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 reticulum has been measured in reconstituted, sealed vesicles as a function of lipid composition. Measurements were performed in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to eliminate any effects of H⁺ transport; in the presence of FCCP, addition of valinomycin had no effect on the level or rate of accumulation of Ca²⁺ showing that, in the presence of FCCP, no electrical potential built up across the membrane. Levels of accumulation were low when the phospholipid was dioleoylphosphatidylcholine (DOPC), even though DOPC supports high ATPase activity. Inclusion of 10 mol% anionic phospholipid [dioleoylphosphatidic acid (DOPA) or dioleoylphosphatidylserine (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 within the reconstituted vesicles to precipitate calcium phosphate. Rates of passive leak of Ca²⁺ from the reconstituted vesicles were slow. The Ca²⁺-accumulation process was simulated assuming either simple passive leak of Ca²⁺ from the vesicles or assuming slippage on the ATPase, a process in which the phosphorylated intermediate of the ATPase releases bound Ca²⁺ on the cytoplasmic rather than the lumenal side of the membrane. The experimental data fitted to a slippage model, with anionic phospholipids decreasing the rate of slippage.