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.