Batrachotoxinin-A-ortho-azidobenzoate: a photoaffinity probe of the batrachotoxin binding site of voltage-sensitive sodium channels.
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-alpha-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 degree C to 37 degrees 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.
Casebolt
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Department of Nutrition Sciences, University of Alabama at Birmingham 35294.
Brown
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D000345:Affinity Labels; D000818:Animals; D001496:Batrachotoxins / Q000138:chemical synthesis; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D004594:Electrophysiology; D066298:In Vitro Techniques; D008297:Male; D008968:Molecular Conformation; D009498:Neurotoxins; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D015222:Sodium Channels / Q000187:drug effects*; D013056:Spectrophotometry, Ultraviolet; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D014701:Veratridine / Q000494:pharmacology
D000345:Affinity Labels; D001496:Batrachotoxins; D009498:Neurotoxins; D015222:Sodium Channels; C041417:batrachotoxin receptor; D014701:Veratridine; C031775:batrachotoxinin A 20-alpha-benzoate