Comparison of Na-K-Cl cotransporters. NKCC1, NKCC2, and the HEK cell Na-L-Cl cotransporter.

The Journal of Biological Chemistry
Paul IsenringBiff Forbush

Abstract

The Na-K-Cl cotransporter (NKCC) mediates the coupled movement of ions into most animal cells, playing important roles in maintenance of cell volume and in epithelial Cl transport. Two forms of NKCC have been described: NKCC1, the "housekeeping" isoform that is also responsible for Cl accumulation in secretory epithelial cells, and NKCC2, which mediates apical Na+K+Cl entry into renal epithelial cells. Here we examine the kinetic properties of NKCC1, NKCC2, and the endogenous HEK-293 cell cotransporter. Stable expression of rabbit NKCC2A was obtained in HEK-293 cells utilizing a chimera (h1r2A0.7) in which the 5'-untranslated region and cDNA encoding 104 amino acids of the N terminus are replaced by the corresponding sequence of NKCC1. h1r2A0.7 exhibits Na and Cl affinities near those of NKCC1, but it has a 4-fold lower Rb affinity, and a 3-fold higher affinity for the inhibitor bumetanide. The activity of h1r2A0.7 is increased on incubation in low [Cl] media as is NKCC1, but the resting level of activity is higher in h1r2A0.7 and activation is more rapid. h1r2A0.7 exhibits an appropriate volume response, unlike NKCC1 for which concomitant changes in [Cl]i appear to be the overriding factor. These results support a model in whi...Continue Reading

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