Transmembrane diffusion of fluorescent phospholipids in human erythrocytes

The outside-inside passage and transmembrane equilibrium distribution of several amphiphilic fluorescent phospholipids were examined in human erythrocytes. The results were compared with previous kinetic data obtained with spin-labeled phospholipids and with the equilibrium distribution of endogenous lipids in erythrocytes. When a nitro benzoxadiazole (NBD) was at the terminal position of a 6 carbon beta-chain, the outside-inside diffusion of the fluorescent phosphatidylserine (PS) analogue was slower, and the plateau lower than with long chain radioactive PS or spin-labeled PS. The corresponding phosphatidylethanolamine (PE) did not flip nor did the phosphatidylcholine (PC) analogue. With a NBD at the 12th carbon of a 18C alpha-chain, the amino-derivatives behaved more like endogenous PS and PE, i.e. they accumulated rapidly on the inner monolayer; however, the phosphatidylcholine analogue reached a plateau corresponding to 50% inside within 2 h at 37 degrees C, indicative of an abnormal rapid diffusion. In the latter case, changing the beta-chain from four to eight carbons had no influence on this rapid diffusion. We conclude that when the NBD is close to the glycerol moiety, it diminishes the affinity of the aminophospholipids for the aminophospholipid translocase. When it is close to the methyl terminal of an acyl chain, there is an acceleration of the spontaneous flip-flop. Presumably the polarity of the NBD is responsible for an unconventional orientation of the flexible acyl chain, thereby causing the transmembrane destabilization of the phospholipid. Overall these results illustrate the respective roles of spontaneous diffusion and translocase activity on transmembrane equilibrium distribution of phospholipids. They also show that NBD derivatives should be used cautiously as indicators of endogenous phospholipids.