A progressive familial intrahepatic cholestasis type 2 mutation causes an unstable, temperature-sensitive bile salt export pump

• Published in: Journal of hepatology Vol. 40; no. 1; pp. 24 - 30
• Main Authors: PLASS, Jacqueline R. M, MOL, Olaf, HEEGSMA, Janette, GEUKEN, Mariska, DE BRUIN, Joost, ELLING, Geeske, MÜLLER, Michael, FABER, Klaas Nico, JANSEN, Peter L. M
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier 2004
• Subjects: Adenosine Triphosphatases - metabolism; Amino Acid Sequence; Animals; Aspartic Acid; atp; ATP Binding Cassette Transporter, Subfamily B, Member 11; ATP-Binding Cassette Transporters - chemistry; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - metabolism; Bile Canaliculi - metabolism; Biological and medical sciences; canalicular membrane-vesicles; Cell Line, Tumor; Cholestasis, Intrahepatic - genetics; Cholestasis, Intrahepatic - metabolism; Cholestasis, Intrahepatic - physiopathology; dependent transport; Disease Progression; Drug Stability; expression; Gastroenterology. Liver. Pancreas. Abdomen; Glycine; Glycosylation; Green Fluorescent Proteins; Humans; Indicators and Reagents; Liver. Biliary tract. Portal circulation. Exocrine pancreas; Luminescent Proteins - genetics; Medical sciences; Mice; Molecular Sequence Data; mrp1; Mutation, Missense; Other diseases. Semiology; p-glycoprotein; Protein Transport; rat-liver; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacokinetics; substrate-specificity; taurocholate; Taurocholic Acid - pharmacokinetics; Temperature; transmembrane conductance regulator; Temperature; ATP-binding cassette transporters; Temperature sensitivity; Enzyme; Bile salt; Genetic cholestatic disease; Adenosinetriphosphatase; Hepatic disease; Biliary tract disease; Genetic disease; Progressive; Intrahepatic cholostasis; Sensitivity; Missense mutation; ATPase activity; Cause; Gastroenterology; Digestive diseases; Hydrolases; Mutation; Transport; ABC transporter
• ISSN: 0168-8278; 1600-0641
• DOI: 10.1016/s0168-8278(03)00483-5

Progressive familial intrahepatic cholestasis type 2 (PFIC-2) patients have a defect in the hepatocanalicular bile salt secretion. The disease is caused by mutations in the bile salt export pump (BSEP). Ten different missense mutations have been described. In this study, we analysed the effect of the D482G PFIC-2 mutation on BSEP function. Adenosine triphosphatase (ATPase) and taurocholate transport assays were performed with full-length mouse Bsep (mBsep) with and without the D482G mutation. The effect on expression and subcellular sorting was studied in HepG2 cells, stably expressing enhanced green fluorescent protein (EGFP)-tagged mBsep proteins. The D482G mutation did not significantly affect the taurocholate transport activity of mBsep, even though the bile salt-inducible ATPase activity of the mutant protein was slightly reduced. Protein expression and canalicular sorting were strongly affected by the D482G mutation. Mutant EGFP-mBsep protein was only partly glycosylated and detected in both the canalicular membrane and the cytoplasm. At 30 degrees C, the mutant mRNA and protein levels were strongly increased, and the protein was predominantly glycosylated and efficiently targeted to the canalicular membrane. These data suggest that PFIC-2 patients with the D482G mutation express a functional, but highly unstable, temperature-sensitive bile salt export pump.