Functionally intact acetylcholine receptors can be solubilized from electric organ membranes of Torpedo californica and incorporated into liposomes by the cholate dialysis technique. Freezing and thawing of the reconstituted preparation appears to seal a population of initially leaky vesicles and leads to vesicle fusion. Inclusion of supplementary cholesterol at an optimal concentration of 20% (w/w) greatly enhances vesicle fusion during the freeze-thaw cycle. Size analysis by electron microscopy of negatively stained preparations indicates that fusion is accompanied by shifts in size and volume distributions of the vesicle population. Liposomes formed in the absence of acetylcholine receptors are distributed over a substantially smaller size range than liposomes containing receptors. Acetylcholine receptors appear in those liposomes as dimers of 80 A doughnut-shaped particles. Freeze-fracture replicas of reconstituted preparations reveal the presence of large vesicles containing particles which correspond in size to acetylcholine receptors and smaller liposomes devoid of particles. The distribution of particles in the reconstituted membranes is sparse compared to their dense packing in native electric organ membranes. The activation and desensitization of reconstituted acetylcholine receptors mediated by acetylcholine or carbamylcholine is dose dependent. The reconstituted receptors distinguish between these agonists in terms of binding affinity in a way similar to receptors in the native membrane. Correlation of the fractional occupancy of ligand binding sites by cobratoxin with inhibition of receptor function is used to demonstrate that in the reconstituted system the doubly liganded acetylcholine receptor prevails in controlling channel gating. The potential experimental advantages as well as limitations of this reconstituted system are discussed.

Animals↗

PubMed1981

Stabilization of acetylcholine receptor channels by lipids in cholate solution and during reconstitution in vesicles.

Acetylcholine receptors were solubilized from electric organ membranes of Torpedo californica in mixed micelles of sodium cholate and soybean lipids. Sodium cholate, when supplemented with relatively low amounts of soybean lipids (cholate:lipid, 20:1, molar ratio), was effective in solubilizing receptors without denaturing their agonist-regulated cation channels. Another dialyzable detergent, octylglucoside, denatured the ion channel even in the presence of excess lipids. Reassembly of receptors and lipids into vesicles was achieved by cholate dialysis. About 70% of the receptors were oriented with their toxin binding sites on the external surface of the vesicles. Evidence suggests that all of the receptors in a single vesicle were oriented either right side out or inside out. During the reassembly process about 10-fold greater lipid concentrations were required for the preservation of channel function. At lipid/protein ratios greater than 16:1 (w/w), receptors reassembled into vesicles at a constant protein/lipid ratio. These vesicles contained approximately 7% receptors by weight, 5-fold less than the native membrane. The remainder of the lipid assembled into small vesicles which did not contain receptors. At lipid/protein ratios less than 16:1 (w/w), receptors reassociated with lipids in higher weight ratios. Irreversible inactivation of a fraction of the acetylcholine receptor channels occurred in proportion to the greater packing density. This channel denaturation was accompanied by a lowered susceptibility of the disulfide bond between the delta subunits of the acetylcholine receptor dimer to reducing agents. Toxin binding and the orientation of the receptors in the reconstituted vesicles was not affected by reduced lipid/protein ratios. The unexpected constant acetylcholine receptor/lipid ratio in the reconstituted vesicles and the unexpected uniform orientation of the acetylcholine receptors within a vesicle are discussed in terms of the interactions occurring during the initial nucleation events of the reassembly process.

Acetylcholine↗

PubMed1980

Purification of acetylcholine receptors, reconstitution into lipid vesicles, and study of agonist-induced cation channel regulation.

We report the purification of acetylcholine receptors with active agonist-regulated cation channels from Torpedo californica electric organ tissue by five methods. In one method, previously used by others, contaminating proteins were removed from partially purified membranes by alkaline extraction, preserving membrane integrity throughout the procedure. In the other four methods, acetylcholine receptors were purified after solubalization with sodium cholate. The continual presence of soybean lipid in mixed micelles with cholate was required to prevent irreversible inactivation of the cation channel. Solubilized receptors were purified by affinity chromatography using either Naga naja siamensis toxin III or concanavalin A coupled to agarose. Sucrose gradient centrifugation was also used to purify solubilized receptors. The best method combined affinity chromatography on toxin-agarose and concanavalin A agarose. Receptors purified by all five methods were incorporated into soybean lipid vesicles by the cholate dialysis technique. The agonist-regulated cation channels of the receptors were equally active after reconstitution, independent of the method used for purification. All reconstituted vesicle preparations were similar in preferential orientation of acetylcholine receptor toward the external surface, dose-response to carbamylcholine, carbamylcholine-induced desensitization, and carbamycholine-induced influx of 22Na+ per mol of receptor. Carbamylcholine-induced 22Na+ influx/receptor was greater after reconstitution than in native vesicles. This was because, in native vesicles, carbamylcholine-induced 22Na+ influx was limited by equilibration of the internal volume of the vesicles with the external 22Na+ concentration, whereas in reconstituted vesicles 22Na+ influx was limited by desensitization of the receptor molecule. We demonstrate that only one of the two toxin binding sites on the receptor monomer, the one which can be affinity alkylated with 4-(N-maleimido)benzyltrimethylammonium, controls the carbamylcholine-induced opening of the cation channel.

Acetylcholine↗