Abstract

Multidrug resistance-associated protein 2 (MRP2) transports glutathione conjugates, glucuronide conjugates, and sulfated conjugates of bile acids. In the present study, we examined the role of charged amino acids in the transmembrane domains of rat Mrp2, conserved among MRP families, using the isolated membrane vesicles from Sf9 cells infected with the recombinant baculoviruses. By normalizing the transport activity for compounds by that for estradiol 17β-d-glucuronide (E₂17βG), it was indicated that the site-directed mutagenesis from Lys to Met at 325 (K325M) and from Arg to Leu at 586 (R586L) results in a marked reduction in the transport for glutathione conjugates [2,4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene (LT) C₄] without affecting that for 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridymethyl) benzothiazole glucuronide and taurolithocholate sulfate. In contrast to the reduced affinity for DNP-SG, the affinity for E₂17βG was increased severalfold in these mutant Mrp2s, suggesting the amino acids at 325 and 586 play an important role in distinguishing between glutathione and glucuronide conjugates. The comparable affinity for LTD₄, LTE₄, and LTF₄ in these mutant Mrp2s with that in wild-type Mrp2 indicates that recognition of LTC₄ metabolites by Mrp2 is different from that of LTC₄. The transport activity for glutathione conjugate was retained on R586K, whereas no such complementary cationic amino acid effect was observed in K325R. In addition, R1206M and E1208Q exhibited the loss of transport activity for the tested compounds. The results of the present study demonstrate that the charged amino acids in the transmembrane domain of rat Mrp2 may play an important role in the recognition and/or transport of its substrates.