To study the function of the Na+-K+-2Cl– cotransporter-1 (NKCC1), we inserted the 3.7 kb open reading frame of the mouse NKCC1 cDNA into pBF, a Xenopus laevis oocyte expression vector.
Xenopus frogs were anesthetized using 0.17 % tricaine in water. All surgeries were approved by the Vanderbilt University Institutional Animal Care and Use Committee. NKCC1 function was assessed via 86Rb-uptakes in oocytes exposed to isotonic (200 mosmol l-1), hypertonic (260 mosmol l-1), or isotonic-low Cl– solutions.
Experiments performed 1-6 days post-RNA injection revealed a steady increase of the K+ flux up to day 4, the time at which the flux reaches a plateau. Injection of various amounts of cRNA ranging from 0.03 to 30 ng revealed saturation at 16 ng. K+ flux times ranging from 5 to 180 min revealed that the oocyte volume was large enough for the specific activity not to reach equilibrium after a 3 h flux. When measured in isosmotic condition, the ouabain-resistant K+ flux in water-injected oocytes was relatively small, compared to the flux measured with NKCC1-injected oocytes: 1270 ± 91 (mean ± S.E.M., n = 20) versus 5057 ± 281 pmol K+ oocyte-1 h-1 (n = 20), respectively. Under hypertonic conditions, the K+ flux was markedly activated in NKCC1-injected oocytes (20 693 ± 882 pmol K+ oocyte-1 h-1, n = 20), whereas it was only slightly increased in water-injected oocytes. When the oocytes were incubated for 24 h in 6 mM Cl– compared to 80 mM, the NKCC1 flux in isotonic solution was increased to 12 819 ± 428 pmol K+ oocyte-1 h-1, n = 20. The entire NKCC1-mediated K+ flux was inhibited by 50 mM bumetanide. When the uptake was performed at temperatures ranging from 10 °C to 34 °C, a sharp increase in hypertonically induced NKCC1 flux was observed between 10 °C and 20 °C, followed by a lesser increase thereafter. This temperature effect, leading to a non-linear Arrhenius plot showing an abrupt change in activation energy around 20-23 °C, suggests a significant effect of lipid phase transition. To examine the role of oxidative stress on NKCC1 function, we tested the effects of arsenite (1 mM) and ascorbic acid (500 µM). Arsenite completely abolished NKCC1 flux, whereas ascorbic acid showed no effect on NKCC1 function. Finally, the specific inhibitor of the p38 MAP kinase pathway, SB203580, did not affect NKCC1 function, as measured in isotonic, hypertonic, or low Cl– conditions.
This work was supported by NIH grant NS36758.