The Complex of Phosphatidylinositol 4,5-Bisphosphate and Calcium Ions Is Not Responsible for Ca²⁺-Induced Loss of Phospholipid Asymmetry in the Human Erythrocyte: A Study in Scott Syndrome, a Disorder of Calcium-Induced Phospholipid Scrambling

Elevation of cytoplasmic Ca²⁺ levels in human erythrocytes induces a progressive loss of membrane phospholipid asymmetry, a process that is impaired in erythrocytes from a patient with Scott syndrome. We show here that porcine erythrocytes are similarly incapable of Ca²⁺-induced redistribution of membrane phospholipids. Because a complex of phosphatidylinositol 4,5-bisphosphate (PIP₂) and Ca²⁺ has been proposed as the mediator of enhanced transbilayer movement of lipids (J Biol Chem 269:6347,1994), these cell systems offer a unique opportunity for testing this mechanism. Analysis of both total PIP₂ content and the metabolic-resistant pool of PIP₂ that remains after incubation with Ca²⁺ ionophore showed no appreciable differences between normal and Scott erythrocytes. Moreover, porcine erythrocytes were found to have slightly higher levels of both total and metabolic-resistant PIP₂ in comparison with normal human erythrocytes. Although loading of normal erythrocytes with exogenously added PIP₂ gave rise to a Ca²⁺-induced increase in prothrombinase activity and apparent transbilayer movement of nitrobenzoxadiazolyl (NBD)-phospholipids, these PIP₂-loaded cells were also found to undergo progressive Ca²⁺-dependent cell lysis, which seriously hampers interpretation of these data. Moreover, loading Scott cells with PIP₂ did not abolish their impaired lipid scrambling, even in the presence of a Ca²⁺-ionophore. Finally, artificial lipid vesicles containing no PIP₂ or 1 mole percent of PIP₂ were indistinguishable with respect to transbilayer movement of NBD-phosphatidylcholine in the presence of Ca²⁺. Our findings suggest that Ca²⁺-induced redistribution of membrane phospholipids cannot simply be attributed to the steady-state concentration of PIP₂, and imply that such lipid movement is regulated by other cellular processes.