Facilitated phosphatidylcholine flip-flop across erythrocyte membranes using low molecular weight synthetic translocases

The transmembrane distribution of phospholipids plays an important regulatory role in human erythrocytes. Membrane-bound translocase enzymes maintain an asymmetric phospholipid distribution across the membrane monolayers by promoting transmembrane diffusion or flip-flop. Mechanistic understanding of the flip-flop process is weak at the molecular level. Recently, we discovered that amide and sulfonamide derivatives of tris(aminoethyl)amine facilitate phospholipid flip-flop across vesicle membranes; that is, they act as low molecular weight, synthetic translocases. In this report, NMR evidence is provided that suggests that the synthetic translocases work by forming a hydrogen-bonded complex with the phosphocholine headgroup which decreases headgroup polarity and promotes diffusion across the lipophilic interior of the membrane. Also cell morphology and fluorescence probe methods are used to show that these synthetic translocases facilitate phosphatidylcholine flip-flop across erythrocyte membranes. Addition of a small amount of dilauroylphosphatidylcholine to erythrocytes produces echinocyte morphology which takes days to revert back to the original discocyte shape. The rate of return is significantly accelerated by the presence of the synthetic translocases. The synthetic translocases facilitate inward-translocation (flip) of the fluorescent phosphatidylcholine probe, 1-palmitoyl-2-(N- [7-nitrobenz-2-oxa-1,3-diazol-4-yl]aminohexanoyl)-sn-glycero (PC-NBD).