The alpha-bungarotoxin binding site on the nicotinic acetylcholine receptor: Analysis using a phage-epitope library

The nicotinic acetylcholine receptor (AcChoR) is a ligand-gated ion channel that is activated upon binding of acetylcholine, alpha-Neurotoxins, in particular alpha-bungarotoxin (alpha-BTX), bind specifically and with high affinity to the AcChoR and compete with binding of the natural ligand, We employed a 15-mer phage-display peptide library to select epitopes reacting with alpha-BTX. Phages bearing the motif YYXSSL as a consensus sequence were found to bind with high affinity to alpha-BTX. The library-derived peptide (MRYYESSLKSYPD) bears amino acid sequence similarities to a region of the alpha-subunit of the Torpeda muscle AcChoR, as well as of other muscle and neuronal AcChoRs that bind alpha-BTX. The library-derived peptide and the corresponding peptides containing residues 187-199 of the Torpedo AcChoR alpha-subunit (WVYYTCCPDTPYL), as well as peptides analogous to the above region in the neuronal AcChoR (e.g., human alpha(7); ERFYECCKEPYPD) that binds alpha-BTX, inhibit the binding of alpha-BTX to the intact Torpedo AcChoR with IC50 values of 10(-6) M. A synthetic peptide from a neuronal AcChoR that does not bind alpha-BTX (e.g., human alpha(2); ERKYECCKEPYPD) which differs by just one amino acid from the homologous peptide from the alpha-BTX-binding protein (alpha(7))-i.e., Lys in alpha(2) and Tyr in alpha(7)-does not inhibit the binding of alpha-BTX to Torpedo AcChoR. These results indicate the requirement for two adjacent aromatic amino acid residues for binding to alpha-BTX.