The Neuron-specific K-Cl Cotransporter, KCC2
ANTIBODY DEVELOPMENT AND INITIAL CHARACTERIZATION OF THE
PROTEIN*
Jeffery R.
Williams,
James W.
Sharp,
Vijaya G.
Kumari
,
Martin
Wilson§, and
John A.
Payne¶
From the Departments of Human Physiology and
Cell
Biology and Human Anatomy, School of Medicine and
§ Section of Neurobiology, Physiology, and Behavior, the
Division of Biological Sciences, University of California,
Davis, California 95616
 |
ABSTRACT |
The neuron-specific K-Cl cotransporter (KCC2) is
hypothesized to function as an active Cl
extrusion
pathway important in postsynaptic inhibition mediated by ligand-gated
anion channels, like
-aminobutyric acid type A (GABAA)
and glycine receptors. To understand better the functional role of KCC2
in the nervous system, we developed polyclonal antibodies to a KCC2
fusion protein and used these antibodies to characterize and localize
KCC2 in the rat cerebellum. The antibodies specifically recognized the
KCC2 protein which is an ~140-kDa glycoprotein detectable only within
the central nervous system. The KCC2 protein displayed a robust and
punctate distribution in primary cultured retinal amacrine cells known
to form exclusively GABAAergic synapses in culture. In
immunolocalization studies, KCC2 was absent from axons and glia but was
highly expressed at neuronal somata and dendrites, indicating a
specific postsynaptic distribution of the protein. In the granule cell
layer, KCC2 exhibited a distinct colocalization with the
2/
3-subunits of the GABAA
receptor at the plasma membrane of granule cell somata and at
cerebellar glomeruli. KCC2 lightly labeled the plasma membrane of
Purkinje cell somata. Within the molecular layer, KCC2 exhibited a
distinctly punctate distribution along dendrites, indicating it may be
highly localized at inhibitory synapses along these processes. The
distinct postsynaptic localization of KCC2 and its colocalization with
GABAA receptor in the cerebellum are consistent with the
putative role of KCC2 in neuronal Cl
extrusion and
postsynaptic inhibition.
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INTRODUCTION |
The K-Cl cotransporter mediates an obligatorily coupled,
electroneutral movement of K+ and Cl
ions
across the plasma membrane of many animal cells. The K-Cl cotransporter
is normally a net efflux pathway, using the favorable K+
chemical gradient maintained by the
Na+,K+-ATPase, to drive Cl
out of
the cell. The cotransporter, however, is bi-directional and can mediate
a net efflux or influx, depending upon the prevailing K+
and Cl
chemical gradients. The predominant function of
this transporter is cellular ion and water homeostasis. For example, in
red blood cells a K-Cl cotransporter is involved in cell volume
regulation (1). Additionally, a K-Cl cotransporter may play an
important role in salt transport across certain epithelia (2, 3). In
neurons, a K-Cl cotransporter appears to serve as the active Cl
extrusion mechanism responsible for maintaining the
Cl
reversal potential (ECl) less
than the membrane potential (Em) as required for the
proper function of
GABAA1 and
glycine receptors in postsynaptic inhibition (for reviews see Refs. 4
and 5).
Recently, two isoforms of the K-Cl cotransporter have been identified
and functionally characterized (KCC1, Ref. 6 and KCC2, Refs. 7 and 8).
KCC1 has a ubiquitous tissue distribution, and functional expression
studies demonstrated that it was activated by cell swelling as well as
by application of N-ethylmaleimide. Both of these treatments
are well known activators of the red blood cell K-Cl cotransport system
(6). Furthermore, like the K-Cl cotransporter studied in red blood
cells, exogenously expressed KCC1 exhibited low transport affinity for
both external K+ (Km >25
mM) and external Cl
(Km
>50 mM). These data support the hypothesis that KCC1
represents the "housekeeping" isoform of the K-Cl cotransporter involved in cell volume regulation (6). In contrast, KCC2 was determined to be neuron-specific and displayed unique functional characteristics, including the lack of swelling activation and a
remarkably high apparent affinity for external K+
(Km = 5 mM; Refs. 7 and 8). These
functional characteristics indicated that KCC2 may have a novel
function in the nervous system, distinct from the volume regulatory
role of KCC1. As KCC2 appears to be a neuron-specific isoform, we have
hypothesized that it is involved in active Cl
extrusion.
Based on thermodynamic considerations, an electroneutral K-Cl
cotransporter can function to maintain very low intracellular [Cl
] ([Cl
]i; 5-7
mM) and ECl more negative than
Em. However, with such a low
[Cl
]i, the driving force for net K-Cl
cotransport will be very close to thermodynamic equilibrium (8). Thus,
KCC2 with its high affinity for external K+ can function as
a very efficient neuronal K+ uptake system whenever
external [K+] ([K+]o) becomes
elevated (>5 mM) and reverses the driving force for net
K-Cl cotransport from efflux to influx. With the driving force for net
K-Cl cotransport poised at equilibrium, KCC2 will be very sensitive to
subtle changes in both [Cl
]i and
[K+]o, and therefore, it may function as a
"dynamic buffer" of these two ion concentrations. This hypothesis
is unifying as it provides a cellular mechanism to account for the
functional link between increases in [K+]o and
alterations in neuronal [Cl
]i and
GABAA receptor function, all of which occur after repetitive stimulation (i.e. activity-dependent disinhibition).
Previous in situ hybridization studies supported a
neuron-specific localization of the KCC2 transcript in rat brain (7). Cellular localization, however, of the KCC2 protein in the mammalian nervous system has not been performed. This may provide important information about the role of KCC2 in neuronal function. We
hypothesized that if KCC2 is important in postsynaptic inhibition, it
should colocalize with GABAA receptors in the postsynaptic
membrane. In the present report, we have prepared and characterized
antibodies specific for the KCC2 protein and used these anti-KCC2
antibodies to characterize and localize the protein in the rat
cerebellum. The rat cerebellum was chosen because of its very high
expression of the KCC2 transcript (7). The KCC2 protein was restricted to neuronal somata and dendrites, and in the granule cell layer of the
rat cerebellum it exhibited a distinct colocalization with the
2/
3-subunits of the GABAA
receptor. These data are fully consistent with KCC2 having an important
role in neuronal Cl
extrusion and postsynaptic inhibition.
