The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein

Abstract

Solubilization and purification of the tetrodotoxin (TTX) binding protein of the lobster walking-leg nerve Na + channel were carried out utilizing [ 3H]tetrodotoxin ([ 3H]tetrodotoxin) as a marker. The nerve membrane was solubilized with Lubrol-PX and the Na + channel protein was purified with diethylaminoethyl Bio-Gel A, Bio-Gel hydroxylapatite powder and two Sepharose 6B columns. Care was taken to keep the temperature of the Na + channel preparation as close to 1°C as possible and to use solutions (pH 7.5) that contain Na channel protectors, i.e., egg phosphatidylcholine/Lubrol-PX mixture, TTX, EDTA, EGTA, phenylmethylsulfonyl fluoride, pepstatin A, iodoacetamide, antipain, phosphoramidon, soybean trypsin inhibnitor, leupeptin and bacitracin. From an initial specific binding of 20.1 pmol of [ 3H]TTX/mg protein for the solubilized membrane, the binding increased to 1241 pmol/mg protein for the most active fraction of the lat Sepharose 6B column. The [ 3H]TTX specific binding of the Sepharose 6B fractions correlated with a large peptide, of M r 260 000 (240–280K), although other peptides were also present in lesser amounts.