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(Received for publication, April 3, 1996)
From the Departments of Human Physiology and
§ Biological Chemistry, University of California School of
Medicine, Davis, California 95616
ABSTRACT Using a combination of data base searching,
polymerase chain reaction, and library screening, we have identified a
putative K-Cl cotransporter isoform (KCC2) in rat brain that is
specifically localized in neurons. A cDNA of 5566 bases was
obtained from overlapping clones and encoded a protein of 1116 amino
acids with a deduced molecular mass of 123.6 kDa. Over its full length,
the amino acid sequence of KCC2 is 67% identical to the widely
distributed K-Cl cotransporter isoform (KCC1) identified in rat brain
and rabbit kidney (Gillen, C., Brill, S., Payne, J.A., and Forbush, B.,
III (1996) J. Biol. Chem. 271, 16237-16244) but only
~25% identical to other members of the cation-chloride cotransporter
gene family, including ``loop'' diuretic-sensitive Na-K-Cl
cotransport and thiazide-sensitive Na-Cl cotransport. Based on analysis
of the primary structure as well as homology with other cation-chloride
cotransporters, we predict 12 transmembrane segments bounded by N- and
C-terminal cytoplasmic regions. Four sites for N-linked
glycosylation are predicted on an extracellular intermembrane loop
between putative transmembrane segments 5 and 6. Northern blot analysis
using a KCC2-specific cDNA probe revealed a very highly expressed
~5.6-kilobase transcript only in brain. Reverse
transcriptase-polymerase chain reaction revealed that KCC1 was present
in rat primary astrocytes and rat C6 glioma cells but that KCC2 was
completely absent from these cells, suggesting KCC2 was not of glial
cell origin. In situ hybridization studies demonstrated
that the KCC2 transcript was expressed at high levels in neurons
throughout the central nervous system, including CA1-CA4 pyramidal
neurons of the hippocampus, granular cells and Purkinje neurons of the
cerebellum, and many groups of neurons throughout the brainstem.
INTRODUCTION The ability of cells to maintain water and electrolyte homeostasis
is often dependent upon cation-chloride cotransport mechanisms. These
cotransporters directly couple the movement of cations and
Cl The K-Cl cotransporter was first described as a swelling-activated
K+ efflux pathway in avian red blood cells (2). It has
subsequently been described in numerous single cells, where it
functions to reduce cell volume after swelling by promoting an efflux
of K+ and Cl In addition to volume regulation and net transepithelial salt movement,
the K-Cl cotransporter appears to carry out a unique function in
neurons: the maintenance of low intracellular [Cl In previous studies, we and others have reported the cloning,
sequencing, and functional expression of multiple isoforms of the
Na-K-Cl cotransporter in secretory (NKCC1, also called BSC2; Refs.
23, 24, 25) and absorptive epithelia (NKCC2, also called BSC1; Refs. 26 and
27). These NKCC isoforms along with the thiazide-sensitive Na-Cl
cotransporter (NCC, also called TSC; Ref. 28) make up a new gene family
of membrane transport proteins. Based on similarities in substrate
transport and drug inhibition, the K-Cl cotransporter is a putative
member of this gene family of transport proteins (29, 30). Assuming
that the K-Cl cotransporter would be related to the CCC gene family, we
employed a cloning strategy based on data base searching, PCR, and
library screening to identify cDNAs displaying homology to NKCC.
Significantly, two closely related but distinctly different gene
products were identified in rat brain that are distantly related to
NKCC and NCC. The molecular and functional characterization of one of
these gene products (KCC1) as a K-Cl cotransporter is presented in the
accompanying article (31). In this report, we present the molecular
characterization of the second gene product as a putative K-Cl
cotransporter isoform (KCC2) that is expressed at very high levels in
the brain and is specifically localized in neurons. Based on its high
expression and neuronal specificity, we propose that this putative K-Cl
cotransporter isoform is responsible for the maintenance of low
[Cl EXPERIMENTAL PROCEDURES Expressed sequenced tags (EST)
from human brain (T16107) and human breast (H15821) were identified in
the GenBankTM data base using the BLAST search programs (32) and the
human colonic Na-K-Cl cotransporter (hNKCC1) as the queried sequence.
