Multidrug resistance-associated protein 2 (MRP2/ABCC2) is an ATP-binding cassette efflux transporter that mediates biliary excretion of glutathione, glucuronide and sulfate conjugates of endo- and xenobiotic organic anions. Single nucleotide polymorphisms (SNPs) of MRP2 are recognized as an important source of interindividual variability in drug response. We characterized the transport function of the human wild type MRP2 (WT) and four MRP2 SNPs M3 (S789F), M4 (A1450T), M5 (V4171) and M11 (T1477M) following their expression in Sf9 insect cells using recombinant baculovirus infection. ATP-dependent transport of the endogenous substrates leukotriene C₄ (LTC₄), estradiol-3-glucuronide (E₂3G), estradio1-17β-glucuronide (E217pG) and tauroursodeoxycholic acid (TUDC) was investigated in Sf9 plasma membrane vesicles. We further determined the ATP K_m and ATPase activity of M3 and M4 where the SNPs are located in the catalytic region of MRP2.

Transport activity was similar between WT and M11, except for a modest increase in TUDC transport. M3 and M4 demonstrated decreased transport for all substrates except E₂17βG, together with decreased ATPase activity but no change in the K_m for ATP. M5 demonstrated decreased apparent affinity for LTC₄, E₂3G and E₂17βG. Thus MRP2 polymorphisms can selectively alter the transport of its substrates.

We also investigated the role of MRP2 in the hepatobiliary disposition of a tetrahydroxy bile acid, 6α-hydroxy taurocholic acid (6α-OH-TC). Tetrahydroxy bile acids become major biliary bile acids in Bsep^-/- mice and Fxr^-/- mice, and also occur in patients with cholestasis. Formation of these unusual hydrophilic bile acids are protective against cholestasis in mice, but their hepatobiliary disposition has not been defined. We investigated whether mouse Mrp2 (mMrp2) and P-glycoprotein (mMdr1a) could transport 6α-OH-TC, and characterized its biliary excretion in wild-type and Mrp2^-/- mice in the presence or absence of GF120918, a P-gp and Bcrp inhibitor. Our study indicated that Mrp2 played a key role in the biliary excretion of 6α-OH-TC, while P-glycoprotein played a secondary role. Transport studies in Sf9 plasma membrane vesicles showed that mouse Mrp2, Mdr1a, and Bsep, but not Bcrp, transported 6α-OH-TC, while human MRP2 and P-glycoprotein transported 6α-OH-TC with positive cooperativity.

KEYWORDS: MRP2, P-glycoprotein, single nucleotide polymorphisms, tetrahydroxy bile acids, cholestasis