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

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.

Molecular Cloning of Human Plasma Membrane Phospholipid Scramblase

“…Whereas incubation with Ca 2ϩ ionophore causes a marked acceleration in transbilayer movement of plasma membrane PL in both platelets and erythrocytes, the apparent rate of transbilayer PL migration in platelet exceeds that in erythrocyte by approximately 10-fold, implying either a higher abundance of PL scramblase or the action of another component in platelet with enhanced PL scrambling function (18,19). Zwaal and associates recently reported evidence for the existence of protein(s) in platelet with functional properties similar to that of PL scramblase we isolated from erythrocyte (10 -12).…”

Scott syndrome erythrocytes contain a membrane protein capable of mediating Ca2+-dependent transbilayer migration of membrane phospholipids.

“…Recent studies suggest that intracellular polyamines, including spermine and spermidine, are inhibitory to the Ca 2ϩ -dependent activation of transbilayer PL migration across the erythrocyte membrane (15,19). This raises the possibility that the Scott cell defect is related either to an abnormal increase in one or more polyamines, or, to an increased sensitivity of PL scramblase to inhibition by these compounds.…”

“…Elevated cytosolic Ca 2 ϩ has been shown to initiate rapid bidirectional transbilayer redistribution of all plasma membrane PL (6-8, 14, 15), although evidence for concomitant selective and vectorial egress of PS and PE to the cell surface has also been reported (9,16). Proposed mechanisms for this effect of intracellular Ca 2 ϩ on the movement of PL between plasma membrane leaflets include ( i ) Ca 2 ϩ -induced vesiculation of the plasma membrane (11,17); ( ii ) calpain-mediated proteolysis of the submembrane cytoskeleton, liberating PL headgroups from interaction with endofacial proteins (18); ( iii ) loss of polyamine-membrane associations that might serve to stabilize PL asymmetry (15,19); ( iv ) destabilization of the PL bilayer by the complex of Ca 2 ϩ and the polyanionic PL, phosphatidylinositol 4,5-bisphosphate (19,20); ( v ) action of a Ca 2 ϩ -dependent membrane protein with PL scramblase activity. Recently, an as yet unidentified RBC membrane protein of ‫ف‬ 37 kD was shown to exhibit PL scramblase activity when reconstituted in proteoliposomes containing exogenous PL (21 endofacial surface of the RBC membrane.…”

Polyamine Regulation of Plasma Membrane Phospholipid Flip-Flop during Apoptosis

“…Additionally, the anionic phospholipid phosphatidylinositol bisphosphate may play a role (27,28). Of note, in both the calcium-treated erythrocyte ghost (15) and the phosphatidylinositol bisphosphate-loaded erythrocyte membrane or vesicle (28), exogenously added polyamines, particularly spermine, have been shown to antagonize calcium-induced nonspecific phospholipid flip-flop.…”

“…Additionally, the anionic phospholipid phosphatidylinositol bisphosphate may play a role (27,28). Of note, in both the calcium-treated erythrocyte ghost (15) and the phosphatidylinositol bisphosphate-loaded erythrocyte membrane or vesicle (28), exogenously added polyamines, particularly spermine, have been shown to antagonize calcium-induced nonspecific phospholipid flip-flop. These observations in simplified models of phospholipid flip-flop, prompted us to ask whether polyamines in intact cells might govern the phospholipid flip-flop seen during apoptosis.…”