Identification of the multidrug-resistance protein (MRP) as the glutathione-S-conjugate export pump of erythrocytes

Abstract

The identification of the multidrug resistance protein (MRP) as a conjugate export pump in several cell types suggested its involvement in the long-known glutathione-S-conjugate transport across erythrocyte membranes. We investigated the ATP-dependent transport of glutathione S-conjugates in human erythrocyte and erythroleukemia cell membrane vesicles using the endogenous conjugate leukotriene C4 (LTC4), known to be a high-affinity substrate for MRP, in addition to S-(2,4-dinitrophenyl)glutathione. The kinetic parameters, including the Km value for LTC4 of 118 +/- 5 nM and the inhibition constants for transport of both substrates for the quinoline-based inhibitor MK 571, were similar to those obtained for transport mediated by recombinant MRP. Direct photoaffinity labeling of human erythrocyte membranes with [3H]LTC4 revealed a major binding protein of about 190 kDa which was immunoprecipitated by an anti-MRP serum. The radiolabeling of this protein was specifically suppressed by the transport inhibitor MK 571. Several additional anti-MRP sera detected the protein of about 190 kDa in human erythrocyte and erythroleukemia cell membranes. These data identify for the first time the glutathione-S-conjugate transporting protein in erythrocyte membranes.