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EXPERIMENTAL PROCEDURES |
Production and Purification of KCC2 Fusion Protein and Anti-KCC2
Polyclonal Antibodies--
A KCC2 fusion protein (termed B22) was
prepared, containing a 112-amino acid segment of the carboxyl terminus
of rat KCC2 (amino acids 932-1043). The sequence encoding the fusion
protein was amplified in a polymerase chain reaction (forward
primer
GAATTCAGCATCACAGATGAATCTCG; reverse
primer
CTCGAGTTAGTTCAAGTTTTCCCACTCCG). The reaction volume of 50 µl
contained 5 µl of template cDNA (ERB10 clone at 2 ng/µl; see
Ref. 7), 0.2 µM of each primer, 50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25 °C), 0.1% Triton X-100, 2.5 mM MgCl2, 0.25 mM dNTPs, and 1 unit
of Taq polymerase (Promega, Madison, WI). Thirty 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 cloned into the plasmid pCR2.1 following the
manufacturer's instructions (Invitrogen, Carlsbad, CA). Using the
restriction sites engineered into the forward (EcoRI) and
reverse (XhoI) PCR primers, the insert in the pCR2.1 vector
was subcloned into pET28 and then sequenced in both directions.
The fusion protein was produced in E. coli according to the
manufacturer's instructions (Novagen, Madison, WI). The B22 fusion protein was very soluble in 1× binding buffer (5 mM
imidazole, 500 mM NaCl, 20 mM Tris-HCl (pH 7.9)
containing 0.1% Triton X-100). It was purified using metal chelation
chromatography following the manufacturer's instructions (His-Bind
Resin; Novagen, Madison, WI). The B22 fusion protein was eluted from
the column using a linear imidazole gradient (60 mM to 1 M). Four of the 1-ml fractions containing most of the
eluted protein were combined and concentrated by centrifugation in
microconcentrators (Amicon, Beverly, MA). This method of purification
yields relatively pure B22 fusion protein with no other visible bands
on Coomassie-stained gels (data not shown).
Rabbit polyclonal antibodies were generated against the purified B22
fusion protein by the Rabbit Antibody Production Program (Animal
Resource Service of the School of Veterinary Medicine, University of
California, Davis). Purified B22 fusion protein (250-300 µg) was
injected subcutaneously with complete Freund's adjuvant. All
subsequent boost injections were performed with incomplete Freund's
adjuvant. Preimmune serum was obtained prior to first injection. Immune
sera used in the present study was harvested >30 days after the second
boost injection.
Immune antisera were purified using affinity chromatography. The B22
fusion protein was coupled to 1,1'-carbonyldiimidazole-activated agarose beads following the manufacturer's instructions (Pierce). Immune antisera was incubated 2 days at 4 °C with B22 fusion
protein-coupled agarose beads. Specific antibodies were eluted from the
beads with 50 mM glycine (pH 2.7). Purified antibodies were
stored in phosphate-buffered saline (PBS) containing 0.02% sodium
azide at 4 °C.
Tissue Culture--
The NG-108 and N1E-115 cells were maintained
in growth medium, containing Dulbecco's modified Eagle's medium
(DMEM), 10% fetal bovine serum, 5% hypoxanthine, aminopterin and
thymidine (HAT) supplement, and 2 mM
L-glutamine. All other cultured cells were maintained in
DMEM supplemented with 10% fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 µg/ml). All cells were maintained in
a humidified incubator with 5% CO2 at 37 °C.
A previous report described the development of HEK-293 cell lines
stably expressing rat KCC2 protein (8). These stable HEK-293 cell lines
(KCC2-9 and KCC2-22T) were maintained in growth medium containing 900 µg/ml geneticin (G418; Life Technologies, Inc.). A KCC1-KCC2 chimeric
construct was produced by ligating rat KCC1 and rat KCC2 cDNA at a
common BamHI site. This common BamHI site occurs
9 amino acids (i.e. 28 nucleotides) beyond the last
predicted transmembrane segment of both proteins. The resulting construct had rat KCC2 encoding the hydrophilic amino-terminal region
and transmembrane segments and rat KCC1 encoding the remaining large
carboxyl-terminal domain. This rat KCC1-KCC2 chimeric construct (termed
KCC2-1C) also contained a 10 amino acid c-myc epitope (EQKLISEEDL) at the amino terminus of the protein. A stable HEK-293 cell line expressing the KCC2-1C construct was produced using previously described methods (9).
Dispersed retinal cultures were prepared from embryonic chickens by
methods previously described (10). Briefly, neurons from 8-day-old
chick embryo retinas were dissociated in 0.1% trypsin. Cells were
seeded into culture dishes containing
poly-L-ornithine-coated glass coverslips. Cells were
maintained in DMEM with 5% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 µg/ml), and 400 µM
L-glutamine. Retinal cultures were incubated at 37 °C
under a 5% CO2 humidified atmosphere. Cultured retinal
cells were obtained for immunodetection after 16 days in culture.
