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 language | English (US) |
|---|---|
| Journal | American Journal of Physiology - Cell Physiology |
| Volume | 273 |
| Issue number | 5 42-5 |
| State | Published - 1997 |
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Keywords
- External potassium homeostasis
- Furosemide
- N-ethytmaleimide
- Postsynaptic inhibition
ASJC Scopus subject areas
- Clinical Biochemistry
- Cell Biology
- Physiology
- Physiology (medical)
Cite this
Functional characterization of the neuronal-specific K-Cl cotransporter : Implications for [K+](o) regulation. / Payne, John A.
In: American Journal of Physiology - Cell Physiology, Vol. 273, No. 5 42-5, 1997.Research output: Contribution to journal › Article
}
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
UR - http://www.scopus.com/inward/record.url?scp=0030658056&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030658056&partnerID=8YFLogxK
M3 - Article
C2 - 9374636
AN - SCOPUS:0030658056
VL - 273
JO - American Journal of Physiology - Renal Fluid and Electrolyte Physiology
JF - American Journal of Physiology - Renal Fluid and Electrolyte Physiology
SN - 1931-857X
IS - 5 42-5
ER -