Reconstitution of pure acetylcholine receptor in phospholipid vesicles and comparison with receptor-rich membranes by the use of a potentiometric dye.

Acetylcholine receptor, isolated in Triton X-100 on a cobra alpha-neurotoxin affinity column was incorporated into unilamellar phospholipid vesicles by a detergent depletion method using Amberlite XAD-2. Vesicles of an average diameter of 25 nm were formed, as verified by freeze-fracture electron microscopy and gel filtration. 85 to 95% of the alpha-bungarotoxin binding sites of the reconstituted acetylcholine receptor were oriented towards the outside of the vesicles. In the reconstituted receptor one molecule of residual Triton X-100 per 2.5 alpha-bungarotoxin binding sites on the receptor molecule could be assessed. The reconstituted protein was not accessible to papain digestion, whereas the pure acetylcholine receptor, solubilized by Triton X-100 was split into smaller polypeptides under the same condition. Reconstituted acetylcholine receptor and receptor-rich membranes did not exhibit the same behavior as measured by use of a potentiometric dye. This is interpreted as an irreversible alteration of at least 95% of the receptors purified in the presence of Triton X-100. Furthermore, it could be shown that the fluorescence intensity changes induced by carbamylcholine in receptor-rich membranes did not reflect ion fluxes, but conformational changes of the protein or a displacement of the dye from the protein.