Reconstitution of carbamylcholine-dependent sodium ion flux and desensitization of the acetylcholine receptor from Torpedo californica

Abstract

Membranes rich in acetylcholine receptor were isolated from Torpedo californica by a modification of the procedure of Sobel et al. (Sobel, A., Weber, M., and Changeux, J.-P. (1977) Eur. J. Biochem. 80, 215-224). The receptor was extracted with 2% potassium cholate in the presence of 2.5% soybean phospholipids. After reconstitution by the cholate dialysis procedure, the vesicles exhibited a rapid, carbamylcholine-dependent uptake of 22Na+, which was inhibited by alpha-bungarotoxin and several other known inhibitors. At concentrations above 5 x 10(-5) M carbamylcholine, the fast phase of Na+ influx lasted less than 10 sec. At 5 x 10(-6)M, it lasted 30 sec but was only about 50% of the maximal total uptake observed at optimal agonist concentration. The phenomenon of desensitization was exhibited by the reconstituted vesicles. When 2 x 10(-4)M carbamylcholine was added, 15 sec before 22Na+, the rapid Na+ influx phase was no longer observed.