Using sequence information from these human ESTs, forward
(5 A rat (Sprague-Dawley)
whole brain cDNA library in The rtKCC2 cDNA was sequenced bidirectionally by the dideoxy chain
termination method using a combination of manual sequencing with
Sequenase II (U. S. Biochemical Corp.) and automated sequencing
(Applied Biosystems Inc.) with synthetic oligonucleotide primers and
fluorescent dideoxy terminators. Analysis of the nucleotide sequences
and deduced amino acid sequence were performed with programs from the
Genetics Computer Group software.
Rat C6 glioma cells and PC12 cells were
maintained in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% fetal bovine serum,
L-glutamine (2 mM), penicillin (50 units/ml),
and streptomycin (50 µg/ml). Rat primary astrocytes were prepared
from newborn Sprague-Dawley rats (33) and maintained in modified
Eagle's medium (Life Technologies, Inc.) supplemented with 5% horse
serum, 5% fetal calf serum, L-glutamine (2 mM), and antibiotic-antimycotic solution (Life
Technologies, Inc.). All cultured cells were maintained in a
water-jacketed humidified incubator (5% CO2 at
37 °C).
Total RNA was
isolated from fresh rat tissues and various cultured cells by the
guanidinium thiocyanate method (34). Poly(A)+ RNA was
purified from total RNA using magnetic beads (Poly(A)Ttract, Promega
Corp.). Samples of rat tissue mRNA were denatured by heating to
65 °C in formamide and formaldehyde and size-fractionated on a 1%
agarose gel. Fractionated mRNA was transferred to a nylon membrane
by semidry blotting. The rat tissue blot used in this study was the
same as that used for the KCC1 Northern hybridization in Gillen
et al. (31). This membrane was completely stripped of
previously hybridized KCC1 cDNA probe by incubating in 0.1% SCC
and 0.5% SDS at 75 °C, and confirmation of probe removal was
determined by film exposure (24 h at After
annealing 5 µg of total RNA with oligo(dT), cDNA was prepared
with Superscript II (Life Technologies, Inc.) following the
manufacturer's instructions. Oligonucleotide primers were synthesized
over regions specific for either the KCC1 or KCC2 cDNAs. For KCC1,
forward (5 RESULTS Two ESTs
were identified in the GenBankTM data base that encoded a peptide
~35% identical to hNKCC1, spanning predicted transmembrane segments
9-12 (see ``Experimental Procedures''). Forward and reverse
oligonucleotide primers were synthesized over this region and used to
amplify a 286-bp PCR product from a population of double-stranded
cDNAs prepared from whole rat brain. The 286-bp PCR product was
cloned and sequenced and was 91% identical to the nucleotide sequence
derived from the two human ESTs.
The 286-bp PCR product was 32P-labeled by random priming
and used to screen a whole rat brain cDNA library under low
stringency conditions. Nineteen cDNA clones were identified and
fully characterized from this initial screening. It was clear from
restriction and sequence analysis that the clones represented two
closely related but distinct gene products, KCC1 (7 cDNAs) and KCC2
(12 cDNAs). The molecular characterization of KCC1 is described in
the accompanying article (31). In order to obtain the 5
The KCC2 cDNA includes a full-length open reading frame encoding a
polypeptide of 1116 amino acids (Fig. 2). The initiating
methionine was assigned to an in-frame ATG within a strong Kozak
consensus site (GCCACC
The predicted molecular mass of KCC2 is 123.6 kDa. As shown in the
sequence alignment (Fig. 2), the full-length amino acid sequence of
KCC2 is 67% identical to KCC1, yet displays much lower identity
(~25%) to other members of the CCC gene family that includes both
isoforms of the bumetanide-sensitive Na-K-Cl cotransporter (NKCC1 and
NKCC2) and the thiazide-sensitive Na-Cl cotransporter (NCC).
