Reconstitution of the purified acetylcholine receptor from Torpedo californica

Abstract

and similar sources has been purified in several laboratories using affinity chromatography [l-3]. Previous attempts in our and other laboratories to reconstitute these purified preparations failed to give reproducible results; it was not clear whether the receptor was damaged or had lost a component essen- tial for channel formation [4-61. We have therefore used crude acetylcholine receptor-enriched membrane preparations [7] to develop a biological ion flux assay based on the reconstitution into liposomes [8]. We now report the purification of a solubilized acetylcholine receptor which is fully active in reconsti- tution. We also show that the carbamylcholine-depen- dent ion flux in these reconstituted vesicles is sensi- tive to inhibitors such as a-bungarotoxin and that specific antibodies inhibit the reconstitution of active vesicles. The reconstituted liposomes exhibited the characteristic phenomenon of desensitization. 2. Materials and methods Materials were as described in [8]. 2.1.