Functional characterization of the neuronal-specific K-Cl
cotransporter: implications for
[K+]o
regulation
John A.
Payne
Department of Human Physiology, University of California, School of
Medicine, Davis, California 95616
 |
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 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
 |
INTRODUCTION |
THE POTASSIUM-CHLORIDE COTRANSPORTER is an integral
membrane protein that mediates the obligatorily coupled, electrically neutral movement of K+ and
Cl
across the plasma
membranes of many animal cells. As an electroneutral transport
mechanism, the direction of net movement of
K+ and
Cl
by the cotransporter is
determined solely by the sum of the chemical potential differences of
the two ions. Under normal physiological conditions, the K-Cl
cotransporter is an efflux pathway with the direction of driving force
being dictated by the outwardly directed K+ chemical potential maintained
by the
Na+-K+-ATPase.
The K-Cl cotransporter is characterized by its sensitivity to the
sulfamoylbenzoic acid "loop" diuretics (e.g., furosemide and
bumetanide) and by its activation by cell swelling or the application
of the thiol alkylating reagent
N-ethylmaleimide (NEM). The K-Cl
cotransporter displays a slightly higher affinity for furosemide than
bumetanide; however, its loop diuretic affinities are significantly
lower than that of the Na-K-Cl cotransporter (4, 20). In red blood
cells, activation of K-Cl cotransport by cell swelling and by NEM has
been studied extensively, and both have been shown to depend on a
dephosphorylation event (9, 18, 19).
In most cells where K-Cl cotransport has been described, its primary
function appears to be the regulation of cell volume after swelling by
promoting an efflux of K+ and
Cl
and osmotically obliged
water. The K-Cl cotransporter also appears to be involved in the
vectorial movement of salt and water across certain epithelia (e.g.,
Refs. 12, 31). A unique function of the K-Cl cotransporter has been
proposed in neurons (e.g., Refs. 1, 3, 18a, 34). Postsynaptic
inhibition involves the conductive movement of
Cl
through ligand-gated
anion channels, i.e.,
-aminobutyric
acidA (GABAA) and glycine receptors.
For these receptors to mediate postsynaptic inhibition, an inwardly
directed Cl
electrochemical
gradient must be maintained so that an influx of
Cl
through the
receptor-channel complex hyperpolarizes the postsynaptic membrane
(i.e., inhibitory postsynaptic potential; IPSP) and stabilizes the
membrane potential
(Em) at or near
the equilibrium potential for
Cl
(ECl). A
neuronal K-Cl cotransporter appears to function as the "active"
Cl
extrusion pathway that
maintains ECl < Em, as required
for the IPSP (for reviews, see Refs. 3, 18a).
Recently, we reported the molecular characterization of two distinct
isoforms of the K-Cl cotransporter (KCC1, Ref. 10; KCC2, Ref. 28).
These two K-Cl cotransporters exhibit ~67% amino acid identity over
their full length and display distinctly different tissue expression
patterns. KCC1 was present in all tissues examined and appears to be a
"housekeeping" isoform involved in cell volume regulation.
Because KCC1 is highly expressed in tissues like kidney and lung, it is
likely to be the isoform involved in epithelial salt transport as well.
In contrast, KCC2 was found only in the brain where it is expressed in
great abundance. Reverse transcriptase-polymerase chain reaction (PCR)
and in situ hybridization studies indicate that within the central
nervous system KCC2 is a neuronal-specific isoform (28). Based on its
abundant neuronal expression, we proposed that KCC2 was likely the
outwardly directed "Cl
pump" of neurons (28). Although KCC1 has been clearly shown to
mediate K-Cl cotransport, KCC2 has not yet been functionally characterized. In the present report, I functionally characterize KCC2
in stable human embryonic kidney (HEK-293) cell lines and demonstrate
that it displays the basic transport features of a K-Cl cotransport
system. The functional analysis revealed, however, that the expressed
KCC2 protein exhibits a very high apparent affinity for external
Rb+(K+),
Michaelis constant
(Km) ~5 mM.
This high affinity for external Rb+(K+)
as well as thermodynamic considerations indicate that in addition to
the maintenance of low neuronal
Cl
, KCC2 may serve to help
buffer external K+ concentration
([K+]o)
in the brain.
A preliminary report of these results has been presented in abstract
form (25).
 |
MATERIALS AND METHODS |
Preparation of expression constructs.
Two KCC2 expression constructs were prepared from a full-length rat
brain cDNA clone (5ERB14; see Ref. 28). This cDNA clone (rtKCC2:
nucleotides
69 to 3635) lacks much of the 3'-untranslated region of the KCC2 sequence. One full-length expression construct was
prepared by subcloning the 5ERB14 cDNA into the mammalian expression
vector, pJB20, at EcoR I and
Kpn I restriction sites of the
polylinker. Another full-length expression construct was prepared by
PCR mutagenesis to contain the c-myc
epitope at the NH2 terminus of the
KCC2 protein. The NH2 terminus is
a poorly conserved region between the two K-Cl cotransporter isoforms
as well as between all members of the cation-chloride cotransporter family. We have proposed that this region is not involved in ion translocation (29) and has limited functional significance (10). Therefore, it has proven to be a useful site for epitope addition on
the cation-chloride cotransporters (10).
PCR mutagenesis was performed using 5ERB14 as template and
synthetic oligonucleotide primers. The reverse primer
(5'-GCAGAAAGAGGATAACACCAAAGAT-3') was used without
purification. The 64-mer forward primer (adding an initiating Met and
c-myc peptide epitope, EQKLISEEDL,
directly to the original initiating Met of the KCC2 protein) was double high-performance liquid chromatography purified. The 399-base pair (bp)
fragment was amplified in a reaction volume of 50 µl containing 10 ng
cDNA template, 50 µM forward and reverse primer, 0.25 mM dNTP, 5 mM
KCl, 10 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 9.0 at 25°C), 0.1% Triton
X-100, 2.5 mM MgCl2, and 1.5 U
Taq DNA polymerase (Promega). After
the reaction contents were heated for 1 min at 94°C, 30 cycles of
PCR were performed (each consisting of incubation of 30 s at 94°C,
30 s at 55°C, and 1 min at 72°C). The PCR product was then
cloned into the plasmid pCRII (Invitrogen) and sequenced in both
directions. After confirmation of the sequence, the PCR product and the
5ERB14 clone were ligated at a common
Bsu36 I site and subcloned into the
mammalian expression vector, pJB20, at
EcoR I and
Kpn I restriction sites of the
polylinker.
