Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation

Am J Physiol. 1997 Nov;273(5):C1516-25. doi: 10.1152/ajpcell.1997.273.5.C1516.

Abstract

The neuronal K-Cl cotransporter isoform (KCC2) was functionally expressed in human embryonic kidney (HEK-293) cell lines. Two stably transfected HEK-293 cell lines were prepared: one expressing an epitope-tagged KCC2 (KCC2-22T) and another expressing the unaltered KCC2 (KCC2-9). The KCC2-22T cells produced a glycoprotein of approximately 150 kDa that was absent from HEK-293 control cells. The 86Rb influx in both cell lines was significantly greater than untransfected control HEK-293 cells. The KCC2-9 cells displayed a constitutively active 86Rb influx that could be increased further by 1 mM N-ethylmaleimide (NEM) but not by cell swelling. Both furosemide [inhibition constant (Ki) approximately 25 microM] and bumetanide (Ki approximately 55 microM) inhibited the NEM-stimulated 86Rb influx in the KCC2-9 cells. This diuretic-sensitive 86Rb influx in the KCC2-9 cells, operationally defined as KCC2 mediated, required external Cl- but not external Na+ and exhibited a high apparent affinity for external Rb+(K+) [Michaelis constant (Km) = 5.2 +/- 0.9 (SE) mM; n = 5] but a low apparent affinity for external Cl- (Km > 50 mM). On the basis of thermodynamic considerations as well as the unique kinetic properties of the KCC2 isoform, it is hypothesized that KCC2 may serve a dual function in neurons: 1) the maintenance of low intracellular Cl- concentration so as to allow Cl- influx via ligand-gated Cl- channels and 2) the buffering of external K+ concentration ([K+]o) in the brain.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biological Transport / drug effects
  • Brain / metabolism*
  • Bumetanide / pharmacology
  • Carrier Proteins / biosynthesis
  • Carrier Proteins / chemistry
  • Carrier Proteins / physiology*
  • Cell Line
  • Cell Membrane / physiology
  • Ethylmaleimide / pharmacology
  • Furosemide / pharmacology
  • Homeostasis
  • Humans
  • Hypotonic Solutions
  • Kidney
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Neurons / metabolism*
  • Polymerase Chain Reaction
  • Potassium / metabolism*
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Rubidium / pharmacokinetics
  • Rubidium Radioisotopes
  • Symporters*
  • Thermodynamics
  • Transfection

Substances

  • Carrier Proteins
  • Hypotonic Solutions
  • Recombinant Proteins
  • Rubidium Radioisotopes
  • Symporters
  • potassium-chloride symporters
  • Bumetanide
  • Furosemide
  • Rubidium
  • Ethylmaleimide
  • Potassium