title: Cystinuria-specific rBAT(R365W) mutation reveals two translocation pathways in the amino acid transporter rBAT-b0,+AT.
abstract: Apical reabsorption of dibasic amino acids and cystine in kidney is mediated by the heteromeric amino acid antiporter rBAT/b(0,+)AT (system b(0,+)). Mutations in rBAT cause cystinuria type A, whereas mutations in b(0,+)AT cause cystinuria type B. b(0,+)AT is the catalytic subunit, whereas it is believed that rBAT helps the routing of the rBAT/b(0,+)AT heterodimeric complex to the plasma membrane. In the present study, we have functionally characterized the cystinuria-specific R365W (Arg(365)-->Trp) mutation of human rBAT, which in addition to a trafficking defect, alters functional properties of the b(0,+) transporter. In oocytes, where human rBAT interacts with the endogenous b(0,+)AT subunit to form an active transporter, the rBAT(R365W) mutation caused a defect of arginine efflux without altering arginine influx or apparent affinities for intracellular or extracellular arginine. Transport of lysine or leucine remained unaffected. In HeLa cells, functional expression of rBAT(R365W)/b(0,+)AT was observed only at the permissive temperature of 33 degrees C. Under these conditions, the mutated transporter showed 50% reduction of arginine influx and a similar decreased accumulation of dibasic amino acids. Efflux of arginine through the rBAT(R365W)/b(0,+)AT holotransporter was completely abolished. This supports a two-translocation-pathway model for antiporter b(0,+), in which the efflux pathway in the rBAT(R365W)/b(0,+)AT holotransporter is defective for arginine translocation or dissociation. This is the first direct evidence that mutations in rBAT may modify transport properties of system b(0,+).
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Barcelona Science Park, Barcelona 08028, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Pineda
authors.forename: Carsten A
authors.initials: CA
authors.lastname: Wagner
authors.forename: Angelika
authors.initials: A
authors.lastname: Bröer
authors.forename: Paul A
authors.initials: PA
authors.lastname: Stehberger
authors.forename: Simone
authors.initials: S
authors.lastname: Kaltenbach
authors.forename: Josep Ll
authors.initials: JL
authors.lastname: Gelpí
authors.forename: Rafael
authors.initials: R
authors.lastname: Martín Del Río
authors.forename: Antonio
authors.initials: A
authors.lastname: Zorzano
authors.forename: Manuel
authors.initials: M
authors.lastname: Palacín
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
authors.forename: Stefan
authors.initials: S
authors.lastname: Bröer
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics* / Q000502:physiology; D026962:Amino Acid Transport System ASC / Q000502:physiology; D026905:Amino Acid Transport Systems / Q000235:genetics / Q000502:physiology; D026923:Amino Acid Transport Systems, Basic; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology; D045744:Cell Line, Tumor; D003555:Cystinuria / Q000235:genetics* / Q000503:physiopathology; D005260:Female; D006367:HeLa Cells / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000502:physiology; D015778:Minor Histocompatibility Antigens; D009154:Mutation / Q000235:genetics* / Q000502:physiology; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014364:Tryptophan / Q000235:genetics; D014982:Xenopus laevis
chemical_list: D026962:Amino Acid Transport System ASC; D026905:Amino Acid Transport Systems; D026923:Amino Acid Transport Systems, Basic; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D015778:Minor Histocompatibility Antigens; C420506:SLC1A5 protein, human; C499489:SLC7A9 protein, human; D014364:Tryptophan; D001120:Arginine