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J Biol Chem, Vol. 273, Issue 18, 11295-11301, May 1, 1998
From the Cellular & Molecular Physiology Department, Yale
University, New Haven, Connecticut 06510
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.
Comparison of Na-K-Cl Cotransporters
NKCC1, NKCC2, AND THE HEK CELL Na-K-Cl COTRANSPORTER
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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