Protein Analysis--
Membranes were prepared from freshly
isolated tissue and from cultured cell lines using differential
centrifugation. Briefly, tissue was dissected or scraped off of culture
plates and homogenized in 10-40 ml of homogenization buffer (250 mM sucrose, 10 mM Tris, 10 mM
HEPES, 1 mM EDTA; pH adjusted to 7.2 at 24 °C)
containing protease inhibitors. Following 10 strokes in a glass-Teflon
homogenizer, the homogenate was centrifuged at 7,000 rpm for 10 min at
4 °C (Sorval RC5, SS-34 rotor). The supernatant was centrifuged at 20,000 rpm for 30 min at 4 °C. The final pellet was resuspended in
~100-500 µl of homogenization buffer with protease inhibitors and
stored at
80 °C. Protein concentration was determined using a
Micro-BCA protein kit (Pierce). Membrane proteins were resolved by
SDS-polyacrylamide gel electrophoresis using a 7.5% Tricine gel
system. Gels were electrophoretically transferred from unstained gels
to PVDF membranes (Immobilon P; Millipore, Bedford, MA) in transfer
buffer (192 mM glycine, 25 mM Tris-Cl (pH 8.3),
and 15% methanol) for
5 h at 50 V using a Bio-Rad Trans-Blot tank
apparatus. PVDF-bound protein was visualized by staining with Coomassie
Brilliant Blue R-250. The PVDF membrane was blocked in 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 either preimmune serum, anti-KCC2
immune serum, affinity purified anti-KCC2 antibody, or c-myc
monoclonal antibody overnight at 4 °C or 2 h at 24 °C. After
three 10-min washes in PBS/milk, the PVDF membrane was incubated with
secondary antibody (horseradish peroxidase conjugated goat anti-rabbit
IgG or anti-mouse IgG; Zymed Laboratories Inc.) 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 Life Science Products).
Deglycosylation experiments were performed on membranes (20 µg)
isolated from whole rat brain and HEK-293 stable cells. Membranes were
incubated for 4 h at 37 °C in a medium containing 0.5%
n-octyl glucoside, 20 mM sodium phosphate buffer
(pH 8.0), 50 mM EDTA, protease inhibitors, and
N-glycosidase F (20 units/ml; Boehringer Mannheim). Control
samples were treated similarly, but incubation was carried out in the
absence of N-glycosidase F. Enzymatic treatment was
terminated by addition of electrophoresis sample buffer supplemented with 6 M urea. Control and deglycosylated membrane proteins
were separated on 7.5% Tricine gels, and KCC2 protein was identified by Western blotting.
Immunolocalization of KCC2 Protein--
Immunocytochemical
detection of KCC2, GABAA receptor, and glial fibrillary
acidic protein (GFAP) employed affinity purified rabbit anti-KCC2
polyclonal antibodies, mouse anti-GABAA receptor
-subunit monoclonal antibody (Chemicon; Temecula, CA), and mouse anti-GFAP monoclonal antibody (Boehringer Mannheim). The
anti-GABAA receptor antibody is specific for the
2/
3-subunits (11, 12). Both an
avidin-biotin complex (ABC)-horseradish peroxidase technique and
fluorescent labeling were used for immunocytochemical detection in rat
cerebellum. Male Sprague-Dawley rats (200-275 g) were anesthetized with ketamine (20 mg/100 g intraperitoneally) and xylazine (0.1 mg/100
g intraperitoneally) and injected with heparin (10 units/100 g
intraperitoneally). Their vasculature was immediately flushed with 200 ml of heparinized 0.9% saline and perfused with 500 ml of 4%
paraformaldehyde in PBS via the ascending aorta. Brains were removed,
postfixed in 4% paraformaldehyde for 2 h, frozen in 2-methyl
butane on dry ice, embedded in TBS mounting media (Fisher), and stored
at
80 °C. A Bright Instruments cryostat produced 15-30-µm
frozen coronal sections.
For immunoperoxidase labeling, cerebellar sections were washed 3 times
in PBS and then placed in 2% goat serum/PBS (GS/PBS; PBS containing
2% goat serum, 0.2% Triton X-100, 0.1% bovine serum albumin) for
2 h at room temperature. Sections were incubated at 4 °C for
48 h with primary antibody diluted 1:200 in GS/PBS. After three
PBS washes, sections were incubated for 2 h at room temperature
with either a biotin-conjugated goat anti-rabbit IgG (1:200 dilution;
Vector Laboratories, Burlingame, CA) or goat anti-mouse IgG (1:200
dilution; Vector Laboratories, Burlingame, CA). Sections were washed
three times in PBS and incubated for 3 h in an avidin/horseradish
peroxidase solution prepared from an ABC kit (Vector Laboratories,
Burlingame, CA). Following three washes in PBS, the horseradish
peroxidase reaction was carried out using diaminobenzidine (DAB;
0.015% in PBS, Sigma) and 0.001% hydrogen peroxide. Sections were
triple washed in PBS and mounted on silanized slides. Some sections
were counterstained with hematoxylin. Sections stained with DAB were
taken through an ethanol/xylene dehydration series and coverslipped
with Permount (Fisher). Photographs of DAB-stained sections were taken
with a Zeiss 135 microscope.
Double-labeled fluorescent immunocytochemical detection of KCC2 with
either the
2/
3-subunits of
GABAA receptor or glial fibrillary acidic protein (GFAP)
was conducted on 10-15-µm cryostat sections that were free-floated
in 24-well tissue culture plates or mounted on slides. Sections were
blocked in 20% GS/PBS (PBS containing 20% goat serum, 0.2% Triton
X-100, 0.1% bovine serum albumin) for 3 h at 24 °C and then
incubated for 3 h at 24 °C with either
anti-GABAA-receptor antibody or anti-GFAP antibody. After 3 washes in PBS, sections were incubated for 2 h at 24 °C with a
fluorescein (FITC)-conjugated goat anti-mouse IgG secondary antibody
(1:200, Jackson Labs, West Grove, PA). Sections were washed 3 times in
PBS and stored overnight in PBS at 4 °C. Sections were again blocked
for 3 h in 20% GS/PBS and then incubated with anti-KCC2
antibodies (1:200) for 3 h at 24 °C. Following three PBS
washes, sections were incubated for 2 h with Cy3-conjugated goat
anti-rabbit IgG (1:200, Jackson Laboratories). Sections were then
washed 3 times in PBS, once in distilled water, and mounted on
acid-washed slides with Gel Mount (Biomedia, Foster City, CA). Cerebellar sections were examined, and digital images were obtained using laser scanning confocal microscopy (Zeiss 510 or Leica
TCS-NT).
Immunoperoxidase labeling of KCC2 in retinal cultures employed the same
basic procedures outlined above with the following modifications.
