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

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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 ~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 i mM N-ethylmaleimide (NEM) but not by cell swelling. Both furosemide [inhibition constant (K(i)) ~25 μM] and bumetanide (K(i) ~55 μM) 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 (K(m)) = 5.2 ± 0.9 (SE) mM; n = 5] but a low apparent affinity for external Cl- (K(m) >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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume273
Issue number5 42-5
StatePublished - 1997

Fingerprint

HEK293 Cells
Ethylmaleimide
Cells
Protein Isoforms
Bumetanide
Furosemide
Diuretics
Neurons
Swelling
Epitopes
Brain
Glycoproteins
Ligand-Gated Ion Channels
Thermodynamics
Ligands
Kinetics
Maintenance
potassium-chloride symporters
Kidney
Cell Line

Keywords

  • External potassium homeostasis
  • Furosemide
  • N-ethytmaleimide
  • Postsynaptic inhibition

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Physiology (medical)

Cite this

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title = "Functional characterization of the neuronal-specific K-Cl cotransporter: Implications for [K+](o) regulation",
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 ~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 i mM N-ethylmaleimide (NEM) but not by cell swelling. Both furosemide [inhibition constant (K(i)) ~25 μM] and bumetanide (K(i) ~55 μM) 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 (K(m)) = 5.2 ± 0.9 (SE) mM; n = 5] but a low apparent affinity for external Cl- (K(m) >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.",
keywords = "External potassium homeostasis, Furosemide, N-ethytmaleimide, Postsynaptic inhibition",
author = "Payne, {John A}",
year = "1997",
language = "English (US)",
volume = "273",
journal = "American Journal of Physiology - Renal Fluid and Electrolyte Physiology",
issn = "1931-857X",
publisher = "American Physiological Society",
number = "5 42-5",

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TY - JOUR

T1 - Functional characterization of the neuronal-specific K-Cl cotransporter

T2 - Implications for [K+](o) regulation

AU - Payne, John A

PY - 1997

Y1 - 1997

N2 - 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 ~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 i mM N-ethylmaleimide (NEM) but not by cell swelling. Both furosemide [inhibition constant (K(i)) ~25 μM] and bumetanide (K(i) ~55 μM) 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 (K(m)) = 5.2 ± 0.9 (SE) mM; n = 5] but a low apparent affinity for external Cl- (K(m) >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.

AB - 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 ~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 i mM N-ethylmaleimide (NEM) but not by cell swelling. Both furosemide [inhibition constant (K(i)) ~25 μM] and bumetanide (K(i) ~55 μM) 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 (K(m)) = 5.2 ± 0.9 (SE) mM; n = 5] but a low apparent affinity for external Cl- (K(m) >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.

KW - External potassium homeostasis

KW - Furosemide

KW - N-ethytmaleimide

KW - Postsynaptic inhibition

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