Functional and molecular characterization of the K-Cl cotransporter of Xenopus laevis oocytes

1 Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tlalpan 14000, Mexico City, Mexico; and 2 Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232 The K-Cl cotransporters (KCCs) have a broad range of physiological roles, in a number of cells and species. We report here that Xenopus laevis oocytes express a K-Cl cotransporter with significant functional and molecular similarity to mammalian KCCs. Under isotonic conditions, defolliculated oocytes exhibit a Cl -dependent 86 Rb + uptake mechanism after activation by the cysteine-reactive compounds N -ethylmaleimide (NEM) and mercuric chloride (HgCl 2 ). The activation of this K-Cl cotransporter by cell swelling is prevented by inhibition of protein phosphatase-1 with calyculin A; NEM activation of the transporter was not blocked by phosphatase inhibition. Kinetic characterization reveals apparent values for the Michaelis-Menten constant of 27.7 ± 3.0 and 15.4 ± 4.7 mM for Rb + and Cl , respectively, with an anion selectivity for K + transport of Cl = PO = Br > I > SCN > gluconate. The oocyte K-Cl cotransporter was sensitive to several inhibitors, including loop diuretics, with apparent half-maximal inhibition values of 200 and 500 µM for furosemide and bumetanide, respectively. A partial cDNA encoding the Xenopus K-Cl cotransporter was cloned from oocyte RNA; the corresponding transcript is widely expressed in Xenopus tissues. The predicted COOH-terminal protein fragment exhibited particular homology to the KCC1/KCC3 subgroup of the mammalian KCCs, and the functional characteristics are the most similar to those of KCC1 (Mercado A, Song L, Vazquez N, Mount DB, and Gamba G. J Biol Chem 275: 30326-30334, 2000). potassium-chloride cotransport; cell volume; cell swelling