Retinal cultures grown on coverslips were fixed in 4% paraformaldehyde
in PBS for 30 min at 24 °C. They were washed 3 times in PBS and
blocked with GS/PBS for 3 h at 24 °C. Cell cultures were
incubated with anti-KCC2 antibodies for 3 h, washed 3 times in
PBS, and then incubated at 24 °C for 2 h with biotin-conjugated goat anti-rabbit (Vector Laboratories). After 3 PBS washes, cells were
incubated for 3 h in the avidin/horseradish peroxidase solution (ABC kit), and the DAB reaction was carried out as outlined above. Cultured cells were mounted on slides and then examined and
photographed with a Zeiss 135 microscope.
Two types of controls were performed for immunocytochemistry. For each
experiment, every third slide was incubated without primary antibody.
None of the control slides run without primary antibody showed
significant staining or fluorescent signal. A second control was
performed with the B22 fusion protein in an antigen adsorption
experiment, similar to that presented in Fig. 2B for Western
blots. Various amounts of the B22 fusion protein (0, 300 ng, 1, 3, or
100 µg) were diluted into 490 µl of GS/PBS. Affinity purified
anti-KCC2 polyclonal antibody (10 µl) was mixed with the B22 fusion
protein samples and incubated for 24 h at 4 °C. After
incubation, the tubes were spun at 11,000 rpm for 10 min. A 250-µl
aliquot of the supernatant was drawn off and diluted with GS/PBS to a
1-ml final volume. This was used as the diluted primary antibody for
brain sections and paralleled the KCC2 polyclonal antibody dilution
factor of 1:200 for immunocytochemistry. Five brain sections were
incubated at 4 °C for 48 h or at 24 °C for 3 h with
each fusion protein-KCC2 antibody reaction supernatant employing the
immunocytochemical protocol outlined above. The B22 fusion protein (>1
µg) was able to prevent specific staining of the anti-KCC2 antibodies
in rat brain sections (data not shown).
 |
RESULTS |
Development and Characterization of Anti-KCC2 Antibodies--
We
targeted the development of KCC2 antibodies to the predicted
intracellular carboxyl-terminal domain, as this represents a large
hydrophilic region of the protein containing a number of areas with
high antigenicity (PEPTIDESTRUCTURE program, Genetics Computer Group,
Madison, WI). Since both KCC1 and KCC2 are known to be present in the
rat brain (6, 7), our goal was to develop antibodies that were specific
for the KCC2 protein. To this end, we generated a fusion protein (B22)
that contained a small segment of the carboxyl terminus of KCC2. The
112 amino acids contained within the B22 fusion protein are very poorly
conserved between the KCC1 and KCC2 isoforms as follows: 1) only 61 of
the 112 amino acids align with KCC1 and they display very low identity
to KCC1 (31%), and 2) the remaining 51 amino acids represent residues unique to the KCC2 sequence (i.e. deleted from KCC1).
Therefore, use of this region of KCC2 as antigen had a high likelihood
of producing KCC2-specific antibodies.
Western blots of rat brain membranes using sera from animals immunized
with the B22 fusion protein showed strong reactivity to a broad
~140-kDa band that was not observed with preimmune sera (Fig.
1). A number of additional bands were
observed at high membrane loads; however, these bands were present at
much lower intensity than the ~140-kDa band. In order to purify the
B22 antisera, we cross-linked the B22 fusion protein to agarose beads
in an affinity column (see "Experimental Procedures"). Affinity
purification of the B22 antisera completely removed all nonspecific
bands on the Western blot of rat brain membranes, demonstrating the
specificity of the antibodies for the ~140-kDa band (Fig.
2A). These data provide strong
evidence that the ~140-kDa band represents the KCC2 protein. In all
subsequent experiments, we refer to affinity purified antisera as
anti-KCC2 antibodies. The specificity of the anti-KCC2 antibodies for
the ~140-kDa band was tested further in an immunoadsorption
experiment. B22 fusion protein was incubated at increasing amounts with
a fixed amount of antibody. The B22 fusion protein at levels
1 µg
was able to prevent completely any reactivity of the anti-KCC2
antibodies when tested against rat brain membranes on strip blots (Fig.
2B).

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Fig. 1.
Western blot analysis of the KCC2 protein in
rat brain membranes using antisera of rabbits immunized with the B22
fusion protein. Rat brain membranes were loaded onto a 7.5%
Tricine-SDS gel at 10-200 µg per lane as noted. The separated
proteins were transferred to PVDF membranes and probed with either
immune sera (antisera) or preimmune (Pre) sera.
Both preimmune and immune sera were used at a 1:1,000 dilution.
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Fig. 2.
Affinity purification of the anti-KCC2
antibodies (A) and immunoadsorption of the anti-KCC2
antibodies with B22 fusion protein (B).
A, polyclonal antibodies were affinity purified by
incubating the B22 antisera with B22 fusion protein coupled to
1,1'-carbonyldiimidazole-activated agarose beads (2 days with rotation
at 4 °C). Following 4 washes with PBS, the specific anti-KCC2
antibodies were eluted with 50 mM glycine (pH 2.7) and then
stored in PBS. Strip blots of rat brain membranes were probed with B22
antisera (1:1,000), postbead fraction (1:1,000), or purified anti-KCC2
antibodies (1:2,000). B, 10 µl of the purified anti-KCC2
antibodies were preadsorbed with B22 fusion protein (1 ng-10 µg) in
PBS/milk overnight at 4 °C. Strip blots of rat brain membranes were
probed with each of the preadsorbed fractions.
