ATP-dependent glutathione disulphide transport mediated by the MRP gene-encoded conjugate export pump

Abstract

We have previously shown that the multidrug resistance protein (MRP) mediates the ATP-dependent membrane transport of the endogenous glutathione conjugate leukotriene C₄ (LTC₄) and of structurally related anionic conjugates of lipophilic compounds [Jedlitschky, Leier, Buchholz, Center and Keppler (1994) Cancer Res. 54, 4833–4836; Leier, Jedlitschky, Buchholz, Cole, Deeley and Keppler (1994) J. Biol. Chem. 269, 27807–27810]. We demonstrate in the present study that MRP also mediates the ATP-dependent transport of GSSG, as shown in membrane vesicles from human leukaemia cells overexpressing MRP (HL60/ADR cells) or HeLa cells transfected with an MRP expression vector (HeLa T5 cells) in comparison with the respective parental or control cells. The K_m value for ATP-dependent transport of GSSG was 93±26 μM (mean value±S.D., n = 5) in membrane vesicles from HeLa T5 cells. GSH, at a concentration of 100 μM, was not a substrate for any significant ATP-dependent MRP-mediated transport. The transport of GSSG was competitively inhibited by LTC₄, by the leukotriene D₄ receptor antagonist 3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethylamino-3-oxopropyl)-thio}-methyl]thio)propanoic acid (MK571) and by S-decylglutathione, with K_i values of 0.3, 0.6 and 0.7 μM respectively. These studies identify MRP as the membrane glycoprotein which mediates the ATP-dependent export of GSSG from these cells.