Protein-lipid interactions and Torpedo californica nicotinic acetylcholine receptor function. 2. Membrane fluidity and ligand-mediated alteration in the accessibility of gamma subunit cysteine residues to cholesterol
Narayanaswami, V.; McNamee, M.G.
Biochemistry 32(46): 12420-12427
Fluorescence-quenching and energy-transfer measurements were carried out to further characterize lipid-protein interactions involving the nicotinic acetylcholine receptor (AChR) from Torpedo californica in reconstituted membranes. To assess the fluidity of the receptor microenvironment, cis- and trans-parinaric acids were used to take advantage of the preferential partitioning behavior of the trans isomer for the gel phase. A relatively higher extent of energy transfer from the intrinsic tryptophan fluorescence of AChR in dielaidoylphosphatidylcholine bilayers to cis-parinaric acid in both the gel and the fluid phase suggests that the AChR is surrounded by a relatively fluid annulus of lipids. The ability of AChR to accommodate and interact with specific lipids such as cholesterol and fatty acids in the vicinity of pyrene-labeled cysteine residues in the membranous domain and/or the membrane-water interface region of the gamma subunit was assessed. Pyrene-labeled AChR prepared in (6,7-dibromostearoyl)phosphatidylcholine showed a 25% decrease in fluorescence as sites accessible to phospholipids were occupied; subsequent addition of dibromocholesterol hemisuccinate (DiBrCHS) caused further quenching by about 25%. This result is consistent with the presence of sites accessible to cholesterol, but not accessible to phospholipids, in the vicinity of the cysteine-bound pyrene in the membranous domain of the AChR. Quenching by DiBrCHS was sensitive to the presence of an AChR activator (carbamylcholine) but not a competitive antagonist (alpha-bungarotoxin). The Stern-Volmer quenching constant was 0.123 in the absence of added ligands and 0.167 and 0.134 in the presence of carbamylcholine and alpha-bungarotoxin, respectively, corresponding to accessibilities of 65%, 90%, and 70%.