Reconstitution of the voltage-sensitive sodium channel of rat brain from solubilized components.
The voltage-sensitive sodium channel of rat brain synaptosomes was solubilized with sodium cholate. The solubilized sodium channel migrated on a sucrose density gradient with an apparent S20,w of approximately 12 S, retained [3H]saxitoxin ([3H]STX) binding activity that was labile at 36 degrees C but no longer bound 125I-labeled scorpion toxin (125I-ScTX). Following reconstitution into phosphatidylcholine vesicles, the channel regained 125I-ScTX binding and thermal stability of [3H]STX binding. Approximately 50% of the [3H]STX binding activity and 58% of 125I-ScTX binding activity were recovered after reconstitution. The reconstituted sodium channel bound STX and ScTX with KD values of 5 and 10 nM, respectively. Under depolarized conditions, veratridine enhanced the binding of 125I-ScTX with a K0.5 of 20 microM. These KD and K0.5 values are similar to those of the native synaptosome sodium channel. 125I-ScTX binding to the reconstituted sodium channel, as with the native channel, was voltage dependent. The KD for 125I-ScTX increased with depolarization. This voltage dependence was used to demonstrate that the reconstituted channel transports Na+. Activation of sodium channels by veratridine under conditions expected to cause hyperpolarization of the reconstituted vesicles increased 125I-ScTX binding 3-fold. This increased binding was blocked by STX with K0.5 = 5 nM. These data indicate that reconstituted sodium channels can transport Na+ and hyperpolarize the reconstituted vesicles. Thus, incorporation of solubilized synaptosomal sodium channels into phosphatidylcholine vesicles results in recovery of toxin binding and action at each of the three neurotoxin receptor sites and restoration of Na+ transport by the reconstituted channels.
D029845:Amphibian Proteins; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism; D004355:Drug Stability; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008564:Membrane Potentials; D051381:Rats; D012530:Saxitoxin / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism*; D014701:Veratridine / Q000494:pharmacology
D029845:Amphibian Proteins; D002352:Carrier Proteins; D007473:Ion Channels; D008081:Liposomes; C027525:saxitoxin-binding protein, Rana catesbeiana; D012530:Saxitoxin; D014701:Veratridine; D012964:Sodium