Nicotinic acetylcholine receptor contains multiple binding sites: Evidence from binding of α-dendrotoxin

Abstract

We have studied the stoichiometry of the binding of the long α-neurotoxins from the venom of Dendroaspis viridis (α-dendrotoxin) and Naja naja siamensis (α-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-α-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 α-cobratoxin was 2.1 ± 0.2 (n = 4), in agreement with the accepted view that α-cobratoxin, like α-bungarotoxin, binds to the two α subunits, which are constituent polypeptides of the AcChoR molecule. α-Dendrotoxin gave a stoichiometry of 4.1 ± 0.5 (n = 12); therefore, the AcChoR molecule contains four binding sites for this α-neurotoxin, two of which are recognized by α-cobratoxin. In support of this contention we have also found that when the AcChoR is saturated with α-bungarotoxin, addition of α-dendrotoxin markedly accelerates the dissociation of the bound α-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 pseudoxymmetric 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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