Functional reconstitution of the purified sodium channel protein from rat sarcolemma.

Abstract

The purified saxitoxin (STX) binding component of the rat sarcolemmal Na channel (SBC) was reconstituted into phospholipid vesicles. The reconstituted SBC displays the pharmacological properties and the ability to control Na fluxes expected of a functional Na channel. Batrachotoxin (BTX) increased 22Na+ influx into reconstituted SBC vesicles by > 100% over control at early time points. The BTX-stimulated 22Na+ influx was specifically and quantitatively blocked by STX. Veratridine and aconitine also stimulated Na+ flux, although less effective than BTX, in the order: TXB > veratridine > aconitine. The log dose-response curves for BTX and veratridine were sigmoidal with a K0.5 of 1.5 and 35 .mu.M, respectively. Vesicles containing the reconstituted SBC demonstrated 3H-labeled STX binding to a single class of high affinity sites with a Kd of 5-7 nM at 0.degree. C; the thermal stability of the STX receptor was markedly enchanged by reconstitution. The purified STX binding component from rat sarcolemma constitutes the Na channel itself and contains at least those components sufficient for channel activation, transmembrane ion movement and inhibition by STX.