WHERE DOES CHOLESTEROL ACT DURING ACTIVATION OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR

Why agonist-induced activation of the nicotinic acetylcholine receptor (nAcChoR) fails completely in the absence of cholesterol is unknown. Affinity-purified nAcChoRs from Torpedo reconstituted into 1,2-dioleoy l-sn-glycero-3-phosphatidycholine/ 1,2-dioleoyl-sn-glycero-3-phosphate /steroid bilayers ar mole ratios of 58:12:30 were used to distinguish between three regions of the membrane where cholesterol might act: the Lipid bilayer, the lipid-protein interface, or sites within the prote in itself. In the bilayer, the role of fluidity has been ruled out and certain neutral lipids can substitute for cholesterol [C. Sunshine, M .G. McNamee, Biochim. Biophys. Acta 1191 (1994) 59-64], therefore, we first tested the hypothesis that flip-flop of cholesterol across the m embrane is important; a plausible mechanism might be the relief of mec hanical bending strain induced by a conformation change that expands t he two leaflets of the bilayer asymmetrically. Cholesterol analogs pre vented from flipping by charged groups attached to the 3-position's hy droxyl supported channel opening, contrary to this hypothesis. The sec ond hypothesis :is that interstitial cholesterol binding sites exist d eep within the nAcChoR that must be occupied for channel opening to oc cur. When cholesterol hemisuccinate was covalently 'tethered' to the g lycerol backbone of phosphatidylcholine, channel opening was still sup ported, Thus, if there are functionally important cholesterol sites, t hey must be very close to the lipid-protein interface and might be ter med periannular. (C) 1998 Elsevier Science B.V.