Reconstitution of functional membrane-bound acetylcholine receptor from isolated Torpedo californica receptor protein and electroplax lipids.

Abstract

Purified acetyicholine receptor and total lipids, both extracted from Torpedo californica electroplax, were utilized to reconstitute chemically excitable membrane vesicles. Reconstitution was achieved by dialysis of the extraction detergent, octyl P-D-glucoside, from protein/lipid incubation mixtures. The reconstituted preparations could be fractionated by sucrose density gradient centrifugation and consisted of vesicular structures visible in electron micrographs. In addition, the reconstituted vesicles exhibited the following properties characteristic of native receptor-enriched membranes: (i) an external distribution of ct-bungarotoxin-binding sites, (ii) a time-dependent binding of a-bungarotoxin that is depressed by preincubation with the cholinergic agonist carbamoylcholine ("desensitization"), (iif) an ability to retain 22Na+ that is lost in the presence of detergents or gramicidin A, and (iv) a carbamoylcholine-induced acceleration of 22Na+ efflux that can be blocked by a-bungarotoxin. The purified acetylcholine receptor that was utilized in the reconstitution experiments apparently does not require other protein components for ligand recognition or ion translocation.

Quantitation of cation transport by reconstituted membrane vesicles containing purified acetylcholine receptor.

“…Studies of this cation translocation using both slow (13) and fast (14) kinetic methods have led to the conclusion that this single molecular complex is the only protein component necessary for full function in terms of cation transport. Preliminary reports of cation transport by reconstituted preparations derived from purified AcChoR (15)(16)(17)(18) have shown qualitatively that the AcChoR complex is capable of conducting cation transport. However, these studies have not allowed the quantitation of cation translocation necessary for determination of whether the reconstituted preparations retain the full physiological properties of the receptor in vivo.…”

Subunit structure of the acetylcholine receptor from Electrophorus electricus.

“…1). Its reconstitution into artificial membrane systems restores the physiological action of the native AcChoR both qualitatively (7)(8)(9)(10)(11)(12)(13) and quantitatively (14)(15)(16) Torpedo (a marine elasmobranch) and Electrophorus (a freshwater teleost) are highly diverged species whose evolution arose separately from the primordial vertebrate stock (=400 million years) and accordingly the presence of electric organs in these two species is due to convergent evolution. Sufficient AcChoR can be isolated from both animals to conduct structural analyses of their constituent subunits.…”

Structural models of the nicotinic acetylcholine receptor and its toxin-binding sites

“…Progress is also being made in the development of methods to predict the secondary and tertiary structure of proteins from their sequences (Cohen et al, 1980), in modeling structures of other membrane proteins (Engelman et al. 1980: Guy, 1980, and in predicting more precisely how drugs bind to proteins (Gund et al, 1980). Thus it may be possible to predict the structure of the AChR complex without having precise X-ray diffraction data.…”