Hydropathy analysis of KCC2 predicts a
large central hydrophobic region bounded by N- and C-terminal
hydrophilic domains (Fig. 3A), similar to the
structure predicted for the previously identified CCC proteins (NKCC
and NCC). Characteristic of this gene family, we predict 12 membrane-spanning
The N terminus is the most divergent region between the two KCC
isoforms, and KCC2 is truncated 21 amino acids at this end of the
primary sequence when compared to KCC1 (Fig. 2). Except for this
truncation, as well as a single amino acid insertion in KCC2 within the
predicted extracellular loop between putative transmembrane segments 5 and 6 and 51 amino acids that are inserted in KCC2 within the predicted
intracellular C-terminal domain, KCC1 and KCC2 align very well over
their entire length. The central core hydrophobic domain containing the
12 putative transmembrane (TM) segments is the region that displays the
highest identity among the two KCC isoforms (Fig.
4A). Seven of the putative TM segments share
The expression of KCC2 in various
rat tissues was examined by Northern blot analysis (Fig.
5). An isoform-specific cDNA probe was prepared that
contained sequence from the 3
In an attempt to examine the distribution of KCC1 and KCC2 expression
within different cell types derived from the rat central and peripheral
nervous system, we used RT-PCR with isoform-specific oligonucleotide
primers (rtKCC1 and rtKCC2) and total RNA isolated from whole rat
brain, rat primary astrocytes, a rat glioma cell line (C6 cell), and a
rat pheochromocytoma cell line (PC12 cell). As shown in Fig.
6, each of the tissue samples displayed a RT-PCR product
of the correct size (233 bp) in the reaction containing primers
specific for the widely distributed KCC1 isoform; however, only whole
rat brain RNA exhibited a RT-PCR product of the correct size (399-bp)
in the reaction containing primers specific for the KCC2 isoform.
In order to examine in greater
detail the distribution of the KCC2 transcript within the rat brain, we
used in situ hybridization with a KCC2-specific
35S-labeled riboprobe. Film autoradiographic analysis of
sagittal brain sections hybridized with the KCC2-specific riboprobe
showed strong expression of mRNA in all layers of the cortex, all
areas of the hippocampus and the granular layer of the cerebellum (Fig.
7A). Expression is also evident in areas of
the brainstem. Signal was not detected in sections treated with RNase A
before hybridization (data not shown).
Microautoradiography of the brain sections showed specific
hybridization signal in neurons in many areas of the brain (Fig. 7,
B-E). White matter in the corpus callosum was devoid of
signal, supporting a neuronal localization for KCC2 mRNA. High
expression of KCC2 mRNA was evident in all layers of the cortex,
all regions of the hippocampus (CA1-CA4 pyramidal neurons, and dentate
gyrus), and Purkinje neurons of the cerebellum. Granular cells in the
cerebellum also expressed KCC2 mRNA but at lower levels than the
Purkinje neurons. Additionally, many groups of neurons throughout the
brainstem showed high expression of KCC2 mRNA.
DISCUSSION This paper describes the molecular characterization of a cDNA
from rat brain, which is proposed to encode a neuronal-specific isoform
of the K-Cl cotransporter (KCC2). While KCC2 has not yet been
functionally expressed, over the full length it displays high amino
acid identity (67%) to the K-Cl cotransporter (KCC1) identified and
functionally characterized in the accompanying article (31). Together,
these two KCC proteins share distant relationship (~25% amino acid
identity) to a gene family of cation-chloride cotransporter proteins
(see Fig. 2 in Ref. 31), which currently includes two isoforms of the
bumetanide-sensitive Na-K-Cl cotransporter (NKCC1 and NKCC2; Refs.
23, 24, 25, 26, 27), the thiazide-sensitive Na-Cl cotransporter (NCC; Refs. 27 and
28), and a number of uncharacterized gene products from cyanobacteria
(36), yeast (), Caenorhabditis elegans
(37, 38), and Manduca sexta (). Based on
the similarities in substrate transport (K+ and
Cl Analysis of the primary structures of the KCC proteins indicates that
overall they are similar to the other members of the CCC gene family,
i.e. hydrophilic N- and C-terminal regions bounding a
central core hydrophobic domain containing 12 putative transmembrane
segments. As expected for a family of membrane proteins, the central
hydrophobic domain containing the TM segments is the region displaying
the highest identity among all of the CCC proteins, including KCC1 and
KCC2. Significantly, the KCC proteins display high identity to hNKCC1
(~80%) over a short 28-amino acid intermembrane loop between
putative TM segments 2 and 3, which is the most highly conserved region
among all members of the CCC gene family. The function of this highly
conserved region of these proteins is unknown, but it is significant
that in the renal Na-K-Cl cotransporter (NKCC2) a portion of this
intermembrane loop is encoded by a cassette exon that is alternatively
spliced in a mutually exclusive fashion, leading to three distinctly
different splice variants of this protein (26, 39).
