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 (Ca(2+)) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5-triphosphate receptor (InsP(3)R2) regulates Ca(2+) release in the canalicular region of hepatocytes. However, the role of InsP(3)R2 and of Ca(2+) signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP(3)R2-mediated Ca(2+) signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild-type (WT) and InsP(3)R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time-lapse imaging of Ca(2+) 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. InsP(3)R2 was concentrated in the canalicular region of WT mice but absent in InsP(3)R2 KO livers, whereas expression and localization of InsP(3)R1 was preserved, and InsP(3)R3 was absent from both WT and KO livers. Ca(2+) signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP(3)R2. 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 InsP(3)R2 KO mice. Finally, ATP increased GFP-Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA.


CONCLUSIONS
InsP(3)R2-mediated Ca(2+) signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane.
Cruz
Laura N
LN
Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520-8019, USA.
Guerra
Mateus T
MT
Kruglov
Emma
E
Mennone
Albert
A
Garcia
Celia R S
CR
Chen
Ju
J
Nathanson
Michael H
MH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001646:Bile / Q000378:metabolism; D001663:Bilirubin / Q000097:blood; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D013655:Taurochenodeoxycholic Acid / Q000494:pharmacology
D000255:Adenosine Triphosphate; D001663:Bilirubin; D002118:Calcium; D004533:Egtazic Acid; D053496:Inositol 1,4,5-Trisphosphate Receptors; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D020033:Protein Isoforms; D013655:Taurochenodeoxycholic Acid; C031655:ursodoxicoltaurine; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid