title: A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.
abstract: The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain.
authors.affiliation: Institute of Biochemistry, Heinrich Heine University, 40225 Düsseldorf.
authors.forename: Marianne
authors.initials: M
authors.lastname: Kluth
authors.affiliation: Department of Gastroenterology, Hepatology and Infectiology, University Hospital, 40225 Düsseldorf.
authors.forename: Jan
authors.initials: J
authors.lastname: Stindt
authors.affiliation: Department of Gastroenterology, Hepatology and Infectiology, University Hospital, 40225 Düsseldorf.
authors.forename: Carola
authors.initials: C
authors.lastname: Dröge
authors.affiliation: Department of Gastroenterology, Hepatology and Infectiology, University Hospital, 40225 Düsseldorf.
authors.forename: Doris
authors.initials: D
authors.lastname: Linnemann
authors.affiliation: Department of Gastroenterology, Hepatology and Infectiology, University Hospital, 40225 Düsseldorf.
authors.forename: Ralf
authors.initials: R
authors.lastname: Kubitz
authors.affiliation: Institute of Biochemistry, Heinrich Heine University, 40225 Düsseldorf; Cluster of Excellence on Plant Sciences, Heinrich Heine University, 40225 Düsseldorf, Germany. Electronic address: lutz.schmitt@hhu.de.
authors.forename: Lutz
authors.initials: L
authors.lastname: Schmitt
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry*; D019943:Amino Acid Substitution; D064751:Ammonium Compounds; D001608:Beryllium / Q000737:chemistry; D002780:Cholestasis, Intrahepatic / Q000201:enzymology / Q000235:genetics; D005459:Fluorides / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D020125:Mutation, Missense; D010843:Pichia / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D064751:Ammonium Compounds; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; C035982:beryllium fluoride; C024822:ammonium fluoride; D000255:Adenosine Triphosphate; C095122:multidrug resistance protein 3; D000251:Adenosine Triphosphatases; D001608:Beryllium; D005459:Fluorides
keywords: ABC Transporter; ATPase; Lipid Transport; Liver Injury; MDR1; MDR3; Multidrug Transporter; PFIC-3; Transmission Interface; X Loop