title: Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6).
abstract: Mutations in the ABCC6 (MRP6) gene cause pseudoxanthoma elasticum (PXE), a rare heritable disorder resulting in the calcification of elastic fibers. In the present study a cDNA encoding a full-length normal variant of ABCC6 was amplified from a human kidney cDNA library, and the protein was expressed in Sf9 insect cells. In isolated membranes ATP binding as well as ATP-dependent active transport by ABCC6 was demonstrated. We found that glutathione conjugates, including leukotriene C(4) and N-ethylmaleimide S-glutathione (NEM-GS), were actively transported by human ABCC6. Organic anions (probenecid, benzbromarone, indomethacin), known to interfere with glutathione conjugate transport of human ABCC1 and ABCC2, inhibited the ABCC6-mediated NEM-GS transport in a specific manner, indicating that ABCC6 has a unique substrate specificity. We have also expressed three missense mutant forms of ABCC6, which have recently been shown to cause PXE. MgATP binding was normal in these proteins; ATP-dependent NEM-GS or leukotriene C(4) transport, however, was abolished. Our data indicate that human ABCC6 is a primary active transporter for organic anions. In the three ABCC6 mutant forms examined, the loss of transport activity suggests that these mutations result in a PXE phenotype through a direct influence on the transport activity of this ABC transporter.
authors.affiliation: Institute of Enzymology, Hungarian Academy of Sciences, 1113 Budapest, Hungary.
authors.forename: Attila
authors.initials: A
authors.lastname: Iliás
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Urbán
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Seidl
authors.forename: Olivier
authors.initials: O
authors.lastname: Le Saux
authors.forename: Emese
authors.initials: E
authors.lastname: Sinkó
authors.forename: Charles D
authors.initials: CD
authors.lastname: Boyd
authors.forename: Balázs
authors.initials: B
authors.lastname: Sarkadi
authors.forename: András
authors.initials: A
authors.lastname: Váradi
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015723:Gene Library; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007313:Insecta; D007700:Kinetics; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D020125:Mutation, Missense; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011561:Pseudoxanthoma Elasticum / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic
chemical_list: D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D018076:DNA, Complementary; D005978:Glutathione; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009711:Nucleotides; C000715087:ABCC2 protein, human; C102029:ABCC6 protein, human