedo AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4–6 residues, and corresponded to the complete sequence of Torpedo AChR α subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to al66-203, were synthesized. α-BTX bound to a peptide containing the sequence αl81-200 and also, albeit to a lesser extent, to a peptide containing the sequence α55-74. WF6 bound to αl81-200 and to a lesser extent to α55-74 and αl34-153. The two other mAbs predominantly bound to α55-74, and to a lesser extent to αl81-200. Peptides αl81-200 and α55-74 both inhibited binding of ¹²⁵I-α-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound α-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR a subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the “discontinuous epitopes” observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].