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

Abstract

The purified saxitoxin (STX) binding component of the rat sarcolemmal sodium channel (SBC) has been reconstituted into phospholipid vesicles. The reconstituted SBC displays the pharmacological properties and the ability to control sodium fluxes expected of a functional sodium channel. Batrachotoxin (BTX) increases 22Na' influx into reconstituted SBC vesicles by >100% over control at early time points. The BTX-stimulated 22Na' influx is specifically and quantitatively blocked by STX. Veratridine and aconitine also stimulate Na+ flux-although less effectively than BTX-in the order: BTX > veratridine > aconitine. The logarithmic dose-response curves for BTX and veratridine are sigmoidal with a K0.5 of 1.5 ,M and 35 ,uM, respec-tively. Vesicles containing the reconstituted SBC demonstrate 3H-labeled STX binding to a single class of high affinity sites with a kd of 5-7 nM at 0°C; the thermal stability of the STX receptor is markedly enhanced by reconstitution. Our results confirm that the purified STX binding component from rat sarcolemma constitutes the sodium channel itself and contains at least those components sufficient for channel activation, transmembrane ion movement, and inhibition by STX.The action potential in excitable membranes typically arises from rapid and transient voltage-dependent increases in membrane permeability to Na+ and K+ (1). These permeability changes are produced by separate, ion-selective channels that are believed to be formed by integral proteins that span the bilayer to provide gated polar pathways for cation movement (2). Recently, rapid progress has been made in the solubilization and purification of components of the voltage-dependent sodium channel. This effort has been aided by the availability of neurotoxins that interact specifically with the channel in a manner that can be defined both electrophysiologically and biochemically (3).Radiolabeled saxitoxin (STX) and tetrodotoxin (TTX) have been used to detect sodium channel components solubilized in non-ionic detergents, and a solubilized STX/TTX binding protein has been purified from eel electroplax (4), rat synaptosomes (5), and rat skeletal muscle sarcolemma (6). Prior to this report, the question of whether these purified proteins were intact sodium channel complexes, and whether they survived solubilization and purification in a functional form, remained unanswered.Several groups have reported reconstitution of unpurified sodium channels into lipid vesicles after partial disruption or complete solubilization of nerve membranes in sodium cholate (7-10). In most studies, crude membrane fragments or solubilized membrane preparations were used for reconstitution and no preliminary fractionation procedures were performed. We report here the successful reconstitution of a purified mammalian sodium channel protein. (11) and stored at -60'C. The LiBr extraction solution contained 1 mM iodoacetamide, 0.1 mM phenylmethylsulfonyl fluoride, pepstatin at 0.1 ,ug/ml, and 0.2 mM EGTA. Solubilization and purification of the SBC wer...

Molecular morphology of the tetrodotoxin-binding sodium channel protein from Electrophorus electricus in solubilized and reconstituted preparations.

“…The Lubrol-solubilized material was reconstituted into PC vesicles by a modification of the method of Weigele and Barchi (13), making use of adsorbtion of the nonionic detergent by SM2 BioBeads (Bio-Rad Laboratories, Richmond, CA). Protein purified in Lubrol-PX (40 mg/ml protein) was supplemented with a concentrated phosphatidylcholine :Lubrol solution (36 mg/ml PC, 9% Lubrol-PX) to a final concentration of6 mg/ml egg PC, 1 .6% detergent wt/vol, and 34 pg/ml protein .…”

Messenger RNA coding for only the alpha subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes.

“…In adult rat brain, the a and ,82 subunits are covalently attached by disulfide bonds (9). Both the rat and eel protein complexes have been reconstituted into phospholipid vesicles, with restoration of toxin-induced activation of Na permeability (13)(14)(15)(16).…”

Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax of Electrophorus electricus.

“…To determine whether the isolated proteins are functionally intact, the TTX/STX-binding components of muscle (10) and brain (11,12) were previously reconstituted into phospholipid vesicles and alkaloid-stimulated, TTX-blocked 22Na+ influx was demonstrated. In this communication we report the incorporation of the Na channel purified from electroplax into lipid vesicles.…”