Canalicular multispecific organic onion transporter multidrug resistance protein 2 mediates low-affinity transport of reduced glutathione

The canalicular multispecific organic anion transporter (cMOAT), a member o f the ATP-binding cassette transporter family, mediates the transport of a broad range of non-bile salt organic anions from liver into bile, cMOAT-def icient Wistar rats (TR-) are mutated in the gene encoding cMOAT, leading to defective hepatobiliary transport of a whole range of substrates, includin g bilirubin glucuronide. These mutants also have impaired hepatobiliary exc retion of GSH and, as a result, the bile flow in these animals is reduced. In the present work we demonstrate a role for cMOAT in the excretion of GSH both in vivo and in vitro. Biliary GSH excretion in rats heterozygous for the cMOAT mutation (TR/tr) was decreased to 63% of controls (TR/TR) (114+/- 24 versus 181+/-20 nmol/min perkg body weight). Madin-Darby canine kidney ( MDCK) II cells stably expressing the human cMOAT protein displayed >10-fold increase in apical GSH excretion compared with wild-type MDCKII cells (141 +/- 6.1 pmol/min per mg of protein versus 13.2 +/- 1.3 pmol/min per mg of protein in wild-type MDCKII cells). Similarly, MDCKII cells expressing the human multidrug resistance protein 1 showed a 4-fold increase in GSH excret ion across the basolateral membrane. In several independent cMOAT-transfect ants, the level of GSH excretion correlated with the expression level of th e protein. Furthermore, we have shown, in cMOAT-transfected cells, that GSH is a low-affinity substrate for the transporter and that its excretion is reduced upon ATP depletion. In membrane vesicles isolated from cMOAT-expres sing MDCKII cells, ATP-dependent S-(2,4-dinitrophenyl)glutathione uptake is competitively inhibited by high concentrations of GSH (K-i approximate to 20 mM). We concluded that cMOAT mediates low-affinity transport of GSH. How ever, since hepatocellular GSH concentrations are high (5-10 mM), cMOAT mig ht serve an important physiological function in maintenance of bile flow in addition to hepatic GSH turnover.