The KCC proteins do exhibit a distinct structural feature that sets
them apart from the previously identified CCC proteins. KCC1 and KCC2
both have a large predicted extracellular intermembrane loop harboring
four N-linked glycosylation sites between putative TM
segments 5 and 6. In the Na+-dependent CCC
proteins (i.e. NKCC and NCC) the extracellular intermembrane
loop containing glycosylation sites is predicted between TM segments 7 and 8 (23, 25, 26, 27, 28).
Previous molecular studies of the CCC proteins have noted that the N
terminus is the region of greatest divergence among the different
proteins (NKCC to NCC) and also among isoforms of the same protein
(NKCC1 to NKCC2; Refs. 25 and 26). This divergence at the N terminus is
also observed between the two KCC isoforms, where they display only
51% amino acid identity and an additional 21 amino acids are found at
the N terminus of KCC1. We have suggested that this N-terminal
variability in the Na-K-Cl cotransporter indicates that this region is
not likely to be directly involved in ion translocation (25). In direct
contrast to the N-terminal region, the large hydrophilic C terminus is
more highly conserved between the KCC isoforms (70% identical). The
K-Cl cotransporter of red blood cells has been shown to be regulated by
phosphorylation-dephosphorylation events (6, 7, 8, 9, 10); however, the
kinase-phosphatase enzymes involved have not yet been clearly
identified. A number of protein kinase C consensus sites are localized
within the C terminus of the KCC proteins, but only one is common to
both isoforms (rtKCC1-Thr748 and
rtKCC2-Ser728). Additionally, KCC2 has a tyrosine kinase
consensus phosphorylation site that is not present in KCC1.
Significantly, in comparing the KCC isoforms, KCC2 has a large
insertion within the C terminus, which is exceedingly rich in serines
and negatively charged amino acids (Asp and Glu) and also contains a
potential protein kinase C phosphorylation site (Ser940).
Based on these findings, KCC1 and KCC2 may display very distinct
differences in their regulation.
Northern blot analysis was used to examine the tissue distribution of
the KCC2 transcript in the rat. In direct contrast to KCC1, which
displays a very wide tissue distribution in rat (31), the KCC2
transcript was found only in brain. Consistent with a specific
distribution of KCC2 within the nervous system is the finding that
highly homologous nucleotide sequences have been identified in the
GenBankTM EST data base that are derived either from whole brain or
retinal tissues. As the K-Cl cotransporter has been described in
various cells and tissues, we propose that KCC1 is likely the
``housekeeping'' form of this protein responsible for cell volume
regulation (31). Reverse transcriptase-PCR was used to examine the
distribution of KCC1 and KCC2 in rat primary astrocytes, rat C6 glioma
cells, and rat PC12 pheochromocytoma cells. Each of these cells
displayed a RT-PCR product of the correct size only for the
oligonucleotide primers specific for KCC1. Only the whole rat brain RNA
exhibited correctly sized RT-PCR products for both the KCC1 and
KCC2-specific primers. These data support the role of KCC1 as the
``housekeeping'' isoform and indicate that KCC2 probably is not of
glial cell origin.
In situ hybridization with a 35S-labeled
antisense cRNA probe specific for the KCC2 transcript demonstrated that
the KCC2 transcript was found in neurons throughout the rat central
nervous system, including pyramidal neurons of the hippocampus and
Purkinje neurons and granular cells of the cerebellum. The fact that
the white matter of the corpus callosum was devoid of signal is
consistent with a neuronal-specific localization. The importance of
this putative electroneutral cotransporter in neurons is underscored by
its tremendous expression throughout the entire central nervous system
as evidenced by Northern blot analysis (Fig. 5) and by in
situ hybridization (Fig. 7A).
