Reconstitution of acetylcholine receptor function using purified receptor protein.
Membrane preparations containing only the four acetylcholine receptor polypeptide subunits (40, 50, 60, and 65 x 10(3) daltons) were purified from Torpedo californica electroplax. The receptor protein was extracted from these membranes with 2% aqueous sodium cholate, and complete dissolution into discrete molecular species was confirmed by sedimentation analysis. The solubilized preparation reassociated with exogenous phospholipids when the detergent was removed by dialysis and formed spherically sealed vesicles 400--600 A in diameter. The reconstituted receptor preparations had nearly 90% of their alpha-bungarotoxin binding sites exposed on the exterior surface of the vesicles. In a reproducible manner, the reconstituted acetylcholine receptor responded to carbamoylcholine by exhibiting a rapid efflux of 22Na+ from within the vesicles. Such preparations were successfully reconstituted only from acetylcholine receptor stabilized by asolectin above a minimal level upon dissolution of the original membrane preparation by detergent. In addition to the response to carbamoylcholine, the reconstituted preparations also exhibited pharmacological characteristics that resemble those observed for the original electroplax membranes. In terms of the carbamoylcholine-induced signal, a significantly large fraction of the total receptor was functionally reconstituted. The results also confirm the notion that only the four polypeptides considered to constitute the receptor are essential for acetylcholine-mediated cation translocation and rule out possible roles for other polypeptide species.
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids; D004557:Electric Organ; D005399:Fishes; D050356:Lipid Metabolism; D008081:Liposomes / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D008854:Microscopy, Electron; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D012911:Snakes; D012995:Solubility
D002038:Bungarotoxins; D002793:Cholic Acids; D008081:Liposomes; D011950:Receptors, Cholinergic; D000109:Acetylcholine