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In order to provide further evidence of antibody specificity and to
test potential antibody cross-reactivity with the KCC1 protein, we used
the anti-KCC2 antibodies on Western blots of membranes prepared from
various HEK-293 cell lines. Membranes were prepared from control
untransfected HEK-293 cells and HEK-293 cells stably expressing either
a full-length KCC2 construct (KCC2-22T) or a chimeric construct of
KCC1 and KCC2 (KCC2-1C). We have previously described the development
and functional expression of the KCC2-22T cell line that expresses the
full-length KCC2 protein (8). The chimeric protein expressed by the
KCC2-1C cell line contains the amino-terminal and transmembrane
domains of KCC2 and the carboxyl-terminal domain of KCC1. In this
chimera, the region over which the B22 fusion protein was prepared has
now been replaced with KCC1 sequence. The KCC2-1C chimera is
functional as the KCC2-1C cell line expressed a significantly elevated
furosemide-sensitive 86Rb influx relative to control cells
(data not shown). Both the KCC2 and KCC2-1C constructs were
epitope-tagged with an amino-terminal c-myc peptide. The
c-myc peptide monoclonal antibody displayed no reactivity in
control HEK-293 cells but recognized a broad ~150-kDa protein in the
two stably transfected cell lines (Fig. 3A). The exogenously expressed
KCC2 protein consistently ran ~10 kDa larger than that from rat
brain. Data presented below will show that this discrepancy in size is
due to differences in glycosylation of the native and exogenously
expressed KCC2 protein. The lower ~125-kDa band observed in both
stably transfected cell lines, but most prominently in the KCC2-22T
cell line, represents intracellular protein prior to addition of
oligosaccharides. This is commonly observed in overexpressing cells
(9). In contrast to the c-myc peptide antibody, the
anti-KCC2 antibodies displayed reactivity only with membranes prepared
from cells stably expressing the full-length KCC2 protein (Fig.
3B). Immunocytochemical studies revealed similar specificity
of the anti-KCC2 antibodies for the KCC2-22T cells (data not shown).
These data clearly demonstrate the specificity of the anti-KCC2
antibodies for the KCC2 isoform with no detectable KCC1
cross-reactivity.

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Fig. 3.
Specificity of the affinity purified B22
antibodies. Membranes (50 µg each lane) were prepared from
untransfected HEK-293 cells (Control) and HEK-293 cells
stably expressing either the KCC2 protein (KCC2-22T) or a
KCC2-KCC1 chimeric protein (KCC2-1C; see text). Each of the
expression constructs for the KCC2 and KCC2-1C chimeric protein were
epitope-tagged with the 10-amino acid c-myc peptide. Western
blot panels were probed with either the c-myc peptide
monoclonal antibody (1:2,000) or the anti-KCC2 antibodies
(1:2,000).
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Characterization of the KCC2 Protein--
In the following
studies, we used the anti-KCC2 antibodies purified from the B22
antisera to characterize the KCC2 protein in the rat nervous system. We
have previously demonstrated that KCC2 is a glycoprotein when it is
exogenously expressed in stably transfected HEK-293 cells (8).
Deglycosylation experiments confirmed this post-translational
modification of the native KCC2 protein in rat brain (Fig.
4). As noted above, exogenously expressed KCC2 protein migrated on SDS-polyacrylamide gel electrophoresis ~10-kDa larger than from native tissue. The deglycosylation
experiment presented in Fig. 4 demonstrates that this is the result of
differences in N-linked glycosylation of the protein as both
native and exogenously expressed KCC2 protein migrate at ~125 kDa
following treatment with N-glycosidase F. Significantly,
this core ~125-kDa KCC2 protein is similar in size to that predicted
from the cDNA (7).

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Fig. 4.
Deglycosylation of the KCC2 protein.
Membranes prepared from whole rat brain and stable HEK cells expressing
KCC2 (KCC2-22T) were incubated with (+) or without ( )
N-glycosidase F for 4 h at 37 °C. Western blot was
probed with anti-KCC2 antibodies (1:2,000).
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Previous studies examining the KCC2 transcript by Northern blot and
in situ hybridization indicated that KCC2 was restricted to
but widely distributed throughout the central nervous system (7).
Western blot analysis with the anti-KCC2 antibodies was fully
consistent with these findings. KCC2 protein was undetectable outside
the central nervous system but was found throughout all regions of the
brain and spinal cord (Fig. 5). Although
retinal tissue was not present on this blot, there are numerous
expressed sequence tags in the GenBankTM data base derived
from mammalian retinal cDNA libraries, indicating high abundance of
KCC2 in this tissue. KCC2 may be distributed largely on neuronal somata
and dendrites, as it was not observed in the sciatic nerve by Western
blot (Fig. 5) or by immunocytochemistry (data not shown).

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Fig. 5.
Western blot analysis of membranes prepared
from rat brain regions (100 µg), spinal cord
(100 µg), sciatic nerve (200 µg), and various rat tissues (200 µg). KCC2 protein was detected with anti-KCC2
antibodies (1:2,000).
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The KCC2 antibodies displayed a very broad cross-reactivity among
vertebrates. Although the anti-KCC2 antibodies were developed against
protein deduced from the rat cDNA sequence, they recognized a
~140-kDa protein in whole brain membranes prepared from every vertebrate species tested, including spiny dogfish, winter flounder, frog, chicken, rabbit, rat, and mouse (Fig.
6). This indicates that the B22 fusion
protein represents a very well conserved region of the KCC2
protein.

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Fig. 6.
Western blot analysis of membranes prepared
from whole brains of shark (spiny dogfish, Squalus
acanthias), teleost fish (winter flounder,
Pseudopleuronectes americanus), amphibian (leopard
frog, Rana pipiens), bird (chicken, Gallus
domesticus), and various mammals, rabbit (New Zealand
White), rat (Sprague-Dawley), and mouse (BALB/c). All membranes
were loaded at 100 µg. KCC2 protein was detected with anti-KCC2
antibodies (1:2,000).
|
|
By using Western blot analysis, we examined KCC2 expression in
membranes prepared from a number of cultured neuronal cell lines
derived from mammalian central or peripheral nervous system (Fig.
