The sodium channel from rat brain. Role of the beta 1 and beta 2 subunits in saxitoxin binding.
Procedures are described for the selective removal of the beta 1 or the beta 2 subunits from the detergent-solubilized channel from rat brain, and the functional integrity of the resulting protein complex is examined. Treatment of the channel with 1.0 M MgCl2 followed by sedimentation through sucrose gradients results in complete separation of beta 1 from the alpha-beta 2 complex and complete loss of [3H]saxitoxin (STX) binding activity. At intermediate MgCl2 concentrations, the loss of beta 1 and the loss of [3H]STX binding activity are closely correlated. Tetrodotoxin (TTX) quantitatively stabilizes the solubilized complex against both the loss of beta 1 and the loss of [3H]STX binding activity. This indicates that association of the alpha and beta 1 subunits is required to maintain the STX/TTX binding site in a conformation with high affinity for STX and TTX in the detergent-solubilized state. Treatment of the solubilized sodium channel with dithiothreitol in the presence of TTX causes specific release of the beta 2 subunit, without significantly removing beta 1. There is little or no correlation between the amount of beta 2 in the sodium channel complex and the ability of the preparation to bind [3H]STX. We conclude from these studies that the presence of beta 1, but not beta 2, is required for the integrity of the STX/TTX binding site of the solubilized and purified rat brain sodium channel.
D J
DJ
Messner
W A
WA
Catterall
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007473:Ion Channels / Q000378:metabolism*; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D015636:Magnesium Chloride; D011487:Protein Conformation; D051381:Rats; D012530:Saxitoxin / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D012995:Solubility; D013779:Tetrodotoxin / Q000378:metabolism
D007473:Ion Channels; D046911:Macromolecular Substances; D015636:Magnesium Chloride; D012530:Saxitoxin; D013779:Tetrodotoxin; D012964:Sodium; D008274:Magnesium; D004229:Dithiothreitol