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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Sep;83(17):6646–6650. doi: 10.1073/pnas.83.17.6646

Nicotinic acetylcholine receptor contains multiple binding sites: evidence from binding of alpha-dendrotoxin.

B M Conti-Tronconi, M A Raftery
PMCID: PMC386561  PMID: 3462717

Abstract

We have studied the stoichiometry of the binding of the long alpha-neurotoxins from the venom of Dendroaspis viridis (alpha-dendrotoxin) and Naja naja siamensis (alpha-cobratoxin) to the membrane-bound acetylcholine receptor (AcChoR) from Torpedo californica electric organ. The number of toxin molecules bound to one AcChoR molecule was determined by simultaneous-quantitative gas-phase microsequencing of all the amino acid sequences present in AcChoR-alpha-neurotoxin complexes. This method permits the use of homogeneous (nonradiolabeled) preparations of native toxins to obtain molar ratios of neurotoxin-receptor complexes. The stoichiometry obtained for alpha-cobratoxin was 2.1 +/- 0.2 (n = 4), in agreement with the accepted view that alpha-cobratoxin, like alpha-bungarotoxin, binds to the two alpha subunits, which are constituent polypeptides of the AcChoR molecule. alpha-Dendrotoxin gave a stoichiometry of 4.1 +/- 0.5 (n = 12); therefore, the AcChoR molecule contains four binding sites for this alpha-neurotoxin, two of which are recognized by alpha-cobratoxin. In support of this contention we have also found that when the AcChoR is saturated with alpha-bungarotoxin, addition of alpha-dendrotoxin markedly accelerates the dissociation of the bound alpha-bungarotoxin, demonstrating that the occupancy of the additional two sites by the latter toxin influences and decreases the affinity of the former toxin for its two binding sites. The fact that the AcChoR molecule is a pseudosymmetric complex of five highly homologous peptides suggests the possibility that as many as five binding sites for cholinergic ligand could be present, one on each subunit.

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Selected References

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