title: A progressive familial intrahepatic cholestasis type 2 mutation causes an unstable, temperature-sensitive bile salt export pump.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  These data suggest that PFIC-2 patients with the D482G mutation express a functional, but highly unstable, temperature-sensitive bile salt export pump.
authors.affiliation: Department of Gastroenterology and Hepatology, Center for Liver, Digestive and Metabolic Diseases, University Hospital Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands.
authors.forename: Jacqueline R M
authors.initials: JR
authors.lastname: Plass
authors.forename: Olaf
authors.initials: O
authors.lastname: Mol
authors.forename: Janette
authors.initials: J
authors.lastname: Heegsma
authors.forename: Mariska
authors.initials: M
authors.lastname: Geuken
authors.forename: Joost
authors.initials: J
authors.lastname: de Bruin
authors.forename: Geeske
authors.initials: G
authors.lastname: Elling
authors.forename: Michael
authors.initials: M
authors.lastname: Müller
authors.forename: Klaas Nico
authors.initials: KN
authors.lastname: Faber
authors.forename: Peter L M
authors.initials: PL
authors.lastname: Jansen
mesh_terms: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001648:Bile Canaliculi / Q000378:metabolism; D045744:Cell Line, Tumor; D002780:Cholestasis, Intrahepatic / Q000235:genetics* / Q000378:metabolism* / Q000503:physiopathology; D018450:Disease Progression; D004355:Drug Stability; D005998:Glycine; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000493:pharmacokinetics; D013656:Taurocholic Acid / Q000493:pharmacokinetics; D013696:Temperature
chemical_list: C093491:ABCB11 protein, human; D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; C490549:Abcb11 protein, mouse; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D013656:Taurocholic Acid; D000251:Adenosine Triphosphatases; D005998:Glycine