REQUIREMENT FOR PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE IN THE CA2-INDUCED PHOSPHOLIPID REDISTRIBUTION IN THE HUMAN ERYTHROCYTE-MEMBRANE()

In order to investigate how calcium on the cytosolic side of human ery throcytes induces the transmembrane redistribution of phospholipids, w e studied the effect of this cation on the transmembrane movements of spin-labeled phospholipids (phosphatidylserine (PS) and phosphatidylch oline (PC)) incorporated into inside-out vesicles derived from human e rythrocytes. We found that the extent of the Ca2+-induced lipid scramb ling was dependent upon the level of phosphatidylinositol 4,5-bisphosp hate (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 th e original erythrocytes or by incorporation of exogenous PIP2. Similar ly 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 time s higher concentration. The effect was specific for PIP2, the other ph osphoinositides being unable to induce the lipid redistribution. The s hape change, but not the scrambling, required a normal ATP level. Thes e results show that Ca2+ or Mg2+ trigger the lipid redistribution eith er from the internal or the external side of the membrane, provided th at enough PIP, is present on that side. Thus, no specific protein is r equired 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 translo case which relocates PS and phosphatidylethanolamine to the inner mono layer without simultaneous outward diffusion of PC and sphingomyelin.