7A). The cell lines tested
included two mammalian neuroblastomas (human SH-SY5Y cells and murine
N1E-115 cells), a murine neuroblastoma-glioma hybrid (NG-108), a clonal
murine hypothalamic neuron (GT1-7 cells), and a pheochromocytoma cell
derived from rat adrenal (PC12 cells). None of these cultured cells
expressed detectable amounts of the KCC2 protein. Also, as expected for
a neuron-specific protein, KCC2 was not detectable in membranes from
primary cultured rat astrocytes or rat glioma C6 cells. The failure to
detect KCC2 in cultured neuronal cell lines indicated that neuronal
differentiation might be required for KCC2 expression. In support of
this hypothesis, we have identified two expressed sequence tags derived
from a differentiated postmitotic human neuron (hNT) that clearly
represent sequence from KCC2 (GenBankTM accession numbers
AA16760 and AA166885). Furthermore, we examined the expression of KCC2
in a number of primary cultured neurons. Low levels of KCC2 protein
were detected by immunocytochemistry in both rat hippocampal neurons
and rat cerebellar granule cells during the first 7 days in culture
(data not shown). In contrast, we observed significant KCC2 protein
expression in retinal neurons grown 16 days in a dispersed culture
system prepared from chicken (Fig. 7B). This dispersed
retinal culture has been well characterized and is composed of amacrine
cells, photoreceptors, bipolar cells, and glial cells, all of which are
distinguishable on the basis of morphology and antibody staining (10,
13). Amacrine cells in these cultures have been shown to form synapses
from one cell to another (10) as well as autapses from one cell to
itself (14). Significantly, these synapses and autapses are exclusively GABAAergic (10). As shown in Fig.
8, amacrine cells in the retinal cultures
abundantly expressed the KCC2 protein. In contrast, glial cells that
form a monolayer upon which neurons were often found were never seen to
express KCC2, although antibodies to the Na-K-Cl cotransporter stained
glial cells strongly (data not shown). Among the neurons within
cultures examined at embryonic equivalent day 16 or older, many
although not all labeled for KCC2. Amacrine cells and some apparent
bipolar cells showed the heaviest KCC2 staining. In those cells that
stained, weak and homogeneous staining of the cell body cytoplasm was
observed together with punctate staining of the plasma membrane both at
the cell body and along the dendrites (arrows in Fig. 8). In
general, the greatest density of stained spots occurred at the places
where cell bodies or dendrites touched each other, consistent with the
idea that KCC2 is found predominantly at synapses.

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Fig. 7.
Western blot analysis of membranes prepared
from whole rat brain and various cultured cells (100 µg) with anti-KCC2 antibodies (upper
panels, 1:2,000) and anti- -actin
antibodies (lower panels, 1:2,000). The cell
lines include the following: A, rat primary astrocytes, rat
C6 glioma cells, human neuroblastoma SH-SY5Y cells, murine hypothalamic
GT1-7 neurons, murine neuroblastoma N1E-115 cells, murine
neuroblastoma-glioma hybrid NG-108 cells, and rat pheochromocytoma PC12
cells; B, primary cultured retinal cells prepared from
chicken.
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Fig. 8.
Differential interference contrast images of
primary cultures of chick retinal cells at embryonic equivalent day
16. ABC-horseradish peroxidase technique was used for
immunocytochemical detection. Peroxidase reaction was visualized with
DAB. Top panel shows a typical staining pattern for
anti-KCC2 antibodies. DAB reaction product is absent from underlying
glial cells but is seen as distinct spots in the cell body and
processes of neurons (arrowheads). Secondary-only control,
bottom panel, shows no DAB reaction product. Scale
bar is 10 µm.
|
|
Immunocytochemical Localization in Rat Cerebellum--
Further
confirmation of the neuron-specific nature of the KCC2 protein was
obtained from localization studies with the anti-KCC2 antibodies in rat
cerebellum. Immunocytochemical staining for the KCC2 protein in the
cerebellum was distinct and positive relative to controls (Fig.
9, A and B; data
not shown for controls). KCC2 staining occurred principally in the
granule cell and the molecular layers of the cerebellar cortex. There
was no apparent difference in the distribution or intensity of staining
among the lobules or along the length of the folia. No detectable
immunocytochemical KCC2 staining was observed in axons or in glial
cells (Fig. 9, A and B). In experiments using
double-labeled fluorescent microscopy, the KCC2 protein and a
glial-specific marker, GFAP, exhibited distinctly different
localization patterns (Fig. 10,
A-C). The anti-KCC2 antibodies (in red) brightly
labeled the plasma membranes of neuronal somata and dendrites
throughout the rat cerebellum, indicating that KCC2 is largely
postsynaptic in its distribution. In contrast, the anti-GFAP antibody
(in green) labeled Bergmann fibers that predominate in the
molecular layer as well as some glial processes that occur within the
granule cell layer. These data are consistent with the neuron-specific
nature of the KCC2 protein and confirm our earlier findings based on
in situ hybridization experiments (7).

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Fig. 9.
Immunocytochemistry with anti-KCC2 polyclonal
antibodies and anti-GABAA receptor
2/ 3-subunit
monoclonal antibody in rat cerebellum. Diaminobenzidine reaction
product was used for immunocytochemical detection on coronal sections
(15-20 µm). KCC2 immunostaining in rat cerebellum at low
(A, scale bar is 200 µm) and high power
(B, scale bar is 100 µm). Immunostaining of the
GABAA receptor 2/ 3-subunits
in rat cerebellum at low (C, scale bar is 200 µm) and high power (D, scale bar is 100 µm).
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Fig. 10.
Double-labeled immunofluorescent microscopy
in the rat cerebellum of KCC2 and GFAP (A-C) and KCC2
and
2/ 3-subunits
of the GABAA receptor (D-F). Digital
images were obtained from confocal laser scanning microscopy. The KCC2
protein (A, red) exhibits no distinct colocalization with
GFAP (B, green) in the superimposed digital picture
displayed in C. In contrast, distinct colocalization of KCC2
protein (D, red) and the
2/ 3-subunits of the GABAA
receptor (E, green) is clearly observed in the granule cell
layer as a yellow signal when the digital images were superimposed in
F. Scale bars are 45 µm.
|
|
The granule cell layer of the cerebellum exhibited the most intense
immunocytochemical staining for KCC2 as well as for the GABAA receptor
2/
3-subunits
(Fig. 9, A-D). Using double-labeled fluorescent microscopy,
these two proteins exhibited a distinct colocalization in the granule
cell layer (Fig. 10, D-F). There was no
stratification of KCC2 staining through the depth of the granule cell
layer. Fluorescent labeling for KCC2 and
2/
3-subunits of the GABAA
receptor brightly outlined the plasma membranes of granule cell somata
(Fig. 10, D-F). Structures that stained most intensely for KCC2 and the
2/
3-subunits
of the GABAA receptor did not show nuclear counterstaining
with hematoxylin, indicating they were cerebellar glomeruli.
