ANTAGONIST EFFECTS OF CA2-BISPHOSPHATE-MEDIATED TRANSMEMBRANE REDISTRIBUTION OF PHOSPHOLIPIDS IN LARGE UNILAMELLAR VESICLES AND IN ERYTHROCYTES( AND SPERMINE ON PHOSPHATIDYLINOSITOL 4,5)

We have previously suggested the involvement of a Ca2+-phosphatidylino sitol 4,5-bisphosphate (PIP2) complex in the phospholipid transmembran e redistribution triggered by cytosolic Ca2+ in erythrocytes. Indeed, the lipid scrambling was induced by extracellular Ca2+ in erythrocytes loaded with PIP2 and was abolished in inside-out vesicles prepared fr om PIP2-depleted erythrocytes (Sulpice, J. C., Zachowski, A., Devaux, P. F., & Giraud, F. (1994) J. Biol. Chem. 269, 6347-6354). Here, we sh ow that Ca2+ triggers a partial redistribution of spin-labeled phospho lipids in protein-free large unilamellar vesicles (LUVs), only when th ey contain PIP2. Spermine, a polyamine known to interact with PIP2 and reported to inhibit lipid scrambling in resealed ghosts, was found to inhibit also the Ca2+-induced scrambling in LUVs and in PIP2-loaded e rythrocytes, presumably by interacting with PIP2 and preventing the fo rmation of Ca2+-PIP2 complexes. A similar mechanism can account for sp ermine inhibition in natural membranes, confirming the role of PIP2 in the scrambling process without excluding the participation of protein s. In erythrocytes, activation of the phosphoinositide phospholipase C (PLC) or a 20 h ATP depletion, which both led to a reduction in the P IP2 content by 40-60%, did not affect Ca2+-induced phospholipid scramb ling. In contrast, longer ATP depletion, resulting in a 80% reduction in the PIP2 content, did induce a significant decrease in lipid scramb ling, suggesting that only the PIP2 pool resistant to the PLC was invo lved. Spermine was able to inhibit hydrolysis of this pool by an exoge nous PLA(2). It is thus likely that spermine antagonized the Ca2+-indu ced scrambling in resealed ghosts by interacting with the PLC-resistan t pool of PIP2.