Fine localization of the major alpha-bungarotoxin binding site to residues alpha 189-195 of the Torpedo acetylcholine receptor. Residues 189, 190, and 195 are indispensable for binding

Tzartos, S.J.; Remoundos, M.S.

Journal of Biological Chemistry 265(35): 21462-21467


ISSN/ISBN: 0021-9258
PMID: 2254308
Accession: 040146713

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alpha-Bungarotoxin blocks acetylcholine-mediated ion channel opening of peripheral acetylcholine receptors (AChR). A major binding region for alpha-bungarotoxin has been recently identified within parts of the segment 170-204 of the alpha-subunit. We used the Pepscan systematic peptide synthesis system to determine the minimum Torpedo AChR segment required for alpha-bungarotoxin binding and to investigate the role of each residue within this segment. Continuously overlapping decapeptides within alpha 179-203 and several decapeptides covering other alpha-subunit sequences showed that alpha 188-197 and alpha 189-198 exhibited the best 125I-alpha-bungarotoxin binding activity (KD = 7.3 x 10(-8) and 4.3 x 10(-8) M, respectively). Several continuously overlapping nona-, octa-, hepta-, hexa-, and tetrapeptides showed that the heptapeptide alpha 189-195 was the minimum sequence with high binding activity (KD = 5.6 x 10(-8)M). d-Tubocurarine, but not carbamylcholine, blocked toxin binding. Twenty-six analogs of the alpha 188-197, most having 1 residue substituted by Ala or Gly, showed that Tyr189, Tyr190, and especially Asp195 were indispensable for 125I-alpha-bungarotoxin binding. Cys192 and Cys193 could be substituted by other amino acids, proving that the disulfide bond between alpha 192-193 was not required for alpha-bungarotoxin binding. The decreased alpha-bungarotoxin binding capacity of the equivalent human muscle AChR alpha 188-197 peptide was the result of substitution of Tyr by Thr at alpha 189.