The
K-Cl cotransporter is a major Cl It is interesting to note that in some neurons GABA causes a membrane
depolarization with an efflux of Cl In the present study, we have identified a putative K-Cl cotransporter
that is specifically localized to neurons and is highly expressed
throughout the central nervous system. We proposed that this putative
neuronal K-Cl cotransporter may be the so-called ``Cl FOOTNOTES The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U55816[GenBank]. Acknowledgments We are indebted to Drs. Peter Cala, Chris
Lytle, Vijaya Kumari, Traci Yerby, Dandan Sun, Chris Gillen, and Bliss
Forbush III for many helpful discussions and for reading the
manuscript. Cell lines and rat primary astrocytes were kindly provided
by Dandan Sun and Lee Anne McLean. Automated sequencing was performed
by the W. M. Keck Foundation Nucleic Acid Facility.
REFERENCES
Volume 271, Number 27,
Issue of July 5, 1996
pp. 16245-16252
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A NEURONAL-SPECIFIC ISOFORM*
,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
ions in an electrically neutral fashion, and
therefore, net ion movement is dependent solely on the chemical
gradients of the transported ions. Two such cation-chloride
cotransporters (CCC)1 include the Na-K-Cl
and K-Cl cotransporters. Both of these transporters are sensitive to
the sulfamoylbenzoic acid ``loop'' diuretics furosemide and
bumetanide; however, the K-Cl cotransporter has a much lower affinity
for these drugs than the Na-K-Cl cotransporter (1).
and osmotically obliged water
(for recent review, see Ref. 3). The mechanism and regulation of K-Cl
cotransport has been extensively studied in vertebrate red blood cells.
For example, the direct coupling and 1:1 stoichiometry of
K+ and Cl fluxes have been clearly
established in human red blood cells (4, 5), and a net
dephosphorylation event has been shown to be necessary for activation
of the red blood cell K-Cl cotransporter (6, 7, 8, 9, 10). A K-Cl cotransporter
has also been reported in secretory and absorptive epithelia, where, in
addition to regulating cell volume, it may participate in net
transepithelial salt and water movement (11, 12, 13, 14, 15).
]
([Cl]i). Evidence supporting the role of a K-Cl
cotransporter as a Cl efflux pathway maintaining low
[Cl]i has been reported in both invertebrate
and vertebrate neuronal preparations (16, 17, 18, 19, 20, 21, 22).
Chloride-dependent synaptic inhibition in many neurons
relies upon the maintenance of low [Cl]i and a
favorable inwardly directed Cl electrochemical gradient.
The inhibitory postsynaptic potential (IPSP) elicited by inhibitory
neurotransmitters such as 
-aminobutyric acid (GABA) and glycine is
dependent upon the conductive movement of anions through a ligand-gated
channel in the membrane. For example, the GABAA receptor
functions as an anion channel that is highly selective for
Cl. The IPSP results from an influx of Cl
through the ligand-gated channel that causes a hyperpolarization of the
membrane, and thus drives the membrane potential away from its
threshold value. In order to prevent the collapse of the
Cl electrochemical gradient, an ``active''
Cl extrusion mechanism must be present in these GABA- and
glycine-sensitive neurons. As a secondary active transport mechanism,
the K-Cl cotransporter appears to carry out this important transport
function in neurons.
]i in neurons and the proper function of
Cl-sensitive channels (inhibitory neurotransmitter
receptors) in postsynaptic inhibition.
-CATCCTCATCGCCTCCCTCG-3) and reverse (5-CGTCACCCCACTCCTTCTCAGC-3)
oligonucleotide primers were synthesized and used to amplify a 286-bp
fragment from double-stranded cDNA prepared from 1 µg of
poly(A)+ RNA isolated from whole rat brain (Marathon
cDNA amplification kit, Clontech). The reaction volume of 50 µl
contained 5 µl of a 1:250 dilution of the double-stranded rat brain
cDNA, 0.2 µM of each primer, 50 mM
Tris-HCl (pH 9.2, 25 °C), 16 mM
(NH4)2SO4, 1.75 mM
MgCl2, 0.2 mM dNTP and 3 units of ExpandTM long
template DNA polymerase (Boehringer Mannheim). Twenty-eight cycles of
PCR were performed (each consisting of incubation of 30 s at
94 °C, 30 s at 60 °C, and 2 min at 68 °C). The 286-bp PCR
product from five reaction vials was combined, precipitated, and
isolated on a 1.5% agarose gel. After purification with glass beads
(Geneclean), the 286-bp PCR product was cloned into the plasmid pCRII
(Invitrogen) and sequenced in both directions.
