title: Electrophysiologic characterization of an organic anion transporter cloned from winter flounder kidney (fROAT).
abstract: The two-electrode voltage clamp technique was used to demonstrate translocation of p-aminohippurate (PAH) and related compounds such as loop diuretics in Xenopus laevis oocytes expressing the renal organic anion transporter from winter flounder kidney (fROAT). In fROAT-expressing oocytes, PAH (0.1 mM) induced a depolarization of 4.2 +/- 0.4 mV and at a holding potential of -60 mV an inward current of -22.6 +/- 3.5 nA. PAH-induced current and the current calculated from [3H]-PAH uptake were of similar magnitude. Depolarization, inward current, and current-to-uptake relation indicated exchange of the monovalent PAH with a divalent anion, possibly alpha-ketoglutarate (alpha-KG), causing net efflux of one negative charge. The kinetic analysis of PAH-induced currents revealed that translocation is dependent on membrane potential, saturable with an apparent Km of 58 +/- 8 microM, and sensitive to probenecid and furosemide. In contrast to probenecid and furosemide, the loop diuretics bumetanide, ethacrynic acid, and tienilic acid and the nephrotoxic mycotoxin ochratoxin A elicited inward currents indicating translocation through fROAT. Substrate-dependent currents provide a tool to elucidate the structure/function relationship of the renal organic anion transporter.
authors.affiliation: Zentrum Physiologie und Pathophysiologie, Georg-August Universität Göttingen, Göttingen, Germany.
authors.forename: Birgitta Christina
authors.initials: BC
authors.lastname: Burckhardt
authors.affiliation: Zentrum Physiologie und Pathophysiologie, Georg-August Universität Göttingen, Göttingen, Germany.
authors.forename: Natascha A
authors.initials: NA
authors.lastname: Wolff
authors.affiliation: Zentrum Physiologie und Pathophysiologie, Georg-August Universität Göttingen, Göttingen, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Burckhardt
mesh_terms: D000818:Animals; D027321:Anion Transport Proteins; D001693:Biological Transport, Active / Q000502:physiology; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000187:drug effects* / Q000378:metabolism*; D002478:Cells, Cultured; D002999:Clone Cells; D029382:Dicarboxylic Acid Transporters; D004232:Diuretics / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins; D005432:Flounder; D005665:Furosemide / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D026901:Membrane Transport Proteins / Q000187:drug effects / Q000378:metabolism; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D011339:Probenecid / Q000494:pharmacology; D013379:Substrate Specificity; D014982:Xenopus laevis; D010130:p-Aminohippuric Acid / Q000493:pharmacokinetics* / Q000494:pharmacology*
chemical_list: D027321:Anion Transport Proteins; D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters; D004232:Diuretics; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D002034:Bumetanide; C068628:KgtP protein, E coli; D005665:Furosemide; D011339:Probenecid; D010130:p-Aminohippuric Acid