TY - JOUR
T1 - Transbilayer movement of NBD-labeled phospholipids in red blood cell membranes
T2 - Outward-directed transport by the multidrug resistance protein 1 (MRP1)
AU - Dekkers, David W C
AU - Comfurius, Paul
AU - Schroit, A. J.
AU - Bevers, Edouard M.
AU - Zwaal, Robert F A
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1998/10/20
Y1 - 1998/10/20
N2 - The outward movement (flop) of fluorescently labeled analogues of phosphatidylserine (PS) and phosphatidylcholine (PC) in human and murine red blood cells (RBC) was examined. 1-Oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol- 4-yl)amino]caproyl (C6-NBD) analogues of PS and PC were incorporated in the inner leaflet of the plasma membrane through the action of aminophospholipid translocase or through equilibration upon prolonged incubation, respectively. After removal of noninternalized probe, externalization of C6-NBD-PS or C6- NBD-PC from the inner to outer leaflet was monitored by continuous incubation of the cells in the presence of bovine serum albumin. Flop rates for both probes in intact human RBC were virtually identical (t(1/2) ~ 1.5 h), confirming earlier findings by Bitbol et al. [Bitbol, M., et al. (1988) Proc. Natl Acad. Sci. U.S.A. 85, 6783-6787] and Connor et al. [Connor, J., et al. (1992) J. Biol. Chem. 267, 19412-19417] Flop activity in resealed RBC ghosts could only be found upon coinclusion of both ATP and oxidized glutathione (GSSG). Furthermore, flop in intact cells was sensitive to verapamil (IC50 = 5-7 μM), vincristine (IC50 = 20 μM), and indomethacin (IC50 = 50 μM), suggesting the involvement of proteins conferring multidrug resistance (MDR). Experiments with RBC from knockout mice for multidrug resistance P- glycoproteins (Mdr1a/1b-/- and Mdr2-/-) and multidrug resistance protein 1 (Mrp1-/-) revealed that Mrp1 is responsible for the observed flop of the fluorescent lipid analogues. We found no indications for outward transport of endogenous PS by any of these drug-transporting proteins as measured by a sensitive prothrombinase assay. Neither aminophospholipid translocase nor Ca2+-induced lipid scramblase activities were affected in RBC of these knock-out mice. We conclude that lipid floppase activity, as detected with lipid probes, reflects the activity of MRP1 recognizing the modified lipid analogues as xenobiotics to be expelled from the cell.
AB - The outward movement (flop) of fluorescently labeled analogues of phosphatidylserine (PS) and phosphatidylcholine (PC) in human and murine red blood cells (RBC) was examined. 1-Oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol- 4-yl)amino]caproyl (C6-NBD) analogues of PS and PC were incorporated in the inner leaflet of the plasma membrane through the action of aminophospholipid translocase or through equilibration upon prolonged incubation, respectively. After removal of noninternalized probe, externalization of C6-NBD-PS or C6- NBD-PC from the inner to outer leaflet was monitored by continuous incubation of the cells in the presence of bovine serum albumin. Flop rates for both probes in intact human RBC were virtually identical (t(1/2) ~ 1.5 h), confirming earlier findings by Bitbol et al. [Bitbol, M., et al. (1988) Proc. Natl Acad. Sci. U.S.A. 85, 6783-6787] and Connor et al. [Connor, J., et al. (1992) J. Biol. Chem. 267, 19412-19417] Flop activity in resealed RBC ghosts could only be found upon coinclusion of both ATP and oxidized glutathione (GSSG). Furthermore, flop in intact cells was sensitive to verapamil (IC50 = 5-7 μM), vincristine (IC50 = 20 μM), and indomethacin (IC50 = 50 μM), suggesting the involvement of proteins conferring multidrug resistance (MDR). Experiments with RBC from knockout mice for multidrug resistance P- glycoproteins (Mdr1a/1b-/- and Mdr2-/-) and multidrug resistance protein 1 (Mrp1-/-) revealed that Mrp1 is responsible for the observed flop of the fluorescent lipid analogues. We found no indications for outward transport of endogenous PS by any of these drug-transporting proteins as measured by a sensitive prothrombinase assay. Neither aminophospholipid translocase nor Ca2+-induced lipid scramblase activities were affected in RBC of these knock-out mice. We conclude that lipid floppase activity, as detected with lipid probes, reflects the activity of MRP1 recognizing the modified lipid analogues as xenobiotics to be expelled from the cell.
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U2 - 10.1021/bi981011w
DO - 10.1021/bi981011w
M3 - Article
C2 - 9778357
AN - SCOPUS:0032553014
SN - 0006-2960
VL - 37
SP - 14833
EP - 14837
JO - Biochemistry
JF - Biochemistry
IS - 42
ER -