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Electrophysiologic Characterization of an Organic Anion Transporter Cloned from Winter Flounder Kidney (fROAT)

Zentrum Physiologie und Pathophysiologie, Georg-August Universität Göttingen, Göttingen, Germany.

Correspondence to Dr. Birgitta-Christina Burckhardt, Zentrum Physiologie und Pathophysiologie, Georg-August Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany. Phone: +49 551 395880; Fax: +49 551 395883; E-mail: bcburckhardt{at}veg-physiol.med.uni-goettingen.de

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 -ketoglutarate (-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 K_m of 58 ± 8 µM, 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.

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