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

Paul Isenring, Steven C. Jacoby, John A Payne, Bliss Forbush

Research output: Contribution to journalArticle

104 Citations (Scopus)

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 (h1r(2A)0.7) in which the 5'-untranslated region and cDNA encoding 104 amino acids of the N terminus are replaced by the corresponding sequence of NKCC1. h1r(2A)0.7 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 h1r(2A)0.7 is increased on incubation in low [Cl] media as is NKCC1, but the resting level of activity is higher in h1r(2A)0.7 and activation is more rapid. h1r(2A)0.7 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.

Original languageEnglish (US)
Pages (from-to)11295-11301
Number of pages7
JournalJournal of Biological Chemistry
Volume273
Issue number18
DOIs
StatePublished - May 1 1998
Externally publishedYes

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Sodium-Potassium-Chloride Symporters
HEK293 Cells
Bumetanide
Protein Isoforms
Epithelial Cells
Housekeeping
5' Untranslated Regions
Polymerase chain reaction
RNA-Directed DNA Polymerase
Reverse Transcriptase Polymerase Chain Reaction
Cell Size
Animals
Complementary DNA
Chemical activation
Cells
Maintenance
Ions
Rabbits
Kidney
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Comparison of Na-K-Cl cotransporters : NKCC1, NKCC2, and the HEK cell Na- K-Cl cotransporter. / Isenring, Paul; Jacoby, Steven C.; Payne, John A; Forbush, Bliss.

In: Journal of Biological Chemistry, Vol. 273, No. 18, 01.05.1998, p. 11295-11301.

Research output: Contribution to journalArticle

Isenring, Paul ; Jacoby, Steven C. ; Payne, John A ; Forbush, Bliss. / Comparison of Na-K-Cl cotransporters : NKCC1, NKCC2, and the HEK cell Na- K-Cl cotransporter. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 18. pp. 11295-11301.
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