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

Abstract

The binding of the monoiodinated a-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 Kdl = 7 4 pM and Kd2 = 51 & 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-l,l = 25 x l o p 6 s-' and k -l . 2 = 623x s-l respectively), but not in their rate of formation of the toxin-receptor complex ( k + , = 11.7 x lo6 M-' S-' ). From these rate constants the same two values of dissociation constant were deduced (&I = 2 pM and Kdz = 53 pM). All the specific binding was prevented by the cholinergic antagonists abungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 pM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and a-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 a-neurotoxin are selectively distinguishable.

Crystal structure of a Cbtx–AChBP complex reveals essential interactions between snake α-neurotoxins and nicotinic receptors

“…The short a-neurotoxin NmmI was purified from the venom of Naja mossambica mossambica (Marchot et al, 1988). The long a-neurotoxins Cbtx (N. naja siamensis toxin 3) (Latoxan, France) and Bgtx (Sigma-Aldrich) were analyzed by native-PAGE with migration toward the cathode and SDS-PAGE (20% homogenous gels) (Marchot and Bougis, 2000), and by mass spectrometry.…”

Biochemical and Pharmacological Characterization of a Depressant Insect Toxin from the Venom of the Scorpion Buthacus arenicola

“…Samples were hydrolyzed with trypsin, pronase, carboxypeptidase Y, microsomal leucine aminopeptidase and prolidase in a 1 :2: 1 : 1 : 1 (by mass) ratio; the carboxypeptidase Y:toxin ratio was 1:35 (by mass). Reverse-phase HPLC analysis was performed as described (Marchot et al, 1988).…”

Subunit Interface Selectivity of the α-Neurotoxins for the Nicotinic Acetylcholine Receptor

“…Mutagenesis studies have also identified candidate residues in the principal loops of the ␣ (16) and non-␣ subunits (17) that contribute to ␣-toxin binding. Although most ␣-toxins do not distinguish between the two sites on the receptor, an ␣-toxin from the venom of Naja mossambica mossambica (NmmI) distinguishes between the two sites of the Torpedo receptor (18). Thus NmmI emerges as a potentially valuable ligand for determining regions of close approach between ␣-toxins and the non-␣ subunits at the binding site.…”