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Vol. 273, Issue 5, C1516-C1525, November 1997
Department of Human Physiology, University of California, School of Medicine, Davis, California 95616
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 1 mM
N-ethylmaleimide (NEM) but not by cell
swelling. Both furosemide [inhibition constant (Ki) ~25
µM] and bumetanide (Ki
~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
(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.
furosemide; N-ethylmaleimide; external potassium homeostasis; postsynaptic inhibition
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