Functional Reconstitution of the Purified Sodium Channel Protein from Rat Sarcolemma

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 79; no. 11; pp. 3651 - 3655
• Main Authors: Weigele, J. B., Barchi, R. L.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01.06.1982; National Acad Sciences
• Subjects: Animals; Binding sites; Biological Transport; Eggs; Ethanol; Freeze Fracturing; Hot Temperature; Ion Channels - drug effects; Ion Channels - physiology; Liposomes; Muscle Proteins - isolation & purification; Phospholipids; Rats; Receptors; reconstitution; Sarcolemma; Sarcolemma - analysis; Saxitoxin - pharmacology; saxitoxin-binding protein; Sodium; Sodium - physiology; Sodium channels; Solubilization; Toxins; vesicles
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.79.11.3651

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) increases22Na+influx into reconstituted SBC vesicles by >100% over control at early time points. The BTX-stimulated22Na+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 K0.5of 1.5 μ M and 35 μ M, respectively. Vesicles containing the reconstituted SBC demonstrate3H-labeled STX binding to a single class of high affinity sites with a Kdof 5-7 nM at 0 degrees 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.