Binding of scorpion toxin to sodium channels in vitro and its modification by beta-bungarotoxin.

1. Binding of a purified scorpion toxin to membrane fragments isolated from electroplaque of an electric eel Electrophorus electricus was studied using a radio-iodinated toxin.2. A scorpion toxin was purified from the venom of Leiurus quinquestriatus and iodinated with (125)I in a lactoperoxidase-catalysed reaction. Monoiodinated toxin, isolated by an ion exchange chromatography, retarded the inactivation kinetics of Na current to a similar extent as the native toxin, indicating that radioiodination did not appreciably affect physiological and binding properties of the native toxin.3. Analyses of binding properties by Scatchard plots showed the presence of two classes of binding sites (with low and high affinities) in the membrane preparation from eel electroplaque; similar preparation from an electric skate, of which the electroplaque is known to be devoid of Na channels, possessed only the low affinity sites.4. The number of high affinity sites in the eel preparation was 41.8 +/- 10.5 p-mole/g tissue; the value was within the range reported for tetrodotoxin binding to similar preparations (15-148 p-mole/g tissue).5. A variety of cations (Na(+), Mn(2+) and La(3+)) inhibited the high affinity scorpion toxin binding, as indicated for the toxin binding to Na channels by a previous electrophysiological study. K(D) value in the presence of 120 mM-Na(+) (approx. 8 nM) agreed reasonably with that (approx. 10nM) reported for the scorpion toxin binding to excitable neuroblastoma cells or synaptic nerve ending particles under conditions where membrane potential was depolarized by the addition of 135 mM-KCl.6. Pretreatment of the eel membrane preparation with beta-bungarotoxin (7-44 ng/ml.) in the presence of Ca ions (10-200 muM) resulted in a substantial loss of high affinity binding of scorpion toxin. When phospholipase A(2) activity of the beta-toxin was inactivated by a chemical modification with p-bromophenacyl bromide, the inhibitory action of the beta-bungarotoxin was abolished.7. It is concluded that a high affinity binding of scorpion toxin to the eel electroplaque membrane fragments represents the binding to Na channels in vitro, and that phospholipase A(2) activity of beta-bungarotoxin interferes with the binding of scorpion toxin to Na channels.