Stable expression in HEK-293 cells.
The human embryonic kidney cell line (HEK-293) was maintained in
Dulbecco's modified Eagle's medium (Cellgro) supplemented with 10%
fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 µg/ml)
in a humidified incubator (5% CO2
at 37°C). The two expression constructs were transfected into
separate HEK-293 cells by calcium phosphate precipitation using
previously described methods (29). After 3 wk of growth in 900 µg/ml
Geneticin (GIBCO), single resistant colonies were amplified and
screened by Western blot with a c-myc
epitope monoclonal antibody or by
86Rb influx assay (see
Functional 86Rb
flux assay).
Membrane protein identification and deglycosylation.
Prestained molecular weight markers (Amersham or Bio-Rad) and protein
samples from cell lystates or membrane preparations of stable cells
were boiled in sample buffer [3% sodium dodecyl sulfate (SDS),
50 mM Tris · HCl (pH 8.5), 12% sucrose, 0.01% Serva Blue G, 0.002% phenol red, and 15.5 mg/ml dithiothreitol] and then separated by SDS-polyacrylamide gel electrophoresis using a 7.5%
tricine gel system. Gels were electrophoretically transferred from
unstained gels to polyvinylidene difluoride (PVDF) membranes (Immobilon
P; Millipore) in transfer buffer [192 mM glycine, 25 mM Tris (pH
8.3), and 15% methanol] for 5 h at 50 V using a Bio-Rad Trans-Blot tank apparatus or 1 h at 25 V using a Bio-Rad Trans-Blot semi-dry apparatus. PVDF-bound protein was visualized by staining with
Coomassie brilliant blue R-250. The PVDF membrane was blocked in
phosphate-buffered saline (PBS)-milk (7% nonfat dry milk and 0.1%
Tween 20 in PBS, pH 7.4) for 1 h and then incubated in PBS-milk with
the mouse monoclonal c-myc peptide
antibody either overnight at 4°C or 1 h at 24°C. After three
10-min washes in PBS-milk, the membrane was incubated with secondary
antibody (horseradish peroxidase-conjugated goat anti-mouse
immunoglobulin G; Zymed) for 2 h at 24°C in PBS-milk. After three
washes in PBS-0.1% Tween 20, bound antibody was detected using
an enhanced chemiluminescence assay (NEN Renaissance
system).
To deglycosylate membrane protein, membranes were isolated by
differential centrifugation and denatured by boiling in 0.5% SDS. The
deglycosylation of the c-myc-tagged
KCC2 protein with N-glycosidase F
required prior denaturization with SDS. Deglycosylation was carried out
in a medium containing 0.5%
n-octylglucoside, 20 mM sodium
phosphate buffer (pH 8.0), 50 mM EDTA, protease inhibitors, and
N-glycosidase F (20 U/ml; Boehringer
Mannheim). The sample was incubated for 4 h at 37°C in a thermal
cycler. Enzymatic treatment was terminated by addition of
electrophoresis sample buffer supplemented with 6 M urea.
Functional 86Rb flux assay.
Flux experiments were performed at 24°C on 1-day postconfluent
cells grown in 96-well plates. Before flux measurement, cells were
washed free of growth media with three successive washes in control
flux media [135 mM NaCl, 5 mM RbCl, 1 mM
CaCl2, 1 mM MgCl2, 1 mM
Na2HPO4,
2 mM
Na2SO4,
3 mM glucose, 15 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid titrated to pH 7.4 with
N-methyl-D-glucamine
base, and 0.1 mM ouabain; final osmolality, ~290
mosmol/kgH2O] in the
presence or absence of 1 mM NEM. After being washed, the cells were
then preincubated for 15 min in the presence or absence of 1 mM NEM. After preincubation, the cells were brought up in the same media as in
the preincubation but containing 2 µCi/ml
86RbCl. In transfected cell lines,
the NEM-stimulated 86Rb uptake was
linear for 7 min, and 3-min influx assays were routinely performed to
obtain initial rates. In HEK-293 control cells,
86Rb influx was linear for 30 min,
and 10-min influx assays were performed to obtain initial rates.
86Rb influx was terminated by five
washes in Tris-buffered saline (pH 7.4) containing 2 mM furosemide.
Cells were solubilized in 2% SDS and assayed for
86Rb by Cerenkov radiation and for
protein by the MicroBCA method (Pierce). In ion substitution
experiments,
N-methyl-D-glucamine replaced sodium, whereas gluconate or methanesulfonate replaced chloride.
To assess the effect of swelling on the activity of KCC2, the cells
were exposed to a medium rendered hypotonic (230 mosmol/kgH2O) by the reduction of
NaCl to 95 mM. The isotonic control media was prepared by the addition
of sodium gluconate (final osmolality 290 mosmol/kgH2O). Thus the isotonic
and hypotonic media contained similar final
Cl
concentration
([Cl
]) (104 mM). Cells were preincubated for 15 min in hypotonic or isotonic media
before 86Rb flux analysis.
The ability of the loop diuretics furosemide and bumetanide as well as
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and
[(dihydroindenyl)oxy]alkanoic acid (DIOA) to inhibit the
KCC2-mediated 86Rb influx was
examined. In these experiments, the cells were washed and preincubated
for 15 min in control flux media containing 1 mM NEM and various
concentrations of inhibitor. The initial rate of
86Rb influx was then monitored in
fresh control flux media containing similar NEM and inhibitor
concentrations as in the preincubation media.
Data analysis.
A nonlinear iterative procedure (DeltaGraph, DeltaPoint, Monterey, CA)
was used in Fig. 7 to calculate the inhibition constant (Ki) for
furosemide and bumetanide inhibition. The data from each experiment
were fit to the equation J = Jmax
{(Jmax
Jmin)/(1 + Ki/[Inh])},
where J is the measured
86Rb influx,
Jmax is the
maximal 86Rb influx in the absence
of diuretic, Jmin
is the diuretic-insensitive 86Rb
influx, and [Inh] is the diuretic concentration. The
reported Ki
values are means ± SE of 4 separate experiments.
Results were analyzed statistically using either a
t-test or single-factor analysis of
variance in which experimental values were compared with control
measurements. Error bars are ±SE. Statistical significance was
defined as P < 0.05.
 |
RESULTS |
The neuronal-specific K-Cl cotransporter was functionally characterized
in stably transfected HEK-293 cell lines. Two full-length expression
constructs were prepared in the mammalian expression vector, pJB20. The
insert of one construct was prepared from unmodified KCC2 cDNA. The
other construct was prepared by PCR mutagenesis to contain an epitope
tag (c-myc peptide) at the
NH2 terminus of the KCC2 protein.
