Complementary Functions of the Flippase ATP8B1 and the Floppase ABCB4 in Maintaining Canalicular Membrane Integrity

doi:10.1053/j.gastro.2011.07.042

Gastroenterology

Volume 141, Issue 5, November 2011, Pages 1927-1937.e4

https://doi.org/10.1053/j.gastro.2011.07.042 Get rights and content

Background & Aims

Progressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4^−/− mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts.

Methods

To investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts.

Results

Overexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1–CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice.

Conclusions

ATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1–CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts.

Section snippets

Plasmids

Generation of the pcDNA3.1+ vector (Invitrogen, Carlsbad, CA) expressing human ABCB4 and its K435M derivative (replacing the conserved lysine within the Walker A motif of the first nucleotide binding domain to inhibit adenosine triphosphate binding) was described previously.¹¹ The E558Q derivative (replacing a conserved glutamate in the Walker B motif with a glutamine to prevent adenosine triphosphate hydrolysis) was derived from the wild-type ABCB4 by site-directed mutagenesis using the

Expression of Functional ABCB4 Is Deleterious to HEK293T Cells

To investigate the molecular biochemistry of ABCB4 function, we expressed the protein in the human cell line HEK293T. Despite efficient transient transfection (Supplementary Figure 1), wild-type ABCB4 was only expressed to a low level (Figure 1A). Two forms of the protein were evident that differed in their glycosylation status (Supplementary Figure 2). We also noted that inactive derivatives of ABCB4 carrying a point mutation in either the Walker A (K435M) or Walker B (E558Q) motifs of the

Discussion

Following transient transfection, we found that wild-type ABCB4 expressed poorly in HEK293T cells. Several lines of evidence suggested that this lack of expression was due to cytoxicity of ABCB4 function: we found high levels of LDH in the media of cells expressing wild-type ABCB4, but not with mutant ABCB4; while little of the wild-type ABCB4 could be detected on the plasma membrane, the inactive mutant form of the protein localized very well on the plasma membrane. Intriguingly, the

Conclusions

Our data show that ATP8B1/CDC50A-mediated flipping of PS is particularly important in the canalicular membrane, where ABCB4 flops PC. In the absence of PC flopping, the canalicular membrane appears to be sufficiently rigid to withstand bile salt–mediated extraction without PS flipping by ATP8B1.

Acknowledgments

The authors are grateful to Marjolein Snippe for invaluable assistance with confocal microscopy and to Jan van Marle for electron microscopy.

Drs Groen and Romero contributed equally as joint first authors. Drs Linton and Oude Elferink contributed equally as joint senior authors.

Dr Romero is currently at the Laboratory of Experimental Hepatology and Drug Targeting, CIBERehd, University of Salamanca, Salamanca, Spain.

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: Conflicts of interest The authors disclose no conflicts.

: Funding This study was supported by grants from The Netherlands Organization for Scientific Research (NWO; program grant 912-02–73), a postdoctoral fellowship for MMR from the Spanish Ministry of Science, core funding from the Medical Research Council, UK, and the Imperial College Healthcare NHS Trust Biomedical Research Center.

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Original ResearchBasic and Translational—BiliaryComplementary Functions of the Flippase ATP8B1 and the Floppase ABCB4 in Maintaining Canalicular Membrane Integrity

Background & Aims

Methods

Results

Conclusions

Section snippets

Plasmids

Expression of Functional ABCB4 Is Deleterious to HEK293T Cells

Discussion

Conclusions

Acknowledgments

J Hepatol

J Hepatol

Hepatology

Gastroenterology

Gastroenterology

Cell

Cell

Biochim Biophys Acta

Membrane phosphatidylserine distribution as a non-apoptotic signalling mechanism in lymphocytes

Nat Cell Biol

Hepatocanalicular transport defects: pathophysiologic mechanisms of rare diseases

Gastroenterology

Model systems, lipid rafts, and cell membranes

Annu Rev Biophys Biomol Struct

Hepatic secretion of phospholipid vesicles in the mouse critically depends on mdr2 or MDR3 P-glycoprotein expressionVisualization by electron microscopy

J Clin Invest

Original Research
Basic and Translational—Biliary
Complementary Functions of the Flippase ATP8B1 and the Floppase ABCB4 in Maintaining Canalicular Membrane Integrity