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

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Abstract
The nicotinic acetylcholine receptor (AcChoR) is a ligand-gated ion channel that is activated upon binding of acetylcholine. α-Neurotoxins, in particular α-bungarotoxin (α-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 α-BTX. Phages bearing the motif YYXSSL as a consensus sequence were found to bind with high affinity to α-BTX. The library-derived peptide (MRYYESSLKSYPD) bears amino acid sequence similarities to a region of the α-subunit of the Torpedo muscle AcChoR, as well as of other muscle and neuronal AcChoRs that bind α-BTX. The library-derived peptide and the corresponding peptides containing residues 187–199 of the Torpedo AcChoR α-subunit (WVYYTCCPDTPYL), as well as peptides analogous to the above region in the neuronal AcChoR (e.g., human α7; ERFYECCKEPYPD) that binds α-BTX, inhibit the binding of α-BTX to the intact Torpedo AcChoR with IC50 values of 10−6 M. A synthetic peptide from a neuronal AcChoR that does not bind α-BTX (e.g., human α2; ERKYECCKEPYPD) which differs by just one amino acid from the homologous peptide from the α-BTX-binding protein (α7)—i.e., Lys in α2 and Tyr in α7—does not inhibit the binding of α-BTX to Torpedo AcChoR. These results indicate the requirement for two adjacent aromatic amino acid residues for binding to α-BTX.