The two KCC2 constructs were transfected into separate sets of HEK-293
cells via CaPO4 coprecipitation. After 3 wk of growth in selection media, G418-resistant colonies were
screened by Western blot (c-myc-tagged
construct) or 86Rb influx
(untagged control construct). In screening the cell lines for
86Rb influx, the cells were
pretreated with NEM, an agent known to stimulate K-Cl cotransport in
red blood cells as well as KCC1 expressed in HEK-293 cells (10). Seven
cell lines expressing the unmutated control KCC2 construct and three
cell lines expressing the c-myc-tagged
KCC2 construct were successfully isolated. Due to their high levels of
expression, I chose to characterize more fully the KCC2-9
(untagged control) and KCC2-22T
(c-myc tagged) cell lines. Some of the
cell lines functionally expressing KCC2 are shown in Fig.
1.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Furosemide-sensitive 86Rb influx
of HEK-293 control cells (HEK) (A)
and stable HEK-293 cell lines expressing either epitope-tagged KCC2
protein (KCC2-#T) or unmutated KCC2 protein (KCC2-#)
(B). Influx measurements were made
after 15-min preincubation in media containing 1 mM
N-ethylmaleimide. Values are means ± SE of 6 replicates.
|
|
The KCC2-22T cell line was used to examine protein expression by
Western blot analysis. This cell line expressed a membrane glycoprotein
of 150 kDa that was completely absent from the untransfected control
cells (Fig. 2). The KCC2 glycoprotein was
similar in size to the KCC1 glycoprotein previously expressed in
HEK-293 cells (10). Presumably, the 150-kDa KCC2 glycoprotein is the
mature functional cotransporter delivered to the plasma membrane, since removal of the N-linked
oligosaccharides with N-glycosidase F reduced the protein to a size predicted from the cDNA (~125 kDa).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
Western blot analysis of membranes prepared from HEK-293 control cells
(HEK) or HEK-293 cells stably expressing epitope- tagged KCC2 protein
(KCC2-22T cells). Membranes were incubated with (+) or without
( ) N-glycosidase F for 4 h at
37°C. Epitope- tagged KCC2 protein was detected with
c-myc peptide monoclonal antibody.
|
|
In a number of preliminary experiments, both the KCC2-22T and
KCC2-9 cell lines provided very similar functional data,
substantiating the hypothesis that the
NH2-terminal epitope tag does not
alter the functional expression of the KCC2 protein (data not shown). Under control conditions, the 86Rb
influx in the KCC2-9 cell line was significantly greater than that
of HEK-293 control cells and was completely inhibited by 2 mM
furosemide (Fig.
3A). The
furosemide-sensitive 86Rb influx
was 6.2-fold higher in KCC2-9 cells (35.4 ± 1.8 nmol · mg
protein
1 · min
1)
than untransfected control cells (5.7 ± 1.1 nmol · mg
protein
1 · min
1).
Thus, as reported for KCC1, the KCC2 protein is capable of mediating a
86Rb flux that is active without
exogenous stimulation when transfected in HEK-293 cells.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 3.
86Rb influx of HEK-293 control
cells (HEK) or HEK-293 cells stably expressing KCC2 protein
(KCC2-9 cells) after treatment with 1 mM
N-ethylmaleimide (NEM)
(A) or with hypotonic swelling
(~230 mosmol/kgH2O)
(B) at constant external
[Cl ] (104 mM;
isotonicity was restored with sodium gluconate). Furosemide was applied
at 2 mM. Values are means ± SE of >3 experiments. * Significance from furosemide-sensitive control value
(P < 0.05; t-test).
|
|
The furosemide-sensitive 86Rb
influx in the KCC2-9 cells was stimulated ~1.6-fold by treatment
with 1 mM NEM (Fig. 3A). In
contrast, this flux was not significantly increased by cell swelling
(Fig. 3B). In these latter
experiments, external
[Cl
] was held
constant at 104 mM, and osmolarity was increased to isotonicity with
sodium gluconate (similar data were obtained with the permeant anion
methansulfonate; data not shown). This allowed me to directly compare
initial flux rates in isotonic and hypotonic conditions, but because
the affinity of the K-Cl cotransporter for external
[Cl
] is low
(see below), this resulted in a reduction in
86Rb influx (compare control
fluxes in Fig. 3, A and
B). After treatment with NEM, the
furosemide-sensitive 86Rb influx
was 20 times greater than that monitored in similarly treated
untransfected HEK-293 cells. As previously reported, the NEM
pretreatment significantly reduced the endogenous furosemide-sensitive 86Rb influx of control HEK-293
cells, which is due predominantly to an endogenous Na-K-Cl
cotransporter (10). Because this improved the ability to monitor the
expressed KCC2 protein in HEK-293 cells, NEM pretreatment was used in
all subsequent studies to characterize the furosemide-sensitive
86Rb influx in the KCC2-9
cell line. I operationally defined this NEM-stimulated
86Rb flux in the KCC2-9 cells
as KCC2 mediated.
Figure 4 presents the external ion
dependency of the KCC2-mediated
86Rb influx. In these experiments,
the cells were first preincubated in control flux media containing 1 mM
NEM for 15 min and then quickly washed three times in media lacking
either external Na+ (replaced by
N-methyl-D-glucamine)
or external Cl
(replaced by
gluconate). The furosemide-sensitive
86Rb influx was then monitored in
these same Na+- or
Cl
-free media. As expected
for a K-Cl cotransport system, the KCC2-mediated 86Rb influx required external
Cl
but not external
Na+.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Furosemide-sensitive 86Rb influx
in HEK-293 control cells (HEK) and HEK-293 cells stably expressing KCC2
protein (KCC2-9 cells). Cells were pretreated 15 min with 1 mM NEM
in control flux media and then quickly washed in control flux media
(A),
Na+-free
(N-methyl-D-glucamine
replacement) media (B), or
Cl -free (gluconate
replacement) media (C) immediately
before flux analysis. Values are means ± SE of 12 experiments.
* Significance from control value
(P < 0.05;
t-test).
|
|
Figure 5 shows the dependence of
KCC2-mediated 86Rb influx on
external Rb+ and
Cl
concentrations. We have
previously reported (10) that KCC1 expressed in HEK-293 cells has low
apparent affinities for external Rb+(K+)
[Michaelis constant
(Km) >25
mM] and external Cl
(Km >50 mM).
