肝胆管側膜有機アニオン輸送体MRP2/cMOATの基質認識性に関する解析

抄録

Multidrug resistance-associated protein 2 (MRP2), along with MRPI, transports glutathione and glucuronide conjugates as well as non-conjugated organic anions. In the present study, we examined the role of charged amino acids in the transmembrane domains of rat Mrp2, conserved among MRP families, by means of sitedirected mutagenesis. A single amino acid mutation from Lys to Met at 325 (K325M), from Arg to Leu at 586 (R586L) and from Asp to Asn at 329 (D329N) resulted in a marked reduction in the transport activity of glutathione-S conjugates (2, 4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene C₄ (LTC₄)) without affecting that of the subsequent metabolites of LTC₄ (LTD₄, LTE₄ and LTF₄) or glucuronide conjugates (17β-estradiol 17-β-D-glucuronide (E₂17βG) and E3040-glucuronide). In contrast to the reduced affinity for DNP-SG and LTC₄, the affinity for E₂17βG was increased several-fold in these mutants, suggesting that amino acids at 325 and 586 may play an important role in distinguishing between glutathione and glucuronide conjugates. Furthermore, R1096L acquired the ability to transport TC, which is not transported by Mrp2. Since Mrp3, which transports TC, has a neutral amino acid at 1084 corresponding to this position, the charge-dependence on the transport ability of TC was further investigated in both Mrp2 and Mrp3. In the case of Mrp2, substitution of Arg at 1096 by Lys (R1096K) and Val (RIO96V) also resulted in the acquisition of the ability to transport TC. In contrast, substitution of Leu at 1084 of rat Mrp3 by Lys (L1084K_Mrp3) resulted in the loss of transport activity for both E₂17βG and TC, although transport activity for these compounds was unaffected by substitution by Val (L1084V_Mrp3) or Met (L1084M_Mrp3). This suggests that the charged moieties at 1096 of Mrp2 and 1084 of Mrp3 do not simply govern the transport ability for TC; Arg at 1096 acts as an obstacle to the transport of TC in rat Mrp2, whereas a neutral amino acid at the corresponding position in Mrp3 (Leu at 1084) is required for transport of both TC and E₂17βG.