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.