ZAP II was obtained from Stratagene
(La Jolla, CA). Approximately 7.5 × 105 plaques were
screened with the 286-bp PCR product after labeling with
[32P]dCTP by random priming (Boehringer Mannheim). Plaque
lifts were screened under low stringency conditions (16 h at 35 °C
in 50% formamide, 5 × SSPE, 5 × Denhardt's solution,
0.1% SDS, and 100 µg/ml salmon sperm DNA) with a final stringent
wash at 50 °C in 0.5 × SSC, and 0.1% SDS for 15 min. Positive
clones were plaque-purified, excised into pBluescript SK,
and then characterized by 5- and 3-end sequencing, restriction
mapping, and Southern blotting. Additional clones encoding the 5-end
of rtKCC1 and rtKCC2 were obtained by rescreening the library under
similar conditions as above using PCR products derived from cDNA
clones obtained from the initial screening (rtKCC1: nt 610-950;
rtKCC2: nt 546-1000).
70 °C). A KCC2
isoform-specific cDNA probe was prepared from an 783-bp
PstI-PstI (rtKCC2: nt 4668-5451) fragment
derived from the 3-untranslated region contained in clone ERB10. The
rat tissue Northern blot was prehybridized 4 h at 65 °C in 0.25 M Na2HPO4 (pH 7.2), 1 mM EDTA, and 7% SDS and then hybridized for 16 h in
fresh hybridization solution containing 106 cpm/ml
32P-labeled cDNA probe. The blot was subjected to a
final stringent wash of 20 min at 58 °C in 40 mM
Na2HPO4 (pH 7.2), 1 mM EDTA, and
1% SDS and analyzed by autoradiography at 70 °C with an
intensifying screen.
-CCTGGAGTTGGGTTGTCTAAGA-3) and reverse
(5-CATCAGCCCTCACCAGTCATCTC-3) primers were used to amplify a 233-bp
fragment from KCC1 (rtKCC1: nt 3278-3510). For KCC2, forward
(5-CTCAACAACCTGACGGACTG-3) and reverse
(5-GCAGAAGGACTCCATGATGCCTGCG-3) primers were used to amplify a 399-bp
fragment from KCC2 (rtKCC2: nt 4-402). The PCR reaction volume of 50 µl contained 2 µl of template cDNA (or water for negative
controls), 0.2 µM of forward and reverse primer, 50 mM KCl, 10 mM Tris-HCl (pH 9.0, 25 °C),
0.1% Triton X-100, 1.5 mM MgCl2, 0.2 mM dNTP, and 2.5 units of Taq DNA polymerase
(Promega). After heating the reaction contents for 3 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).
Ten µl of the PCR reactions were then run on a 2.0% agarose gel
containing ethidium bromide.
In situ hybridization was
performed on sections from normal male Wistar rats. Rats were killed by
decapitation, and the whole brain was rapidly removed, frozen on dry
ice, and stored at 80 °C, until processed for in situ
hybridization, as described previously (35). A KCC2-specific probe
similar to that used for Northern blot analysis, was generated by
linearization of clone ERB10 with XhoI (rtKCC2: nt
4712-5451), and cRNA probe transcribed in vitro using T3
RNA polymerase (Boeringer Mannheim), [35S]UTP (800 Ci/mmol, Amersham International, United Kingdom) and unlabeled
nucleotides to a specific activity of 3-5 × 108
Ci/mmol. In situ hybridization was performed on 10-µm
sagittal or coronal sections of brain mounted on
poly-L-lysine subbed slides. Sections were postfixed in 4%
paraformaldehyde in 0.1 M phosphate-buffered saline
solution for 10 min and rinsed once in phosphate-buffered saline and
three times in 2 × SSC. All solutions were treated with
diethylpyrocarbonate (0.02%) and autoclaved. 35S-Labeled
cRNA probes were denatured by heating at 70 °C and added to
hybridization mix (50% deionized formamide, 0.6 M NaCl, 10 mM Tris-Cl (pH 7.5), 0.02% Ficoll, 0.02%
polyvinylpyrrolidone, 0.1% bovine serum albumin, 1 mM
EDTA, 100 µg/ml denatured salmon sperm DNA, 0.05 mg/ml yeast tRNA,
10% dextran sulfate, and 10 mM dithiothreitol) to give
107 counts/ml. Hybridization mix (200 µl) was added to
each slide, and hybridization was carried out overnight in sealed humid
chambers at 50 °C. After hybridization slides were rinsed in 2 × SSC and treated with RNase A (30 µg/ml) for 60 min at 37 °C.
