Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

Am J Physiol. 1999 Feb;276(2):C328-36. doi: 10.1152/ajpcell.1999.276.2.C328.


We have studied the regulation of the K-Cl cotransporter KCC1 and its functional interaction with the Na-K-Cl cotransporter. K-Cl cotransporter activity was substantially activated in HEK-293 cells overexpressing KCC1 (KCC1-HEK) by hypotonic cell swelling, 50 mM external K, and pretreatment with N-ethylmaleimide (NEM). Bumetanide inhibited 86Rb efflux in KCC1-HEK cells after cell swelling [inhibition constant (Ki) approximately 190 microM] and pretreatment with NEM (Ki approximately 60 microM). Thus regulation of KCC1 is consistent with properties of the red cell K-Cl cotransporter. To investigate functional interactions between K-Cl and Na-K-Cl cotransporters, we studied the relationship between Na-K-Cl cotransporter activation and intracellular Cl concentration ([Cl]i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activity than controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells was activated <2-fold by low-Cl hypotonic prestimulation, compared with 10-fold activation in HEK-293 cells and >20-fold activation in cells overexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cells had lower resting [Cl]i than HEK-293 cells; cell volume was not different among cell lines. We found a steep relationship between [Cl]i and Na-K-Cl cotransport activity within the physiological range, supporting a primary role for [Cl]i in activation of Na-K-Cl cotransport and in apical-basolateral cross talk in ion-transporting epithelia.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Carrier Proteins / antagonists & inhibitors
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Line / cytology
  • Cell Line / metabolism
  • Chlorides / administration & dosage
  • Chlorides / metabolism
  • Ethylmaleimide / pharmacology
  • Humans
  • Hypotonic Solutions / pharmacology
  • Intracellular Membranes / metabolism
  • K Cl- Cotransporters
  • Osmolar Concentration
  • Rabbits
  • Rubidium / metabolism
  • Sodium-Potassium-Chloride Symporters
  • Symporters*
  • Transfection


  • Carrier Proteins
  • Chlorides
  • Hypotonic Solutions
  • Sodium-Potassium-Chloride Symporters
  • Symporters
  • Rubidium
  • Ethylmaleimide