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

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

Subunit structure of the acetylcholine receptor from Electrophorus electricus.

The amino-terminal amino acid sequences of the four major peptides (Mr 41,000, 50,000, 55,000, and 62,000) present in purified preparations of Electrophorus electricus nicotinic acetylcholine receptor (AcChoR) have been determined for 24 cycles by automated sequence analysis procedures yielding four unique polypeptide sequences. The sequences showed a high degree of similarity, having identical residues in a number of positions ranging between 37% and 50% for specific pairs of subunits. Comparison of the sequences obtained with those of the subunits of similar molecular weight from Torpedo californica AcChoR revealed an even higher degree of homology (from 46% to 71%) for these two highly diverged species. Simultaneous sequence analysis of the amino termini present in native, purified Electrophorus AcChoR showed that these four related sequences were the only ones present and that they occur in a ratio of 2:1:1:1, with the smallest subunit ("alpha 1") being present in two copies. Genealogical analysis suggests that the subunits of both Torpedo and Electrophorus AcChoRs derive from a common ancestral gene, the divergence having occurred early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as the highly conserved structure of the AcChoR complex along animal evolution, suggest that each of the subunits evolved to perform discrete crucial roles in the physiological function of the AcChoR