Abstract

The ATP-binding cassette multidrug resistance protein 1 MRP1 (ABCC1) mediates the cellular efflux of organic anions including conjugated metabolites, chemotherapeutic agents, and toxicants. We previously described a mutation in cytoplasmic loop 7 (CL7) of MRP1, Pro1150Ala, which reduced leukotriene C₄ (LTC₄) transport but increased 17β-estradiol 17β-d-glucuronide (E₂17βG) and methotrexate (MTX) transport. Vanadate-induced trapping of [α-³²P]8N₃ADP by the Pro1150Ala mutant in the absence of substrate was also greatly reduced compared with wild-type MRP1 suggesting an uncoupling of ATP hydrolysis and transport activity. To determine whether the functional importance of MRP1-Pro¹¹⁵⁰ is conserved, the analogous Pro¹¹⁵⁸ and Pro¹¹⁴⁷ residues in the MRP2 and MRP3 transporters, respectively, were mutated to Ala. Expression levels of the three mutants were unaffected; however, the vesicular transport activity of at least one organic anion substrate was significantly altered. As observed for MRP1-Pro1150Ala, LTC₄ transport by MRP2-Pro1158Ala was decreased. However, E₂17βG and MTX transport was comparable with that of wild-type MRP2 rather than increased as was observed for MRP1-Pro1150Ala. In the case of MRP3-Pro1147Ala, LTC₄ transport was increased, whereas E₂17βG transport was unaffected. MTX transport by MRP3-Pro1147Ala was also increased but to a lesser extent than for MRP1-Pro1150Ala. In contrast, all three mutants showed a marked reduction in levels of vanadate-induced trapped [α-³²P]8N₃ADP. We conclude that MRP1-Pro¹¹⁵⁰, MRP2-Pro¹¹⁵⁸, and MRP3-Pro¹¹⁴⁷ in CL7 differ in their influence on substrate specificity but share a common role in the nucleotide interactions of these transporters.