Reconstitution of a Functional Acetylcholine Receptor

Abstract

The 'reconstitution cycle' is composed of the following sequence of operations. Highly purified receptor-rich membranes prepared from Torpedo marmorata electric organ are exposed to pH 11 to remove the 43000-M, protein and dispersed into solution by sodium cholate under conditions where more than 8 5 % of the receptor protein is in its 9-S form. Elimination of the detergent by filtration on a Sephadex column (or dialysis) yields a 'reconstituted receptor' fraction, under conditions which conserve part of the endogenous lipids, or 'reconstituted vesicles' in the presence of an excess of exogenous lipids.The polypeptide composition of these fractions was analysed by sodium dodecylsulfate gel electrophoresis. Conditions are defined for quantitative measurements of the various polypeptide chains. The 40000-M, chain, which is labelled by the affinity reagent 4-(N-maleimido)phenyI [3H]trimethylammonium and therefore carries the acetylcholine receptor site, is the dominant polypeptide in the alkaline-treated membranes and the reconstituted acetylcholine receptor.Electron microscopy discloses that many of the alkaline-treated membranes no longer form closed vesicles and do not show the transverse asymmetry of the native membranes observed after tannic acid fixation. In the reconstituted receptor fractions, the receptor molecules reaggregate into discs and may be exposed on both faces of the discs. In the reconstituted vesicles, receptor rosettes are integrated to the lipid vesicles.With native membranes, the radioactive local anesthetic [3H]trimethisoquin binds to three classes of sites : non-specific, low-affinity and high-affinity. Carbamylcholine causes an increase in the number of high-affinity sites up to approximately 0.7 times the number of cc-'251-bungarotoxin sites. This ratio, the three classes of binding sites, and their regulation by carbamylcholine are conserved through the reconstitution cycle.Investigations into the molecular mechanism of the physiological action of acetylcholine may, taking a reductionist viewpoint, be organised along two lines. In the first, the acetylcholine-sensitive membrane is resolved into its various components, which are subsequently characterized chemically. These components evidently suffice for the occurrence of the physiological response. The demonstration that they are necessary may be accomplished, following the second approach, by the reconstitution of an acetylcholine-sensitive membrane in which the individual molecular species are reintegrated into an artificial environment.Abbreviations. NaDodS04, sodium dodecyl sulfate; Dns-CsCho, [l-(5-dimethyIaminonaphthalene)-sulfonamido]-n-hexanoic acid-8-N-trimethylammonium bromide ethyl ester.To carry out this demonstration, membrane fragments were purified, on the basis of their high content in acetylcholine receptor sites, from electric organ of(see [4]). The method was developed on a macroscopic scale [2] required for biochemical and biophysical studies and the same membranes were shown to respond in vitro to cholinergic agoni...

Immunofluorescence localization at the mammalian neuromuscular junction of the Mr 43,000 protein of Torpedo postsynaptic membranes.

“…Spectroscopic (12) and photolabeling experiments (14) suggested that the Mr 43,000 protein bound local anesthetics, raising the possibility that it might be part of the AcCho-activated cation channel. However, Neubig et aL (9) demonstrated, and others have confirmed (10,15,16), that the Mr 43,000 protein can be removed from the membranes by treatment at pH 11 without altering either the binding of cholinergic ligands or the control of ion permeability.…”

Stabilization of the high affinity state of the membrane‐bound acetylcholine receptor from Torpedo marmorata by noncompetitive blockers

“…It might account for the heterogeneity of binding reported for [3H] trimethisoquin with ACh-receptor-rich membrane fragments, and for the differential effects of HTX on this binding [15]. It could also explain the results obtained upon covalent labelling by UV irradiation of the polypeptide chains of the ACh-receptor protein with radioactive noncompetitive blockers [34].…”

“…They include, among others, the aminated local anesthetics proadifen and dimethisoquin [3][4][5][6][7], the frog toxin histrionicotoxin (HTX) and its derivatives [8][9][10][11], the hallucinogenic drug phencyclidine [12][13][14]. These pharmacological agents block the physiological response in the/~M range and their binding to saturable sites on ACh-receptor-rich membrane fragments isolated from Torpedo electric organ has been demonstrated with the radiolabelled derivatives [3H]meproadifen [6], [3H] trimethisoquin [ 15 ], [all] perhydro-HTX [ 11,[16][17][18] and [3H]phencyclidine [12,19,20]. A number of structurally unrelated compounds usually considered as primarily interacting with the lipid phase of the membrane, such as the detergents Triton X-100 or Na-cholate [21,22], fatty acids [21,36], phospholipases (reviews [23,27]), general anesthetics and alcohols [7,24[ also block the response to ACh in a noncompetitive manner.…”

A combined ELISA-immunoelectron microscopic approach for topological mapping of membrane protein epitopes: application to the nicotinic acetylcholine receptor.

“…Solubilization of membranes with cholate was performed according to Sobel et al (1980). To 1 volume ofmembranes at 2.5 mg proteinlml in 0.1 M %is, pH 6.8, was added 1/9 volume ofcholate 31%.…”