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 which apical NKCC2 activity is matched to basolateral Cl exit through changes in [Cl]i. Reverse transcriptase-polymerase chain reaction of HEK-293 cell mRNA is positive with NKCC1 primers and negative with NKCC2 primers. Surprisingly, we found that the behavior of the endogenous HEK cell Na-K-Cl cotransporter is unlike either of the two forms which have been described: compared with NKCC1, HEK cell cotransporter has a 2.5-fold lower Na affinity, an 8-fold lower Rb affinity, and a 4-fold higher bumetanide affinity. These results suggest the presence of a novel isoform of NKCC in HEK-293 cells.
Highlights
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
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 N terminus of the cation-chloride cotransporters is very poorly conserved across isoforms and species [3, 24], and we have shown that it does not play a role in sNKCC1/hNKCC1 ion affinity differences [21]
Summary
The Chimera h1r2A0.7—h1r2A0.7 is composed of the entire coding region of the renal Na-K-Cl cotransporter (rNKCC2A), except that two-thirds of the N terminus is replaced by the corresponding region in hNKCC1 (Fig. 1). A common NcoI restriction site in hNKCC1 and rNKCC2A was used to create the chimeric junction 0.7 (Thr217/Met218 in hNKCC1 and Thr104/Met105 in rNKCC2A) This site occurs in a conserved region in the N terminus, 75 amino acids before the first putative transmembrane domain. Cell Lines—Control HEK cells, mock-transfected cells, and lines stably expressing sNKCC1 and hNKCC1 were the same as in Ref. 21. H1r2A0.7 cDNA was transfected into HEK cells by calcium phosphate precipitation, and stable lines were isolated by G-418 resistance, as described previously [6, 21]. Several experiments with hNKCC1 were performed with a different line reported in Ref. 6, with indistinguishable results. All experiments with sNKCC1 were carried out with the cell line described in Ref. 21. Samples of PCR reactions were analyzed on a 1.2% agarose gel stained with ethidium bromide (10 g/ml)
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