Selective distinction at equilibrium between the two α‐neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration

Abstract

The binding of the monoiodinated α‐neurotoxin I from Naja mossambica mossambica to the membrane‐bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar‐sensitive microtitration assay. From equilibrium binding studies a non‐linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants K_d1= 7 ± 4 pM and K_d2= 51 ± 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k_−1.1= 25 × 10⁻⁶ s⁻¹ and k_−1.2= 623 × 10⁻⁶ s⁻¹ respectively), but not in their rate of formation of the toxin‐receptor complex (k₊₁, = 11.7 × 10⁶ M⁻¹ s⁻¹). From these rate constants the same two values of dissociation constant were deduced (K_d1= 2 pM and K_d2= 53 pM). All the specific binding was prevented by the cholinergic antagonists α‐bungarotoxin and d‐tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d‐tubocurarine sites (K_da= 103 nM and K_db= 13.7 μM respectively). Evidence is provided indicating that these two sites are shared by d‐tubocurarine and α‐neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high‐affinity binding sites for this short α‐neurotoxin are selectively distinguishable.