In sections stained by the immunoperoxidase method, there was little
overt concentration of KCC2 around the cell body or axon hillock of
Purkinje cells (Fig. 9B). Immunofluorescent labeling for
KCC2, however, showed clear labeling on the plasma membrane of Purkinje
cell somata (Fig. 10, A and D). As observed in
earlier studies, the
2/
3-subunits of the
GABAA receptor exhibited little reactivity with the
Purkinje cell somata using either immunoperoxidase or immunofluorescent
labeling (Figs. 9D and 10E; Refs. 11 and 15).
Immunostaining for KCC2 in the molecular layer of the rat cerebellum
was distinctly punctate and oriented mostly along neuronal processes
(Figs. 9B and 10, A and D). The
anti-GABAA receptor antibodies exhibited very low level
staining in the molecular layer (Figs. 9, C and
D, and 10E), confirming previous reports for the
2/
3-subunits of the GABAA
receptor in this region (11, 15). Purkinje cell dendrites alone may not
account for all of the KCC2 immunostaining in the molecular layer.
Golgi and basket cells have significant numbers of small dendrites
running perpendicularly and obliquely through the molecular layer.
Staining in the molecular layer may be associated with the dendritic
processes of all these neurons. The plasma membranes of neuronal somata
in the molecular layer were also distinctly outlined with KCC2 (Figs.
9B, and 10, A and D). These neurons
likely included both basket and stellate cells. There was little KCC2
staining of neuronal processes along the long axis of the folia in
transverse sections, indicating the absence of significant KCC2 protein
in the parallel fibers of the granule cells.
 |
DISCUSSION |
Intracellular [Cl
] is an important component in
determining the direction of Cl
movement through
conductive pathways. In order for conductive Cl
movement
to take place, Cl
must be maintained away from
equilibrium through active transport mechanisms. An emerging hypothesis
is that the cation chloride cotransporters (i.e. Na-K-Cl and
K-Cl cotransport) of various cell types are important regulators of
[Cl
]i and, therefore, are important
determinants of the direction and driving force for net
Cl
movement through anion channels (16-19). In neurons,
a K-Cl cotransport system has been implicated as the active
Cl
extrusion mechanism that maintains low
[Cl
]i and an inwardly directed Cl
electrochemical gradient necessary for the inhibitory function of
GABAA receptors (e.g. Refs. 20-24). In the
present study, we have developed antibodies against the neuron-specific
K-Cl cotransporter, KCC2. These immunological probes were used to
characterize and localize the protein to understand better the role of
KCC2 in ion homeostasis in the nervous system.
The anti-KCC2 antibodies were generated against a purified KCC2 fusion
protein, and they specifically recognized the ~140-kDa KCC2 protein
from a number of vertebrate species. Importantly, the antibodies
displayed no cross-reactivity with the KCC1 isoform. Like the
exogenously expressed KCC2 protein in stable HEK-293 cells (8), the
native KCC2 protein is glycosylated. Following treatment with
N-glycosidase F, the apparent molecular mass of the rat
brain KCC2 protein decreased from ~140- to ~125-kDa. The fact that
the protein, following treatment with N-glycosidase F,
displayed a molecular mass very close to that predicted from the
cDNA (123.6-kDa) indicates that the predominant glycans are N-linked. Other members of the cation chloride cotransporter
gene family have been shown to be adorned predominantly with
N-linked oligosaccharides (9, 25, 26). The region of the
KCC2 protein likely harboring the N-linked glycans is the
large predicted extracellular loop between putative transmembrane
segments 5 and 6 where four consensus sites for
N-glycosylation are located.
Expression of KCC2 appears to require neuronal differentiation. We
failed to detect KCC2 protein in a number of undifferentiated neuronal
cell lines. However, we did observe KCC2 protein in primary cultured
neurons, especially in a dispersed retinal culture. In these retinal
cultures, KCC2 protein exhibited a distinctly punctate distribution at
the plasma membrane of amacrine cell somata and dendrites, and the
highest expression was observed in regions where cells contacted each
other. As amacrine cells in culture form synapses and autapses that are
exclusively GABAAergic (10), these findings indicate that
KCC2 might be highly localized at GABAAergic synapses. Such
a model would allow KCC2 to effectively control
[Cl
]i immediately at the inhibitory synapse.
The fact that KCC2 was observed only in differentiated cultured neurons
indicates that synaptic formation may be an important requirement for
KCC2 expression.
In an earlier report, we demonstrated that KCC2 transcript was
specifically localized in neurons throughout the rat central nervous
system and was especially abundant within the cerebellum (7). Western
blot analysis and immunolocalization studies conducted with the
anti-KCC2 antibodies fully support these findings. Distinct KCC2
immunostaining was observed in most cerebellar neurons, and it was
found predominantly if not exclusively at the plasma membranes of
neuronal somata and dendrites. No significant KCC2 staining was evident
along axons or in glial cells of the cerebellum (e.g. Bergmann glial cells). The lack of glial cell staining with the anti-KCC2 antibodies was confirmed in a double label fluorescent experiment with GFAP, which is a specific marker for glial cells (Fig.
10, A-C). These data are consistent with a neuron-specific localization of KCC2 and also provide strong support for a postsynaptic function of KCC2.
