51. Electrophysiologic characterization of an organic anion transporter cloned from winter flounder kidney (fROAT).
- Author
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Burckhardt BC, Wolff NA, and Burckhardt G
- Subjects
- Animals, Anion Transport Proteins, Biological Transport, Active physiology, Bumetanide pharmacology, Cells, Cultured, Clone Cells, Dose-Response Relationship, Drug, Flounder, Furosemide pharmacology, Membrane Potentials drug effects, Membrane Potentials physiology, Membrane Transport Proteins drug effects, Membrane Transport Proteins metabolism, Oocytes physiology, Patch-Clamp Techniques, Probenecid pharmacology, Substrate Specificity, Xenopus laevis, Carrier Proteins drug effects, Carrier Proteins metabolism, Dicarboxylic Acid Transporters, Diuretics pharmacology, Escherichia coli Proteins, Kidney Tubules, Proximal physiology, p-Aminohippuric Acid pharmacokinetics, p-Aminohippuric Acid pharmacology
- 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.
- Published
- 2000
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