Although the furosemide-sensitive
86Rb uptake of the KCC2-9
cells also displayed a low apparent affinity for external
Cl
(Km >50 mM), it
exhibited a relatively high apparent affinity for external
Rb+(K+)
(Km = 5.2 ± 0.9 mM; n = 5). This represents at
least a fivefold higher affinity for external
Rb+(K+)
than that reported for the KCC1 isoform expressed in the same cell line
(10). This high apparent affinity for external
Rb+(K+)
exhibited by KCC2 is an important functional difference between the two
K-Cl cotransporter isoforms.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Dependence of initial rate of furosemide-sensitive
86Rb influx on external
Rb+ concentration
([Rb+]o)
(A) and external
Cl concentration
([Cl ]o)
(B) for HEK-293 cells stably
expressing KCC2 protein (KCC2-9 cells). Cells were pretreated 15 min with 1 mM NEM before flux analysis.
[Rb+]o
was replaced by
N-methyl-D-glucamine
while external Na+ was held at 100 mM.
[Cl ]o
was replaced by gluconate. Curves represent best fits of data to a
model of activation at single sites. Michaelis constant
(Km) values for
representative experiment are shown in each panel (summary data are
presented in text).
|
|
Furosemide and bumetanide inhibited the
86Rb influx in the KCC2-9
cells with Ki
values of 25 ± 3 µM (n = 4) and 55 ± 13 µM (n = 4),
respectively (Fig. 6). Similar
furosemide and bumetanide Ki values were
obtained for inhibition of the
86Rb uptake mediated by KCC1
expressed in HEK-293 cells (furosemide, 40 ± 4 µM; bumetanide, 59 ± 9 µM; Ref. 10). In addition to the loop diuretics,
a number of other compounds are known to inhibit K-Cl cotransport in
red blood cells (6, 35). These include the stilbene disulfonic acid
DIDS and the alkanoic acid DIOA. As shown in Fig.
7, both of these drugs significantly
inhibited the 86Rb uptake of
KCC2-9 cells; however, the inhibition by either drug at 100 µM
was not complete when compared with the furosemide inhibition (~80%
of the furosemide-sensitive influx).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Furosemide (A) and bumetanide
(B) inhibition of intial rate of
86Rb influx into HEK-293 cells
stably expressing KCC2 protein (KCC2-9 cells). Cells were
preincubated with various concentrations of loop diuretic and 1 mM NEM
for 15 min before flux analysis. Results were fit by a model of
inhibition at a single site. Summary data of 4 separate experiments are
shown in each panel (means ± SE).
|
|

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 7.
86Rb influx of HEK-293 cells
stably expressing KCC2 protein (KCC2-9 cells) after treatment with
2 mM furosemide (A) or DIDS or
[(dihydroindenyl)oxy]alkanoic acid (DIOA)
(B). Cells were preincubated 15 min
with 1 mM NEM and various inhibitors before flux analysis. Values are
means ± SE of >4 experiments. * Significance from control value (P < 0.05; analysis of
variance).
|
|
 |
DISCUSSION |
In a previous study, a K-Cl cotransporter (KCC1), displaying a
ubiquitous tissue distribution, was functionally characterized in
stable HEK-293 cells (10). Concurrent with the isolation of KCC1, we
identified a closely related but distinct gene product (KCC2) that was
specifically expressed in neurons throughout the central nervous system
(28). On the basis of its high identity to KCC1 (67%) and unique
tissue distribution, we proposed that this second gene product
represented a neuronal-specific isoform of the K-Cl cotransporter. In
the present study, I have functionally characterized KCC2 as a novel
K-Cl cotransporter and demonstrated that it exhibits unique functional
properties. These unique properties provide insight into potential
roles of KCC2 in ion homeostasis within the central nervous system.
Functional characterization of KCC2.
With the use of an epitope-tagged construct expressed in a stable
HEK-293 cell line, the biochemical characteristics of the KCC2 protein
were first examined. Like KCC1, KCC2 is a glycoprotein with a molecular
mass of ~150 kDa when expressed in HEK-293 cells. Treatment of
membranes prepared from the stable cell line with N-glycosidase F reduced the KCC2
protein to a mass of ~125 kDa, which is the size of the core KCC2
protein predicted from the cDNA. These data demonstrate that KCC2,
although an apparent neuronal-specific protein, is properly processed
and delivered to the plasma membrane of HEK-293 cells. As with all
other members of the cation-chloride cotransporter gene family, both
K-Cl cotransporter isoforms are glycoproteins. The region of the
primary sequence that likely harbors the oligosaccharides is the large
predicted extracellular loop between putative transmembrane segment
(TM) 5 and TM6 where both KCC isoforms contain four putative
glycosylation sites.
Confirmation that KCC2 is expressed at the cell surface and encodes a
K-Cl cotransporter protein was obtained from
86Rb flux analysis of the stable
HEK-293 cell lines. Cells expressing the unmutated KCC2 protein
(KCC2-9) exhibited a constitutively active furosemide-sensitive
86Rb influx that was approximately
sixfold greater than untransfected HEK-293 cells. K-Cl cotransport has
been well studied in vertebrate red blood cells where both cell
swelling and the alkylating reagent NEM have been shown to stimulate
this cation-chloride cotransporter (for review see Ref. 21). Hypotonic
swelling and NEM are also known to activate the KCC1 isoform when
stably expressed in HEK-293 cells (10). Much less is known about the
regulation of the KCC2 isoform. Unlike KCC1, the KCC2 isoform expressed
in HEK-293 cells was not stimulated by osmotic swelling. Like KCC1,
however, KCC2 activity was significantly increased by NEM treatment. It
is interesting to note that in the presence of NEM, the
furosemide-sensitive 86Rb influx
mediated by the KCC2-9 cells (56 ± 3 nmol · mg
protein
1 · min
1)
was substantially larger than that reported by Gillen et al. (10) for
KCC1-expressing cells (~1 nmol · mg
protein
1 · min
1).
This large difference could be explained by differences in protein
expression and/or transport kinetics. Because both studies used
5 mM Rb+ in the flux medium, it is
likely that the high external Rb+
affinity exhibited by KCC2 contributed significantly to its larger 86Rb influx.
