Effects of lipids on acetylcholine receptor. Essential need of cholesterol for maintenance of agonist-induced state transitions in lipid vesicles

Abstract

The effects of lipids on the maintenance of characteristic functional properties of the acetylcholine receptor during the course of reconstitution into lipid vesicles were studied by following the kinetics of agonist-induced state transitions. The requirements for successful preservation of these properties could be dissected into two components: (a) adequate nature and concentration of lipids during detergent solubilization; (b) correct lipid environment during reincorporation into lipid vesicles by the cholate dialysis procedure. Optimal bulk lipid concentrations and lipid:cholate ratios for preserving state transitions during solubilization were studied by using both crude soybean lipids and pure synthetic phospholipids. The latter class of lipids was found to be unsuitable substitutes for the crude soybean lipids, irrespective of their polar head group and/or fatty acyl chain, even when detergent:lipid ratios as high as 1:1 (w/w) were employed. Addition of cholesteryl hemisuccinate was able to make up this deficiency, attaining preservation of acetylcholine receptor state transitions at cholate:steroid ratios of about 6:1 (w/w). The presence of steroid decreased the amount of protein solubilized. The correct choice of lipid type was also essential to the reincorporation step, and higher concentrations of lipid were required--about 20 mg/mL for soybean lipids. Pure phospholipids at similar concentrations, however, were unable to maintain the state transitions. Again, steroid (40-46% cholesteryl hemisuccinate/mol of total lipid) provided the adequate conditions at the reincorporation stage and enhanced the amount of protein reincorporated into the vesicles. A large (70-90%) percentage of the receptor was reincorporated with the correct vectorial sidedness. No specificity could be detected for the phospholipid polar head or alkyl chain in relation to any of these findings. The effect of the protein on the physical state of the lipids in the reconstituted vesicles was studied by diphenylhexatriene fluorescence depolarization. The results may be interpreted as a disordering of the acyl chains in the gel state and an ordering in the liquid-crystalline state in the presence of protein, accompanied by shifts in the transition temperatures of the pure phospholipids to lower values.

Extraction of Cholesterol with Methyl-β-Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles

“…Forty large cell profiles (with a total of 2990 and 4812 golds, respectively) were included in the quantification from each experiment. a Data and calculations based on Hansen et al, 1992. TION, cholesterol is important for the structure and function of membrane proteins (Muller and Shinitzky, 1979;Criado et al, 1982;Bloch, 1991;Kilsdonk et al, 1995), and furthermore, a change in the cholesterol content will affect the membrane viscosity, the exposure of membrane proteins to the surroundings, and therefore also the interaction between membrane proteins and cytosolic proteins (Shinitzky and Inbar, 1974;Shinitzky and Rivnay, 1977). Such changes might be responsible for the inhibition of formation of clathrincoated pits observed here.…”

“…A major fraction of cholesterol is present in the plasma membrane (Lange, 1991), and cholesterol is important for various processes at the cell surface. For instance, there seems to be a direct interaction between the oxytocin receptor and cholesterol (Klein et al, 1995), and also the acetylcholine receptor was found to have a functional requirement for cholesterol (Criado et al, 1982). Furthermore, it has been proposed that annexin II, which is involved in the endocytic pathway (Creutz, 1992;Gruenberg and Emans, 1993), serves as an interface between membranes containing a high amount of cholesterol and the actin cytoskeleton (Harder et al, 1997), and it is clear that cholesterol is essential for the structure and function of invaginated caveolae, including the caveolae-dependent endocytosis that has been reported in some cell types (Rothberg et al, 1990;Parton et al, 1994;Parton, 1996;Smaby et al, 1996;Chang et al, 1998;Hailstones et al, 1998).…”

Heterologous Expression of the Human D2S Dopamine Receptor in Protease‐Deficient Saccharomyces cerevisiae Strains

“…A possible explanation for this discrepancy could be the lipid composition of the membranes surrounding the receptor molecules. It has been well established that the lipid composition, especially the cholesterol concentration, is important for the activity, of some membrane-bound hormone receptors such as the nicotinic acetylcholine receptor and the p2 adrenergic receptor (Criado et al, 1982; Kirilovski and Schramm, 1982). In fact, the affinity of the transferrin receptor for its ligand can be modulated by changing the cholesterol contents of the sourrounding lipid bilayer (Nunez and Glass, 1982).…”

Modulation of Nicotinic Acetylcholine Receptor Conformational State by Free Fatty Acids and Steroids

“…As described in the Introduction, the functionality of the AChR (both the passage of ions across the pore and the transition from the R to the D states of the receptor) is highly sensitive to the composition of the surrounding lipid. Speculative models on the role of membrane fluidity on AChR function have been proposed (55,56). Baenziger and co-workers suggest that membrane fluidity modulates the relative proportion of AChR in the R and D states (45).…”