Requirement for phosphatidylinositol 4,5-bisphosphate in the Ca(2+)-induced phospholipid redistribution in the human erythrocyte membrane.

In order to investigate how calcium on the cytosolic side of human erythrocytes induces the transmembrane redistribution of phospholipids, we studied the effect of this cation on the transmembrane movements of spin-labeled phospholipids (phosphatidylserine (PS) and phosphatidylcholine (PC)) incorporated into inside-out vesicles derived from human erythrocytes. We found that the extent of the Ca(2+)-induced lipid scrambling was dependent upon the level of phosphatidylinositol 4,5-bisphosphate (PIP2) contained in the external leaflet of inside-out vesicles. The level of PIP2 in this leaflet, which normally accounts for 80% of the total membrane PIP2, was manipulated either by ATP depletion of the original erythrocytes or by incorporation of exogenous PIP2. Similarly, loading the outer monolayer of the membrane of intact erythrocytes with exogenous PIP2 caused, in a dose-dependent way, the scrambling of spin-labeled phosphatidylethanolamine, sphingomyelin, PC, and PS and in parallel the stomatocytic conversion of the cells. Both scrambling and stomatocytosis were strictly dependent on the presence of divalent cations in the medium. Mg2+ could replace Ca2+ but required a 10 times higher concentration. The effect was specific for PIP2, the other phosphoinositides being unable to induce the lipid redistribution. The shape change, but not the scrambling, required a normal ATP level. These results show that Ca2+ or Mg2+ trigger the lipid redistribution either from the internal or the external side of the membrane, provided that enough PIP2 is present on that side. Thus, no specific protein is required for this process. We infer that the ATP-dependent shape change of erythrocytes after incubation with PIP2 and Ca2+ results from the bilayer imbalance due to the activity of the aminophospholipid translocase which relocates PS and phosphatidylethanolamine to the inner monolayer without simultaneous outward diffusion of PC and sphingomyelin.