The most intense immunostaining for KCC2 in the cerebellum was observed
in discrete areas within the granule cell layer that appeared to be
cerebellar glomeruli. Importantly, KCC2 exhibited a distinct
colocalization with the
2/
3-subunits of
the GABAA receptor at the cerebellar glomeruli. Golgi cells
are GABAergic, and their axons synapse with granule cells at the
cerebellar glomeruli where granule cell dendrites form complex synaptic
contacts with the terminals of afferent mossy fibers. The cerebellar
glomeruli are very rich in inhibitory synapses and exhibit significant
immunoreactivity for glutamic acid decarboxylase, GABA, and
GABAA receptor (Fig. 9, C and D,
Refs. 15 and 27-29). We propose that the intense KCC2 immunostaining
associated with the cerebellar glomeruli is likely at the postsynaptic
membrane of the granule cell dendrites making up these synaptic
structures. Interestingly, we clearly observed plasma membrane staining
of KCC2 around the cell bodies of granule cells. The plasma membranes
of granule cell somata also stained for the GABAA receptor
2/
3-subunits (Fig. 9, C and
D; Refs. 15, 29, and 30). The somatic localization of GABAA receptors in granule cells is believed to be
extrasynaptic as granule cells are not innervated at their cell body
(31). This extrasynaptic colocalization of GABAA receptor
and KCC2 at the plasma membrane of granule cell somata is consistent
with an hypothesis of inhibitory transmission by GABA spillover which may be important in controlling granule cell excitability (32, 33).
We observed a lower level of KCC2 immunoreactivity within the molecular
layer of the cerebellum. The plasma membranes of neuronal somata within
the molecular layer clearly stained for KCC2, and these likely included
both basket and stellate cells. The KCC2 immunoreactivity of stellate
and basket cell bodies is consistent with the GABAergic inhibition
these cells receive from other stellate and basket cells as well as
reciprocal GABAergic inhibition from Purkinje cells (34). Much of the
KCC2 immunostaining within the molecular layer was punctate, indicating
that KCC2 may localize at specific sites along the numerous dendrites
that occur in this region, especially from Purkinje cells. Although
2/
3-subunits of the GABAA
receptor only weakly stain the dendritic tree of Purkinje cells (Fig.
9, C and D, and see Refs. 15 and 29), significant
immunoreactivity of Purkinje cell dendrites has been observed with
anti-GABAA receptor antibodies prepared to the
2-subunit (35). Purkinje cell dendrites receive
GABAergic synapses predominantly from stellate cells. As proposed for
KCC2 expression in cultured amacrine cells, we hypothesize that KCC2 is
highly localized at GABAAergic inhibitory synapses along
Purkinje cell dendrites. GABAergic inhibition is known to occur at the
soma of Purkinje cells (36, 37). The axons of basket cells surround the
soma and axon hillock of Purkinje cells, forming a synaptic structure called pinceaux (38, 39). Basket cells are GABAergic
inhibitory interneurons as their axons are strongly associated with
both GABA and glutamic acid decarboxylase immunoreactivity (15). Significantly, we observed distinct but low level immunostaining for
KCC2 at the plasma membranes of Purkinje cell somata. A weak staining
pattern was also observed for the GABAA receptor
2/
3-subunit antibodies (Fig. 9,
C and D, and see Refs. 15, 29, and 30). The
plasma membranes of Purkinje cell somata have been reported to exhibit
strong immunoreactivity for the
1- and
2-subunits of the GABAA receptor (30, 35).
Furthermore, electrophysiological studies with outside-out patches have
reported abundant functional GABAA receptors on cultured
Purkinje cell somata (40). These latter studies indicate the presence
of significant GABAAergic inhibition at the soma of
Purkinje cells. Such inhibition would require the colocalization of a
Cl
extrusion mechanism, like KCC2, and the KCC2
immunoreactivity observed in the Purkinje cell somata is consistent
with KCC2 mediating this function.
In this report, we have described the development and characterization
of antibodies to the neuron-specific K-Cl cotransporter, KCC2. These
antibodies are specific for the KCC2 isoform, recognize KCC2 protein
from a broad range of vertebrate species, and are useful in both
Western blot analysis and immunocytochemistry. KCC2 protein exhibited
specific somatic and dendritic neuronal localization in rat cerebellum
and was absent from axons and glial cells. Also, KCC2 exhibited a
distinct colocalization with the
2/
3-subunits of the GABAA
receptor at glomeruli and granule cell somata in the granule cell layer
of the rat cerebellum. These findings are consistent with a significant
postsynaptic as well as extrasynaptic membrane localization of KCC2.
Our KCC2 localization studies coupled with the recent KCC2 functional
studies of Rivera et al. (41) on pyramidal neurons of the
rat hippocampus provide strong support for the hypothesis that KCC2 is
the major neuronal Cl
extrusion pathway, permitting fast
hyperpolarizing postsynaptic inhibition in the brain.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Peter Cala, Chris Lytle, Kai
Kaila, and John Russell for many helpful discussions.
 |
FOOTNOTES |
*
This work was supported in part by grants (to J. A. P.)
from the Hibbard E. Williams Research Funds, NINDS Grant NS-36296 from
National Institutes of Health, the Epilepsy Foundation of America, and
the University of California, Davis, Health System Research Funds.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Recipient of a Research Scholar Award from the Council for
Tobacco Research. To whom correspondence should be addressed: MED Human
Physiology, University of California, One Shields Ave., Davis, CA
95616-8644. Tel.: 530-752-1359; Fax: 530-752-5423; E-mail: japayne{at}hph.ucdavis.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
GABAA,
-aminobutyric acid type A;
ABC, avidin-biotin complex;
DAB, diaminobenzidine;
DMEM, Dulbecco's modified Eagle's medium;
GFAP, glial fibrillary acidic protein;
GS/PBS, goat serum phosphate-buffered
saline;
HEK, human embryonic kidney;
[Cl
]i, intracellular Cl
concentration;
[K+]o, extracellular [K+];
KCC1, ubiquitous K-Cl cotransporter;
KCC2, neuron-specific K-Cl
cotransporter;
PBS, phosphate-buffered saline;
PCR, polymerase chain
reaction;
PVDF, polyvinylidene fluoride;
Tricine, N-[2-hydroxy-1,1-
bis(hydroxymethyl)ethyl]glycine.
 |
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