The KCC2-mediated 86Rb influx
displayed many functional characteristics of a K-Cl cotransport
protein. For example, it exhibited complete dependence on the presence
of external Cl
but not
external Na+. Additionally, the
NEM-stimulated 86Rb influx was
inhibited by the loop diuretics with a greater sensitivity to
furosemide than bumetanide. Other known inhibitors of K-Cl cotransport
such as the stilbene disulfonic acid DIDS (6) and the alkanoic acid
DIOA (35) significantly reduced the KCC2-mediated 86Rb influx. Furthermore, the
KCC2-mediated 86Rb influx
displayed simple Michaelis-Menten kinetics with respect to external
[Rb+] and
[Cl
], and such
data are consistent with the electroneutral cotransport of
1Rb+(K+):1Cl
.
Involvement of putative TM2 in ion binding.
The molecular and functional characterization of numerous members of
the cation-chloride cotransporter gene family has provided important
information concerning structure-function relationships of these
transporters. One region that has received particular attention is
putative TM2 of the Na-K-Cl cotransporter (NKCC). This region has been
implicated in forming part of the cation binding site on the Na-K-Cl
cotransporter (17), and it is reasonable to suspect that structural
differences in TM2 help explain the large measured differences in ion
affinities between the shark rectal gland and human colonic Na-K-Cl
cotransporters (29). Furthermore, this region is encoded by an
alternatively spliced 96-bp cassette exon in the absorptive isoform of
the mammalian kidney, leading to three distinct splice variants with
differing TM2 segments (NKCC2a-b-f; see Refs. 15, 26). Remarkably,
these NKCC2 splice variants are restricted to the thick ascending limb of the loop of Henle (TALH) where they display a distinct axial heterogeneity (15). The presence of variant Na-K-Cl cotransporters possessing different ion affinities within the TALH has important functional implications for NaCl reabsorption in the kidney (see Ref.
27).
In comparing the primary structures of KCC1 and KCC2, not surprisingly
TM2 is the most divergent predicted transmembrane segment, sharing only
60% amino acid identity. This divergence is almost entirely at the
postulated extracellular side of TM2 (Fig.
8). Seven of the remaining eleven TMs
display >90% amino acid identity, including TM1, -3, -6, -8, -10, -11, and -12. The large difference in apparent affinity for external
Rb+(K+)
and the low TM2 identity are consistent with the hypothesis that among
the cation-chloride cotransporters TM2 is involved in cation binding.
However, as the KCC proteins do not transport Na+, TM2 of the K-Cl
cotransporters cannot be involved in
Na+ binding. One residue in KCC2
of particular interest is the glutamic acid predicted to be at the
extracellular side of TM2 (Fig. 8). This residue is replaced by
glutamine in KCC1. Thus it is interesting to speculate that the
negative charge of the glutamic acid side chain might be involved in
providing a K+ binding site in
KCC2 with a significantly higher affinity than that in KCC1.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 8.
Hypothetical model of K-Cl cotransporters and amino acid alignment of
putative transmembrane segment 2 (TM2) of two K-Cl cotransporter isoforms (KCC1 and KCC2). Asterisk above alignment denotes glutamic acid (E) at extracellular "mouth" of predicted TM2 of KCC2 that is replaced by a glutamine (Q) in KCC1.
|
|
Physiological function of K-Cl cotransport.
The existence of two different K-Cl cotransporters begs the obvious
question concerning functional differences. KCC1 shares many functional
characteristics of the vertebrate red cell K-Cl cotransporter that has
been the subject of much study. For example, it is activated by cell
swelling and by NEM, and it has a low apparent affinity for external
Rb+(K+)
and external Cl
(10). These
characteristics, along with its ubiquitous tissue distribution, provide
strong circumstantial evidence that KCC1 is the housekeeping isoform
involved in cell volume regulation (10). There is also evidence that
KCC1 may participate in net transepithelial salt movement, since it is
highly expressed in tissues such as lung and kidney (10). KCC2, on the
other hand, displays some very different functional characteristics,
including the lack of stimulation by cell swelling and a very high
apparent affinity for external
Rb+(K+).
The unique characteristics of KCC2 as well as its neuronal specificity
raise the possibility that the physiological functions of KCC2 differ
from those of KCC1. Furthermore, there are significant differences in
the primary sequence between the two KCC isoforms that may confer
isoform-specific differences in regulation. For example, KCC2 has a
large 44-amino acid insertion within the large COOH-terminal
hydrophilic region that contains numerous negatively charged amino
acids as well as a unique potential protein kinase C phosphorylation
site. This region also contains a potential tyrosine kinase
phosphorylation site that is not present in KCC1.
Regulation of neuronal intracellular
[Cl
].
One potential function of KCC2 might be to maintain the low neuronal
[Cl
] and the
favorable inwardly directed
Cl
electrochemical gradient
required for the proper function of ligand-gated
Cl
channels in postsynaptic
inhibition (28). The hyperpolarization that occurs in postsynaptic
neurons after activation of GABAA receptors is due to an influx of
Cl
through the ligand-gated
channel. This represents the underlying cellular mechanism for the
IPSP. For GABAA receptors to
mediate such a hyperpolarization, the electrochemical potential
difference for Cl
must be
kept more negative than the resting membrane potential. This requires
the presence of a transport mechanism that can move Cl
out of the neuron
against an electrochemical gradient, i.e., an active
Cl
extrusion mechanism.
Under normal physiological conditions, there is enough energy stored in
the K+ chemical gradient to move
Cl
against its chemical
gradient out of the neuron via electroneutral K-Cl cotransport, and
this can help maintain
ECl more negative than Em. The
involvement of a K-Cl cotransporter in such a process has been
supported by studies using a number of different neuronal preparations
(1, 2, 33, 34). Although functional data have not firmly established
the involvement of the KCC2 isoform in neuronal
Cl
regulation, its high
level of neuronal-specific expression within the central nervous system
supports this hypothesis.
[K+]o
buffering in the brain.
One functional characteristic of KCC2 not shared by KCC1 is its high
apparent affinity for external
Rb+(K+).
This feature may allow KCC2 to function in
[K+]o
buffering. Because the extracellular space in the brain is small and
[K+]o
is low, neuronal activity can result in substantial changes in
[K+]o.
Depending on the level of neuronal activity,
[K+]o
in the brain varies between 3 and 10 mM (32). Significantly, studies on
mammalian nervous systems have reported a remarkable constant ceiling
level for
[K+]o
of 10-12 mM (14). This level is exceeded only under a few conditions, including hypoxia, ischemia, and spreading depression (13).
