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

Abstract

The canalicular multispecific organic anion transporter (cMOAT), a member of the ATP-binding cassette transporter family, mediates the transport of a broad range of non-bile salt organic anions from liver into bile. cMOAT-deficient Wistar rats (TR-) are mutated in the gene encoding cMOAT, leading to defective hepatobiliary transport of a whole range of substrates, including bilirubin glucuronide. These mutants also have impaired hepatobiliary excretion 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 per kg 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 excretion across the basolateral membrane. In several independent cMOAT-transfectants, the level of GSH excretion correlated with the expression level of the 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-expressing MDCKII cells, ATP-dependent S-(2,4-dinitrophenyl)glutathione uptake is competitively inhibited by high concentrations of GSH (Ki≈ 20 mM). We concluded that cMOAT mediates low-affinity transport of GSH. However, since hepatocellular GSH concentrations are high (5–10 mM), cMOAT might serve an important physiological function in maintenance of bile flow in addition to hepatic GSH turnover.