Acetylcholine receptor from Torpedo. Preferential solubilization and efficient reintegration into lipid vesicles

Abstract

The acetylcholine receptor has been effectively solubilized from Torpedo californica electroplax under defined conditions with the nonionic detergent, β- d-octylglucopyranoside. Preferential solubilization of the receptor protein, with regard to yield and specific α-bungarotoxin binding activity, occurs in the absence of salt and diminishes when NaCl is present in the solubilization media (⩾50 mM). Conversely, elevated salt concentrations increase the solubilization of bulk membrane proteins including the peripheral membrane enzyme, acetylcholine esterase. Additional selectivity for the solubilization of acetylcholine receptor can be obtained by adjusting the detergent to membrane phospholipid molar ratio within a narrow optimum range (4.1 to 6.7). Purified acetylcholine receptor and electroplax total lipid are utilized to reconstitute chemically excitable membrane vesicles. Reconstitution is achieved by dialysis of octylglucopyranoside from lipid/detergent/receptor protein mixed micelles and the resulting vesicles are analyzed by sucrose density gradient centrifugation. Extensive incorporation of the acetylcholine receptor within the lipid vesicles is obtained at lipid concentrations greater than 18 mg/ml with lipid/protein ratios ranging from 12 1 to 60 1 (w/w). Reconstituted receptor vesicles and native receptor-enriched membranes exhibit similar agonist-induced effluxes of 22Na + with 50% of the maximum response occurring at carbamylcholine concentrations of 1.8·10 −5 M and 3.4 · 10 −5 M, respectively. At saturating carbamylcholine concentrations (10 −2 M) the agonist-induced efflux of 22Na + for both native and reconstituted acetylcholine receptor is (6–7) · 10 13 cpm 22Na + per mol of receptor. The efflux responses exhibited by either preparation can be effectively blocked by preincubation with carbamylcholine (‘desensitization’). The similar behavior of native and reconstituted acetylcholine receptor indicates that octylglucopyranoside-purified receptor retains all of the necessary determinants for proper ligand binding and ion translocation.