Functional and molecular characterization of the K-Cl cotransporter of Xenopus laevis oocytes.
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(4)(3-) = 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 microM 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).
Mercado
A
A
Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Universidad Nacional Autónoma de México, Tlalpan 14000, Mexico City, Mexico.
de los Heros
P
P
Vázquez
N
N
Meade
P
P
Mount
D B
DB
Gamba
G
G
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D000838:Anions / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D027981:Symporters; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*; D000096922:K Cl- Cotransporters
D000838:Anions; D002352:Carrier Proteins; D012333:RNA, Messenger; D027981:Symporters; D000096922:K Cl- Cotransporters