Batrachotoxinin-A-ortho-azidobenzoate: A photoaffinity probe of the batrachotoxin binding site of voltage-sensitive sodium channels

Abstract

T. L. Casebolt and G. B. Brown. Batrachotoxinin-A-ortho-azidobenzoate: a photoaffinity probe of the batrachotoxin binding site of voltage-sensitive sodium channels. Toxicon 31, 1113–1122, 1993.—Batrachotoxin (BTX) is one of a group of potent lipid-soluble neurotoxins which binds voltage-sensitive sodium channels. Here we show that [ 3H]batrachotoxinin-A-ortho-azidobenzoate ([ 3H]BTX-OAB), a photolabile derivative of BTX, binds covalently upon irradiation to the BTX sodium channel site of rat cerebral cortical synaptoneurosomes. Another ligand specific for the BTX sodium channel receptor, batrachotoxinin-A 20-α-benzoate (BTX-B), competitively inhibited the specific binding of [ 3H]BTX-OAB. The specific binding of [ 3H]BTX-OAB was increased by the addition of Leiurus quinquestriatus quinquestriatus scorpion venom (ScTx) and inhibited by veratridine, a member of the same class of sodium channel activators. Examination of the [ 3H]BTX-OAB-labeled components revealed that over 90% of the specifically incorporated [ 3H]BTX-OAB was recovered in lipid extracts of photolabeled synaptoneurosomes. Addition of tetrodotoxin (TTX) to the binding mixture increased the specific incorporation of [ 3H]BTX-OAB into protein components as much as 15-fold. Increasing the incubation temperature from 25°C to 37°C had a similar but less marked effect. We conclude that the BTX binding site lies at a lipid-protein interface and that treatments which induce conformational changes in the sodium channel protein (i.e. addition of TTX) can result in a reorientation of BTX at its binding site relative to the protein and lipid domains of voltage-sensitive sodium channels.