Reconstitution of a functional acetylcholine receptor. Polypeptide chains, ultrastructure, and binding sites for acetylcholine and local anesthetics.

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 43,000-Mr protein and dispersed into solution by sodium cholate under conditions where more than 85% 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 40,000-Mr chain, which is labelled by the affinity reagent 4-(N-maleimido)phenyl [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 alpha-125I-bungarotoxin sites. This ratio, the three classes of binding sites, and their regulation by carbamylcholine are conserved through the reconstitution cycle.

[Preservation of the allosteric properties of the protein receptor for acetylcholine in detergent solution without addition of lipids]. / Conservation des propriétés allostériques de la protéine réceptrice de l'acétylcholine en solution détergente sans addition de lipides.

Solubilization by Na cholate of acetylcholine receptor-rich membrane fragments from Torpedo marmorata, followed by exchange of NA cholate by the neutral detergent "Tween 80" yields the receptor protein in its 9 S soluble light form; under these conditions, without adding lipids, the receptor protein conserves its characteristic binding properties for the fluorescent agonist Dns-C6-Cho as followed by fast kinetic techniques, and the allosteric regulation by non-competitive blockers.

Reconstitution of a functional acetylcholine receptor. Conservation of the conformational and allosteric transitions and recovery of the permeability response; role of lipids.

The 'functional' state of the acetylcholine receptor protein has been followed during reconstitution with the fluorescent agonist [1-(5-dimethylaminonaphthalene)-sulfonamidol]-n-hexanoic acid-beta-N-trimetylammonium bromide ethyl ester (Dns-C6-Cho) and rapid-mixing techniques. Under appropriate conditions, a majority of the acetylcholine receptor sites can be recovered in a low-affinity state(s) for Dns-C6-Cho, similar to that found with the native membrane-bound receptor. This state can be slowly interconverted to a high-affinity state after rapid mixing with the agonist, and the non-competitive channel blockers, like the local anesthetics, still regulate this transition in an allosteric manner. Several experimental conditions commonly used for the solubilization of the receptor and for its purification in the presence of sodium cholate result in the failure of reconstitution: the soluble receptor protein is stabilized in a low-affinity state which can no longer be interconverted to a high-affinity state in the presence of agonists or local anesthetics. On the other hand, it is demonstrated that if the concentration of lipids remains elevated in the presence of sodium cholate, a soluble (9-S) low-affinity form of the receptor protein can be obtained which shows most of the characteristic properties of the membrane-bound receptor and in particular the slow interconversion to the high-affinity state and the effect of local anesthetics on this transition; furthermore, in these conditions the soluble protein can be manipulated ad libitum and submitted, for instance, to column filtrations and sucrose gradient centrifugations in the presence of detergent, without losing its characteristic conformational and allosteric transitions. After elimination of the detergent this form yields a reconstituted receptor which presents binding properties identical to those of the native membrane-bound receptor and leads to the formation of vesicles which exhibit carbamylcholine-sensitive ion fluxes. A necessary and sufficient condition for functional reconstitution is therefore the conservation, in the presence of lipids, of the allosteric properties of the receptor protein in its detergent-soluble form.