Undoubtedly, during neuronal activity, some
K+ will return to the neuron via
neuronal
Na+-K+-ATPase,
and some will diffuse to extracellular regions of lower concentration.
There is strong evidence that large rapid increases in
[K+]o
are removed by current-mediated glial spatial buffering
(K+ siphoning; for review, see
Ref. 36); however, this mechanism has been deemed insufficient to
account entirely for
[K+]o
regulation in the brain after neuronal activity (8). Furthermore, the
K+ released during activity must
eventually be returned to the neuron to prevent the depletion of
neuronal K+. I propose that the
neuronal K-Cl cotransporter can participate in the process of
K+ uptake after neuronal activity.
As an electroneutral transporter, the direction and magnitude of net
K-Cl cotransport will be dictated by the sum of the
K+ and
Cl
chemical potential
differences. In most cells, the K-Cl cotransporter has only been
considered as a net efflux pathway because plasma K+ concentration is tightly
regulated and the K+ and
Cl
chemical gradients
almost always favor net efflux. However, in many neurons where
intracellular
[Cl
]
([Cl
]i)
is quite low (7-9 mM if
ECl < Em), the
driving force for net K-Cl cotransport is very close to thermodynamic
equilibrium. Using typical values reported for neuronal
[K+] (100 mM; Ref. 11)
and external
[Cl
] (145 mM;
Ref. 7), Fig. 9 presents the driving force
for net K-Cl cotransport as a function of
[K+]o
at different neuronal
[Cl
]. In
neurons like pyramidal cells of the hippocampus where
[Cl
]i
has been estimated to be ~7 mM (16),
[K+]o
needs only to increase above ~5 mM to reverse the calculated driving
force for net K-Cl cotransport, thus allowing KCC2 to operate as a net
influx pathway.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 9.
Thermodynamic driving force for K-Cl cotransport (J/mol) as a function
of external [K+]
([K+]o)
at various intracellular Cl
([Cl ]i)
values. Driving force was calculated using constant intracellular [K+] (100 mM) and
external [Cl ]
(145 mM) at 37°C.
|
|
For a K-Cl cotransporter to operate efficiently as a
[K+]o
buffer, it should exhibit a number of important features.
1) Its direction of net transport
should be sensitive to subtle changes in
[K+]o.
Because the driving force for net K-Cl cotransport is situated very
near thermodynamic equilibrium in the brain and KCC2 appears to be
constitutively active (at least as expressed in HEK-293 cells), it can
respond rapidly to any increase in
[K+]o.
2) The affinity of the
cotransporter for external K+
should be compatible with the range of the observed
[K+]o
(3-10 mM), allowing a high rate of cotransport activity. This requirement appears to be a unique feature of the KCC2 isoform, as it
exhibits a rather high affinity for external
Rb+(K+)
(Km = 5.2 mM),
whereas the KCC1 isoform displays a low affinity for external
Rb+(K+)
(Km >25 mM;
Ref. 10). Because KCC2 exhibits an affinity for external
Rb+(K+)
that is near the normal
[K+]o,
it is an excellent candidate for
[K+]o
buffering. In other words, the rate of K-Cl cotransport will approach
maximum velocity as
[K+]o
increases to its ceiling level of 10-12 mM.
3) To be an effective buffer,
responses should be rapid. This is an issue of transport capacity,
suggesting high levels of protein expression. Although KCC2 protein has
not yet been examined in the brain, RNA analysis, including both
Northern blot and in situ hybridization studies, have demonstrated that
the KCC2 transcript is expressed at very high levels in neurons
throughout the central nervous system (28). Thus, on the basis of
thermodynamic considerations as well as unique properties of the KCC2
protein, including its transport kinetics, distribution, and density,
KCC2 is well suited to function as an influx pathway and an effective
[K+]o
buffer under conditions of elevated
[K+]o.
Although the K-Cl cotransporter cannot function alone in
[K+]o
homeostasis, it can complement other neuronal
K+ uptake systems (e.g.,
Na+-K+-ATPase)
and glial spatial buffering and contribute significantly to the overall
process of rapidly returning K+ to
the neuron.
In addition to the K-Cl cotransporter, the Na-K-Cl cotransporter also
exhibits some of the features required for an efficient [K+]o
buffer discussed above; that is, it has a strong net inwardly directed
driving force and a high affinity for external
K+ (29). The Na-K-Cl cotransporter
has been well charaterized by Russell and co-workers (5) in the squid
giant axon, and recently, NKCC1 was localized in neurons of the rat
brain (30). In the rat brain, NKCC1 appears to be much more restricted
in its neuronal distribution and density than KCC2. For example, Plotkin et al. (30) found NKCC1 at high levels only in sensory neurons
and certain neurons of the brain stem, and this may limit its role in
[K+]o
buffering within the neuronal microenvironment. On the other hand,
NKCC1 does exhibit very high expression within the epithelial cells of
the choroid plexus, which are responsible for the production of the
cerebrospinal fluid. Plotkin et al. (30) have suggested that NKCC1
plays an important role in the regulation of
[K+] in this secreted
fluid as well as the regulation of
[Cl
]i
in certain neurons. Therefore, both the K-Cl and Na-K-Cl cotransporters appear to serve important roles in the regulation of neuronal [Cl
]i
and
[K+]o
in the brain.
Functional consequences of KCC2-dependent
[K+]o
buffering.
The involvement of KCC2 in the regulation of neuronal
[Cl
]i
and
[K+]o
in the brain is mutually exclusive. In other words, it can function in
the regulation of only one of these parameters at any given time.
However, as touched on above, because the driving force for K-Cl
cotransport is situated very near thermodynamic equilibrium, KCC2
becomes sensitive to subtle changes in either [Cl
]i
or
[K+]o,
and, therefore, it can function as a "dynamic buffer," responding to and reducing changes in either parameter. However, an important consequence of the neuronal K-Cl cotransporter operating in "reverse mode" as a net influx pathway is that it will contribute to an increase in neuronal
[Cl
]. This will
alter the driving force for
Cl
movement through
conductive pathways
(Em-ECl),
like GABAA and glycine receptors,
and may contribute to the neuronal
Cl
accumulation that is
largely responsible for depression of the GABAA inhibitory response after
repetitive stimulation (22, 34). A well-characterized consequence of
repetitive stimulation even at low frequency is a significant and
transient increase in
[K+]o
that results from the release of neuronal
K+ via conductive pathways (e.g.,
Ref. 14). Although it has been suggested that the elevated
[K+]o
would lead to Cl
accumulation through inhibition of outward K-Cl cotransport (34), this
hypothesis does not properly account for the thermodynamics of K-Cl
cotransport. I hypothesize that the
Cl
accumulation that occurs
after repetitive stimulation is largely a result of the K-Cl
cotransporter operating as a net influx pathway because of the elevated
[K+]o
and subsequent reversal of K-Cl cotransport driving force. The possible
involvement of KCC2 in determining the direction and magnitude of the
driving force for Cl
movement through GABAA receptors
indicates this cotransporter may contribute significantly to the
genesis of epileptiform activity in the brain. Significantly,
furosemide application (24, 33, 34) as well as elevation of
[K+]o
(typically 8.5 mM; see Ref. 23), both of which are expected to result
in the accumulation of neuronal
[Cl
] through
effects on the K-Cl cotransporter, have been shown to cause
epileptiform activity in in vitro preparations.
