Treatment of red blood cells with calcium and ionomycin causes activation of the lipid scramblase, a putative membrane protein catalysing flip-flop of (phospho)lipids. Various fluorescent 1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl (C6-NBD) analogues were tested for transbilayer movement across the plasma membrane of red blood cells. Among these phospholipid analogues were phosphatidylgalactose, phosphatidylmaltose and phosphatidylmaltotriose, which were obtained from C6-NBD-phosphatidylcholine by phospholipase D-catalysed transphosphatidylation. The inward movement after the onset of scrambling was monitored by extraction of the non-internalized probe with BSA. We demonstrate that both the amino group and the size of the headgroup determine the kinetics of lipid scrambling, and that lipids with a ceramide backbone migrate much more slowly than glycerophospholipids with the same headgroup.
Abbreviations used: C6-NBD, 1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl; PC, phosphatidylcholine; PE, phosphatidylethanolamine; MePE, phosphatidyl-N-methyl-ethanolamine; DiMePE, phosphatidyl-N,N-dimethyl-ethanolamine; PS, phosphatidylserine; PGal, phosphatidylgalactose; PMal, phosphatidylmaltose; PTriose, phosphatidylmaltotriose; Spm, sphingomyelin; GalCer, galactosylceramide; RBC, red blood cell.