Binding of [ 3H]batrachotoxinin A-20-α-benzoate to a high affinity site associated with house fly head membranes

Abstract

1. [ 3H]Batrachotoxinin A-20-α-benzoate (BTX-B), a radioligand that labels the alkaloid activator recognition site of the voltage-sensitive sodium channel, was bound specifically to high affinity, saturable sites in a subcellular preparation from house fly ( Musca domestica L.) heads that was shown previously to contain binding sites for other sodium channel-directed ligands. 2. Specific binding of [ 3H]BTX-B was observed in the presence of 140 mM sodium or potassium and was inhibited by choline ion. 3. Saturating concentrations of scorpion ( Leiurus quinquestriatus) venom stimulated the specific binding of [ 3H]BTX-B four-fold, increasing the proportion of specific binding of 10 nM [ 3H]BTX-B from less than 15% to 40%. Equilibrium dissociation studies in the presence of scorpion venom gave an equilibrium dissociation constant ( K D ) for [ 3H]BTX-B of 80 nM and a maximal binding capacity ( B max) of 1.5 pmol/mg protein. 4. Parallel experiments in the absence of venom gave a K D value of 140 nM and a B max of 1.3 pmol/mg protein, indicating that scorpion venom stimulated [ 3H]BTX-B binding by increasing the affinity of this site approximately two-fold. 5. The specific binding of [ 3H]BTX-B was inhibited by the sodium channel activators aconitine and batrachotoxin and, to a lesser extent, by the anticonvulsant diphenylhydantoin. However, several other sodium channel-directed neurotoxins known to exert allosteric effects on the binding of [ 3H]BTX-B to mammalian brain preparations did not affect the binding of [ 3H]BTX-B to house fly head membranes. 6. These studies provide evidence for a high affinity binding site in house fly head membrane preparations that exhibits properties expected of the activator recognition site of the voltage-sensitive sodium channel but does not respond to several compounds known to modify allosterically the binding of [ 3H]BTX-B to sodium channels in mammalian brain.