Regulation of multidrug resistance‐associated protein 2 by calcium signaling in mouse liver

Multidrug resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca2+) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5‐triphosphate receptor (InsP3R2) regulates Ca2+ release in the canalicular region of hepatocytes. However, the role of InsP3R2 and of Ca2+ signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP3R2‐mediated Ca2+ signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild‐type (WT) and InsP3R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time‐lapse imaging of Ca2+ signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)‐Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP3R2 was concentrated in the canalicular region of WT mice but absent in InsP3R2 KO livers, whereas expression and localization of InsP3R1 was preserved, and InsP3R3 was absent from both WT and KO livers. Ca2+ signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP3R2. Canalicular secretion of the organic anion 5‐chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP3R2 KO mice. Finally, ATP increased GFP‐Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA. Conclusion: InsP3R2‐mediated Ca2+ signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane. HEPATOLOGY 2010