Washes consisted of 2 × SSC at room temperature and 0.1 × SSC at 50 °C for 60 min. Sections were then dehydrated in graded
ethanols in 0.3 M ammonium acetate, air-dried, and exposed
to 
-max film (Amersham International) for 1 week for determination
of gross localization. Slides were then dipped in K5 nuclear emulsion
(Ilford, United Kingdom), exposed at 4 °C for 3 weeks, developed in
D19 developer (Eastman Kodak Co.), and counterstained with hemeatoxylin
and eosin. Control sections were pretreated with RNase A (100 µg/ml)
for 60 min at 37 °C prior to hybridization.
-end of the
coding regions of KCC1 and KCC2, the rat brain cDNA library was
rescreened with PCR products derived from the 5-ends of two of the
initial cDNAs (rtKCC1-ERB24 and rtKCC2-ERB10). Ten additional
cDNA clones were obtained from this second screening: KCC1 (2 cDNAs) and KCC2 (8 cDNAs). A full-length overlapping nucleotide
sequence of 5566 bp was identified for rtKCC2 from clones ERB10 and
5ERB12 (Fig. 1). One full-length clone was obtained from
the second library screening (5ERB14; Fig. 1).
Fig. 1.
Schematic diagram of selected clones encoding
the putative K-Cl cotransporter (rtKCC2) isolated from a whole rat
brain cDNA library. The cDNA obtained from overlapping
clones is displayed at top with the open reading frame
(open region) and 5- and 3-untranslated regions
(solid region).
C) contained in the most 5-end
clone (5ERB12; Fig. 1).
Fig. 2.
Sequence alignment of the deduced primary
structure of K-Cl cotransporters in rat brain (rtKCC2 and rtKCC1),
Na-K-Cl cotransporters in human colon (hNKCC1) and rat kidney (rtNKCC2;
F-variant), and thiazide-sensitive Na-Cl cotransporter in rat kidney
(rtNCC). The 12 putative transmembrane segments are
underlined below the alignment. Above the alignment,
predicted N-linked glycosylation sites are identified by
bars, potential phosphorylation sites for protein kinase C
by crosses (+), and one potential phosphorylation site for
tyrosine kinase by double crosses (
).
-helices and the placement of both the hydrophilic
N terminus and large C terminus within the cytoplasm (Fig.
3B). Eleven potential N-linked glycosylation
sites are present within the polypeptide, and four of these sites are
located on the predicted extracellular loop between putative
transmembrane segments 5 and 6 (Figs. 2 and 3). There are no
consensus phosphorylation sites for protein kinase A; however, five
consensus phosphorylation sites for protein kinase C
(Thr34, Ser728, Thr787,
Ser940, and Ser1034) as well as one consensus
tyrosine protein kinase phosphorylation site (Tyr1081) are
located within the hydrophilic domains at the N or C terminus of the
protein.
Fig. 3.
A, hydropathy profile of the putative
K-Cl cotransporter from rat brain (rtKCC2). The hydropathy index was
determined by the Kyte and Doolittle algorithm (49) using a 15-amino
acid window. Above the hydropathy plot is a linear hypothetical model
of the rtKCC2 protein indicating the transmembrane region. The 12 putative transmembrane segments are indicated by numbers
above the line. B, proposed model of the putative K-Cl
cotransporter from rat brain (rtKCC2). Circles symbolize
amino acid residues. A potential site for tyrosine kinase
phosphorylation is highlighted in black, and branched
lines specify those asparagines presumed to be capable of
anchoring oligosaccharide.