In summary, the KCC2 protein has been functionally characterized in
stably transfected HEK-293 cell lines, demonstrating that it exhibits
many of the basic transport features of a K-Cl cotransport system. The
KCC2 protein did, however, display some unique functional characteristics that were distinct from the KCC1 isoform, including the
lack of swelling activation and relatively high apparent affinity for
[K+]o.
The unique properties of the KCC2 protein along with the thermodynamics of K-Cl cotransport support the hypothesis that KCC2 can function efficiently as a net influx pathway under conditions of elevated [K+]o. Because the driving force for K-Cl
cotransport in many neurons is situated very near thermodynamic
equilibrium, the direction of net movement of
K+ and
Cl
through the
cotransporter will be very sensitive to changes in [Cl
]i
and
[K+]o.
Thus KCC2 may play an important role in the homeostasis of both
neuronal [Cl
]
and
[K+]o
in the brain.
 |
NOTE ADDED IN PROOF |
A recent article (K. Kaila, K. Lamsa, S. Smirnov, T. Taira, and J. Voipo. J. Neurosci. 17: 7662-7672, 1997) has addressed the
cellular mechanisms that account for the large positive shift in the
GABAA reversal potential (EGABA-A)
in pyramidal neurons following repetitive stimuli. Data from this study
are fully consistent with a K-Cl cotransporter mediating a significant
increase in [Cl
]i under conditions of
elevated [K+]o and contributing to a large
positive shift in EGABA-A in the postsynaptic
neuron.
 |
ACKNOWLEDGEMENTS |
I am grateful to Jeff Williams and Tamara Stevenson for excellent
technical assistance. I am indebted to Drs. Peter Cala and Chris Lytle
for many helpful discussions on transport kinetics and cation-chloride
cotransport function and for critical readings of the manuscript. I am
also grateful to numerous colleagues for discussions and readings of
the manuscript, including Drs. John Russell, Javier Alvarez-Leefmans,
Kai Kaila, Deepak Kaji, Andrew Ishida, Vijaya Kumari, Martha
O'Donnell, and Dandan Sun.
 |
FOOTNOTES |
This work was supported by grants from the Hibbard E. Williams Research
Funds, by National Institute of Neurological Disorders and Stroke Grant
NS-36296-01, and by the Epilepsy Foundation of America.
Received 8 May 1997; accepted in final form 2 July 1997.
 |
REFERENCES |
1.
Aickin, C. C.,
R. A. Deisz,
and
H. D. Lux.
Ammonium action on post-synaptic inhibition in crayfish neurones: implications for the mechanism of chloride extrusion.
J. Physiol. (Lond.)
329:
319-339,
1982[Medline].
2.
Aickin, C. C.,
R. A. Deisz,
and
H. D. Lux.
Mechanisms of chloride transport in crayfish stretch receptor neurones and guinea pig vas deferens: implications for inhibition mediated by GABA.
Neurosci. Lett.
47:
239-244,
1984[Medline].
3.
Alvarez-Leefmans, F. J.
Intracellular Cl
regulation and synaptic inhibition in vertebrate and invertebrate neurons.
In: Chloride Channels and Carriers in Nerve, Muscle, and Glial Cells, edited by F. J. Alvarez-Leefmans,
and J. M. Russell. New York: Plenum, 1990, p. 109-158.
4.
Aull, F.
Potassium chloride cotransport in steady-state ascite tumor cells.
Biochim. Biophys. Acta
643:
339-345,
1981[Medline].
5.
Breitwieser, G. E.,
A. A. Altamirano,
and
J. M. Russell.
Elevated [Cl
]i and [Na+]i inhibit Na+, K+, Cl
cotransport by different mechanisms in squid giant axons.
J. Gen. Physiol.
107:
261-270,
1996[Abstract].
6.
Delpire, E.,
and
P. K. Lauf.
Kinetics of DIDS inhibition of swelling activated K-Cl cotransport in low K sheep erythrocytes.
J. Membr. Biol.
26:
89-96,
1992.
7.
Dietzel, I.,
U. Heinemann,
G. Hofmeier,
and
H. D. Lux.
Stimulus-induced changes in extracellular Na+ and Cl
concentration in relation to changes in the size of the extracellular space.
Exp. Brain Res.
46:
73-84,
1982[Medline].
8.
Dietzel, I.,
U. Heinemann,
and
H. D. Lux.
Relations between slow extracellular potential changes, glial potassium buffering, and electrolyte and cellular volume changes during neuronal hyperactivity in cat brain.
Glia
2:
25-44,
1989[Medline].
9.
Flatman, P. W.,
N. C. Adragna,
and
P. K. Lauf.
Role of protein kinases in regulating sheep erythrocyte K-Cl cotransport.
Am. J. Physiol.
271 (Cell Physiol. 40):
C255-C263,
1996[Abstract/Free Full Text].
10.
Gillen, C. M.,
S. Brill,
J. A. Payne,
and
B. Forbush III.
Molecular cloning and functional expression of the K-Cl cotransport from rabbit, rat, and human: a new member of the cation-chloride cotransporter family.
J. Biol. Chem.
271:
16237-16244,
1996[Abstract/Free Full Text].
11.
Grafe, P.,
J. Rimpel,
M. M. Reddy,
and
G. ten Bruggencate.
Changes of intracellular sodium and potassium ion concentrations in frog spinal motoneurons induced by repetitive synaptic stimulation.
Neuroscience
7:
3213-3220,
1982[Medline].
12.
Guggino, W. B.
Functional heterogeneity in the early distal tubule of the Amphiuma kidney: evidence for two modes of Cl
and K+ transport across the basolateral cell membrane.
