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

Abstract

We have studied the stoichiometry of the binding of the long a-neurotoxins from the venom of Dendroaspis viridis (a-dendrotoxin) and Naja naja siamensis (a-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-aneurotoxin 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 a-cobratoxin was 2.1 ± 0.2 (n = 4), in agreement with the accepted view that a-cobratoxin, like a-bungarotoxin, binds to the two a subunits, which are constituent polypeptides of the AcChoR molecule. aDendrotoxin gave a stoichiometry of 4.1 ± 0.5 (n = 12); therefore, the AcChoR molecule contains four binding sites for this a-neurotoxin, two of which are recognized by acobratoxin. In support of this contention we have also found that when the AcChoR is saturated with a-bungarotoxin, addition of a-dendrotoxin markedly accelerates the dissociation of the bound a-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.The nicotinic acetylcholine receptor (AcChoR) from Torpedo electric organ is a complex pentameric molecule formed by four highly homologous proteins in a stoichiometry a2Pfy8 (reviewed in ref.

Effects of Chronic Nicotinic Ligand Exposure on Functional Activity of Nicotinic Acetylcholine Receptors Expressed by Cells of the PC12 Rat Pheochromocytoma or the TE671/RD Human Clonal Line

“…Thus, this interpretation allows the prediction that the functional inactivation assay may have usefulness in dissecting sites and modes of nicotinic ligand action, including the long-noted idiosyncrasies (Rang and Ritter, 1970) in the actions of "metaphilic" ligands at nAChRs, and that pancuronium and alcuronium may not have open channel blocking activities found for d-TC and decamethonium. A third alternative is that selected antagonists and agonists share the ability to interact at novel regulatory sites (Takeyasu et al, 1983(Takeyasu et al, , 1986, some of which may interact differentially with neurotoxins (Hanley et al, 1978;Conti-Tronconi and Raftery, 1986;Bradley et al, 1987) or smaller ligands (Conti-Tronconi et al, 1982;Dunn and Raftery, 1982).…”

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

“…We identified low affinity agonist-specific sites, which displayed properties that were consistent with the involvement of these sites in channel activation [12][13][14]. Supporting evidence for multiple binding sites has come from studies of α-dendrotoxin binding [15], photoaffinity labeling [16] and from flux studies of pre-formed receptor-ligand complexes [17]. In this report, we have characterized the binding of an agonist, epibatidine [18], to the Torpedo nAChR and directly demonstrate the presence of four agonist binding sites under equilibrium conditions.…”

Multiple effects of α‐toxins on the nicotinic acetylcholine receptor

“…This group has claimed that agonists can still bind to the low-affinity sites after the high-affinity sites on the AChR have been blocked by curare or covalently labeled with bromoacetylcholine [21]. It has been reported that although the a,-toxin from N. naja siamensis has two binding sites on the AChR (one on each of the two ce-subunits), the a-toxin from Dendroaspis viridis has four binding sites on the AChR [22].…”