Reconstitution of functional membrane-bound acetylcholine receptor from isolated Torpedo californica receptor protein and electroplax lipids

Abstract

Purified acetylcholine receptor and total lipids, both extracted from Torpedo californica electroplax, were utilized to reconstitute chemically excitable membrane vesicles. Reconstitution was achieved by dialysis of the extraction detergent, octyl beta-D-glucoside from protein/lipid incubation mixtures. The reconstituted preparations could be fractionated by sucrose density gradient centrifugation and consisted of vesicular structures visible in electron micrographs. In addition, the reconstituted vesicles exhibited;the following properties characteristic of native receptor-enriched membranes: (i) an external distribution of alpha-bungarotoxin-binding sites, (ii) a time-dependent binding of alpha-bungarotoxin that is depressed by preincubation with the cholinergic agonist carbamoylcholine ("desensitization"), (iii) an ability to retain 22Na+ that is lost in the presence of detergents or gramicidin A, and (iv) a carbamoylcholine-induced acceleration of 22Na+ efflux that can be blocked by alpha-bungarotoxin. The purified acetylcholine receptor that was utilized in the reconstitution experiments apparently does not require other protein components for ligand recognition or ion translocation.