Antagonist Effects of Ca2+and Spermine on Phosphatidylinositol 4,5-Bisphosphate-Mediated Transmembrane Redistribution of Phospholipids in Large Unilamellar Vesicles and in Erythrocytes

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Abstract
We have previously suggested the involvement of a Ca(2+)-phosphatidylinositol 4,5-bisphosphate (PIP2) complex in the phospholipid transmembrane 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 from PIP2-depleted erythrocytes (Sulpice, J.C., Zachowski, A., Devaux, P.F., & Giraud, F. (1994) J. Biol. Chem. 269, 6347-6354). Here, we show that Ca2+ triggers a partial redistribution of spin-labeled phospholipids in protein-free large unilamellar vesicles (LUVs), only when they 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 Ca(2+)-induced scrambling in LUVs and in PIP2-loaded erythrocytes, presumably by interacting with PIP2 and preventing the formation of Ca(2+)-PIP2 complexes. A similar mechanism can account for spermine inhibition in natural membranes, confirming the role of PIP2 in the scrambling process without excluding the participation of proteins. In erythrocytes, activation of the phosphoinositide phospholipase C (PLC) or a 20 h ATP depletion, which both led to a reduction in the PIP2 content by 40-60%, did not affect Ca(2+)-induced phospholipid scrambling. In contrast, longer ATP depletion, resulting in a 80% reduction in the PIP2 content, did induce a significant decrease in lipid scrambling, suggesting that only the PIP2 pool resistant to the PLC was involved. Spermine was able to inhibit hydrolysis of this pool by an exogenous PLA2. It is thus likely that spermine antagonized the Ca(2+)-induced scrambling in resealed ghosts by interacting with the PLC-resistant pool of PIP2.