Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

Accumulation of Ca2+ by the Ca2+-ATPase of skeletal-muscle sarcoplasmic ret iculum has been measured in reconstituted, sealed vesicles as a function of lipid composition. Measurements were performed in the presence of carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP) to eliminate any effects of HC transport; in the presence of FCCP, addition of valinomycin had no e ffect on the level or rate of accumulation of Ca2+ showing that, in the pre sence of FCCP, no electrical potential built up across the membrane. Levels of accumulation were low when the phospholipid was dioleoylphosphatidylcho line (DOPC), even though DOPC supports high ATPase activity. Inclusion of 1 0 mol%, anionic phospholipid [dioleoylphosphatidic acid (DOPA) or dioleoylp hosphatidylserine (DOPS)] led to higher levels of accumulation of Ca2+, 10 mol% being the optimum concentration. Cardiolipin or phosphatidylinositol 4 -phosphate were more effective than DOPA or DOPS in increasing accumulation of Ca2+. Effects of anionic phospholipids were seen in the presence of an ATP-regenerating system to remove ADP, and in the presence of phosphate wit hin the reconstituted vesicles to precipitate calcium phosphate. Rates of p assive leak of Ca2+ from the reconstituted vesicles were slow. The Ca2+-acc umulation process was simulated assuming either simple passive leak of Ca2 from the vesicles or assuming slippage on the ATPase, a process in which t he phosphorylated intermediate of the ATPase releases bound Ca2+ on the cyt oplasmic rather than the lumenal side of the membrane. The experimental dat a fitted to a slippage model, with anionic phospholipids decreasing the rat e of slippage.