Membrane Potential Dependent Binding of Scorpion Toxin to the Action Potential Sodium Ionophore. Studies With a 3‐(4‐hydroxy 3‐[125i] Iodophenyl) Propionyl Derivative

A polypeptide toxin purified 80‐fold from the venom of the scorpion Leiurus quinquestriatus enhances activation of the action potential Na+ ionophore by the alkaloid neurotoxins veratridine, batrachotoxin and aconitine in electrically excitable neuroblastoma cells. The purified toxin can be labelled with [125I] by reaction with N‐succinimidyl 3‐(4‐hydroxy 3‐[125I] iodophenyl) propionate. The [125I] labelled toxin obtained from carboxymethyl Sephadex ion exchange chromatography appears homogeneous by gel electrophoresis and isoelectric focusing. The [125I] labelled toxin binds to a single class of saturable binding sites and also activates the action potential Na+ ionophore in electrically excitable neuroblastoma cells showing identical concentration dependence for both the binding and the activation effects. The labelled toxin does not show any saturable binding or activation of the action potential Na+ ionophore in variant neuroblastoma clones that specifically lack the action potential Na+ ionophore. The results indicate that scorpion toxin binds specifically to the action potential Na+ ionophore. The binding sites have a mean equilibrium dissociation constant of 3 IIH, a mean binding capacity of 46fmol toxin per mg cell protein and a mean density of 24 sites per μm2 of cell surface membrane. A single action potential Na+ ionophore transports 1 × 108 ions per min and has a conductance of 3 psiemens at physiologic ion concentrations. Depolarization of cells by elevated K+ concentration inhibits the saturable binding. Depolarization of cells by incubation in high Na+ medium (130mm‐Na+, 5mm‐K+) with gramicidin A or batrachotoxin also inhibits the saturable toxin binding. These results suggest that scorpion toxin binds specifically to a regulatory component (gate) of the Na+ ionophore. whose conformation is dependent on membrane potential.