Am. J. Physiol.
250 (Renal Fluid Electrolyte Physiol. 19):
F430-F440,
1986[Medline].
13.
Hansen, A. J.
Effect of anoxia on ion distribution in the brain.
Physiol. Rev.
65:
101-148,
1985[Free Full Text].
14.
Heinemann, U.,
and
H. D. Lux.
Ceiling of stimulus induced rises in extracellular potassium concentration in the cerebral cortex of cat.
Brain Res.
120:
231-249,
1977[Medline].
15.
Igarashi, P.,
G. B. Vanden Heuvel,
J. A. Payne,
and
B. Forbush III.
Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F405-F418,
1995[Abstract/Free Full Text].
16.
Inoue, M.,
M. Hara,
X.-T. Zeng,
T. Hirose,
S. Ohnishi,
T. Yasukura,
T. Uriu,
K. Omori,
A. Minato,
and
C. Inagaki.
An ATP-driven Cl
pump regulates Cl
concentrations in rat hippocampal neurons.
Neurosci. Lett.
134:
75-78,
1991[Medline].
17.
Isenring, P.,
R. Behnke,
and
B. Forbush III.
Involvement of the first 2 transmembrane domains in ion binding by the Na-K-Cl cotransporter.
J. Am. Soc. Nephrol.
6:
341,
1995.
18.
Jennings, M. L.,
and
R. K. Schulz.
Okadaic acid inhibition of KCl cotransport. Evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethylmaleimide.
J. Gen. Physiol.
97:
799-818,
1991[Abstract].
18a.
Kaila, K.
Ionic basis of GABAA receptor channel function in the nervous system.
Prog. Neurobiol.
42:
489-537,
1994[Medline].
19.
Kaji, D. M.,
and
Y. Tsukitani.
Role of protein phosphatase in activation of KCl cotransport in human erythrocytes.
Am. J. Physiol.
260 (Cell Physiol. 29):
C178-C182,
1991.
20.
Lauf, P. K.,
N. C. Adragna,
and
R. P. Garay.
Activation by N-ethylmaleimide of a latent K+-Cl
flux in human red blood cells.
Am. J. Physiol.
246 (Cell Physiol. 15):
C385-C390,
1984[Abstract].
21.
Lauf, P. K.,
J. Bauer,
N. C. Adragna,
A. M. M. Zade-Oppen,
K. H. Ryu,
and
E. Delpire.
Erythrocyte K-Cl cotransport: properties and regulation.
Am. J. Physiol.
263 (Cell Physiol. 32):
C917-C932,
1992[Abstract/Free Full Text].
22.
Ling, D. S. F.,
and
L. S. Benardo.
Activity-dependent depression of monosynaptic fast IPSCs in hippocampus: contributions from reductions in chloride driving force and conductance.
Brain Res.
670:
142-146,
1995[Medline].
23.
McBain, C. J.
Hippocampal inhibitory neuron activity in the elevated potassium model of epilepsy.
J. Neurophysiol.
72:
2853-2863,
1994[Abstract/Free Full Text].
24.
Misgeld, U.,
R. A. Deisz,
H. U. Dodt,
and
H. D. Lux.
The role of chloride transport in postsynaptic inhibition of hippocampal neurons.
Science
232:
1413-1415,
1986[Medline].
25.
Payne, J. A.
The neuronal-specific K-Cl cotransporter (KCC2) exhibits high apparent affinity for external K+: implications for external K+ buffering in the brain (Abstract).
FASEB J.
11:
A299,
1997.
26.
Payne, J. A.,
and
B. Forbush III.
Alternatively spliced isoforms of the putative renal Na-K-Cl cotransporter are differentially distributed within the rabbit kidney.
Proc. Natl. Acad. Sci. USA
91:
4544-4548,
1994[Abstract].
27.
Payne, J. A.,
and
B. Forbush III.
Molecular characterization of the epithelial Na-K-Cl cotransporter isoforms.
Curr. Opin. Cell Biol.
7:
493-503,
1995[Medline].
28.
Payne, J. A.,
T. J. Stevenson,
and
L. F. Donaldson.
Molecular characterization of a putative K-Cl cotransporter in rat brain: a neuronal-specific isoform.
J. Biol. Chem.
271:
16245-16252,
1996[Abstract/Free Full Text].
29.
Payne, J. A.,
J.-C. Xu,
M. Haas,
C. Y. Lytle,
D. Ward,
and
B. Forbush III.
Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon.
J. Biol. Chem.
270:
17977-17985,
1995[Abstract/Free Full Text].
30.
Plotkin, M. D.,
M. R. Kaplan,
L. N. Peterson,
S. R. Gullans,
S. C. Hebert,
and
E. Delpire.
Expression of the Na+-K+-2Cl
cotransporter BSC2 in the nervous system.
Am. J. Physiol.
272 (Cell Physiol. 41):
C173-C183,
1997[Abstract/Free Full Text].
31.
Reuss, L.
Basolateral KCl cotransport in a NaCl-reabsorbing epithelium.
Nature
305:
723-726,
1983[Medline].
32.
Sykova, E.
Extracellular K+ accumulation in the central nervous system.
Prog. Biophys. Mol. Biol.
42:
135-189,
1983[Medline].
33.
Thompson, S. M.,
R. A. Deisz,
and
D. A. Prince.
Relative contributions of passive equilibrium and active transport to the distribution of chloride in mammalian cortical neurons.
J. Neurophysiol.
60:
105-124,
1988[Abstract/Free Full Text].
34.
Thompson, S. M.,
and
B. H. Gahwiler.
Activity-dependent disinhibition. II. Effects of extracellular potassium, furosemide, and membrane potential on ECl in hippocampal CA3 neurons.
J. Neurophysiol.
61:
512-523,
1989[Abstract/Free Full Text].
35.
Vitoux, D.,
O. Olivieri,
R. P. Garay,
E. J. Cragoe,
F. Galacteros,
and
Y. Beuzard.
Inhibition of K+ efflux and dehydration of sickle cells by [(dihydroindenyl)oxyl]alkanoic acid: an inhbitor of the K+Cl
cotransport system.
Proc. Natl. Acad. Sci. USA
86:
4273-4276,
1989[Abstract].
36.
Walz, W.
Role of glial cells in the regulation of the brain ion microenvironment.
Prog. Neurobiol.
33:
309-333,
1989[Medline].
AJP Cell Physiol 273(5):C1516-C1525
0363-6143/97 $5.00
Copyright © 1997 the American Physiological Society