Electrophysiologic characterization of an organic anion transporter cloned from winter flounder kidney (fROAT)

• Published in: Journal of the American Society of Nephrology 2000, Vol.11 (1), p.9-17
• Main Authors: BURCKHARDT, B. C, WOLFF, N. A, BURCKHARDT, G
• Format: Article
• Language: English
• Subjects: Animals; Anion Transport Proteins; Biological and medical sciences; Biological Transport, Active - physiology; Bumetanide - pharmacology; Carrier Proteins - drug effects; Carrier Proteins - metabolism; Cells, Cultured; Clone Cells; Dicarboxylic Acid Transporters; Diuretics - pharmacology; Dose-Response Relationship, Drug; Escherichia coli Proteins; Flounder; Fundamental and applied biological sciences. Psychology; Furosemide - pharmacology; Kidney Tubules, Proximal - physiology; Membrane Potentials - drug effects; Membrane Potentials - physiology; Membrane Transport Proteins - drug effects; Membrane Transport Proteins - metabolism; Oocytes - physiology; p-Aminohippuric Acid - pharmacokinetics; p-Aminohippuric Acid - pharmacology; Patch-Clamp Techniques; Probenecid - pharmacology; Substrate Specificity; Vertebrates: urinary system; Xenopus laevis
• ISSN: 1046-6673; 1533-3450
• DOI: 10.1681/ASN.V1119

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.