The human multidrug-resistance-associated protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin

Results of previous studies have suggested that UCB (unconjugated bilirubin) may be transported by MRP1/Mrp1 (multidrug-resistance-associated protein 1). To test this hypothesis directly, [³H]UCB transport was assessed in plasma-membrane vesicles from MDCKII cells (Madin–Darby canine kidney II cells) stably transfected with human MRP1 or MRP2; wild-type MDCKII cells served as controls. As revealed by Western blotting, transfection achieved abundant expression of MRP1 and MRP2. [³H]UCB uptake was measured in the presence of 60 μM human serum albumin at a free (unbound) concentration of UCB (B_F) ranging from 5 to 72 nM and in the presence of 3 mM ATP or 3 mM AMP-PCP (adenosine 5′-[β,γ-methylene]triphosphate). MRP1-transfected vesicles showed transport activity three and five times higher respectively compared with MRP2 or wild-type vesicles, whose transport did not differ significantly. [³H]UCB transport was stimulated 4-fold by 1.5 mM GSH, occurred into an osmotically sensitive space, was inhibited by 3 μM MK571 and followed saturative kinetics with K_m=10±3 nM (B_F) and V_max=100±13 pmol·min⁻¹·(mg of protein)⁻¹. UCB significantly inhibited the transport of LTC4 (leukotriene C4), a leukotriene substrate known to have high affinity for MRP1. Collectively, these results prove directly that MRP1 mediates ATP-dependent cellular export of UCB and supports its role in protecting cells from bilirubin toxicity.