Phosphatidylinositol 4,5-bisphosphate domain inducers promote phospholipid transverse redistribution in biological membranes

Transmembrane phospholipid redistribution (scrambling), leading to exposure of phosphatidylserine on the cell surface, plays a physiological role to induce platelet procoagulant activity and clearance of injured or apoptotic cells. Scrambling is generally attributed to an increase in intracellular Ca(2+) and would be mediated by a protein (scramblase), whose activity could be modulated by cofactors. We reported previously that phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a positive regulator of Ca(2+)-induced scrambling. We show here, using inside-out vesicles from erythrocyte membranes, that a pleckstrin homology (PH) domain, which interacts with high affinity with PIP(2), inhibited Ca(2+)-induced scrambling, confirming the role of PIP(2). As Ca(2+) is known to interact with PIP(2) and to promote the formation of lateral domains of acidic phospholipids in membranes, we investigated whether PIP(2) domain formation could be involved in scrambling. Spermine, polylysine, and MARCKS (151-175) peptide caused scrambling in parallel to their reported ability to form domains of acidic phospholipids, including PIP(2). Similarly, neomycine, another PIP(2)-interacting polycation, induced scrambling. A PIP(2) antibody was also found to induce scrambling, presumably by a similar mechanism, since phospholipid antibodies are known to promote phospholipid capping. In conclusion, Ca(2+) is not the sole inducer of scrambling, and formation of PIP(2) domains could play a critical role in this process.