90% identity; these include: TM1, TM3, TM6, TM8, TM10, TM11, and
TM12. In addition, a number of regions predicted to be outside the TM
segments are very well conserved between KCC1 and KCC2, including a
predicted intracellular intermembrane loop between TM2 and TM3 as well
as a few small regions within the large hydrophilic C-terminal domain
(Figs. 2 and 4A). Interestingly, the predicted intermembrane
loop between TM2 and TM3 is the most highly conserved region among all
members of the CCC gene family, and the high identity displayed by the
KCC isoforms for the NKCC and NCC proteins over this region clearly
places these new KCC proteins within the CCC gene family (Figs. 2 and
4B).
Fig. 4.
Fractional identity of rtKCC2 to rtKCC1
(A) and the Na-K-Cl cotransporter in human colon
(B; hNKCC1). Fractional identity is averaged over a
running window of 21(A) or 15(B) amino acids. Above each plot is a
linear hypothetical model of the rtKCC2 protein indicating the position
of the proposed transmembrane segments and N-linked
glycosylation sites. The dotted line represents the
fractional identity over the full-length amino acid sequence. Note that
in B only the more highly conserved central hydrophobic
domain containing the putative 12 transmembrane segments is
displayed.
-untranslated region of the rtKCC2
cDNA and displayed no significant identity to the rtKCC1 cDNA.
This cDNA probe hybridized with a ~5.6-kilobase transcript in
brain only (Fig. 5). The KCC2 transcript was not detected in any other
tissue even after a longer exposure time (48 h at 70 °C; data not
shown). Thus, the KCC2 transcript is specific for brain tissue. In
other experiments, we also observed this same ~5.6-kilobase
transcript exclusively in brain following hybridization with cDNA
probes prepared from the more highly conserved coding regions of rtKCC1
and rtKCC2 (data not shown). Significantly, the level of KCC2
expression in the brain was very high. A strong signal for the brain
KCC2 transcript was observed after only a 4-h exposure of the Northern
blot as shown in Fig. 5.
Fig. 5.
Northern blot analysis of expression of KCC2
in rat tissues. In upper panel, tissue mRNA (5 µg
each) was hybridized with a 783-bp cDNA probe specific for the KCC2
transcript (see ``Experimental Procedures''). The blot was
rehybridized with a -actin cDNA probe as a control for RNA
integrity (lower panel). The blot was exposed for 4 h
(KCC2 probe) or 6 h (-actin probe) at 80 °C with an
intensifying screen. The second band in some lanes of the -actin
panel reflects a muscle-specific actin transcript of ~1.8
kilobases.
Fig. 6.
Expression of KCC1 and KCC2 in various cell
lines derived from rat nervous system. Reverse transcriptase-PCR
was performed using total RNA from whole rat brain, rat C6 glioma
cells, rat PC12 pheochromocytoma cells, and rat primary astrocytes.
Oligonucleotide primers specific for KCC1 and KCC2 were used to amplify
a 233-bp (KCC1) or 399-bp (KCC2) product. Negative controls for both
sets of primers containing no template cDNA are shown at far
left along with the DNA markers (values in base pairs). The
ethidium bromide-stained gel is depicted as an inverse image.
Fig. 7.
In situ hybridization detection of KCC2
mRNA in rat brain. A, film autoradiograph of 10-µm
section of rat brain hybridized with a KCC2-specific riboprobe (see
``Experimental Procedures'') showing widespread distribution of KCC2
mRNA throughout the brain. Scale bar,= 3 mm.
Cort, cortex; Hipp, hippocampus; Cb,
cerebellum; Bs, brainstem. B and C,
dark field photomicrographs of hippocampus. B shows field
denoted by solid box in A. Hybridization of probe
to the whole hippocampus is shown by silver grains overlying the
neuronal cell bodies. C shows a higher magnification of
neurons in the dentate gyrus. D and E, dark field
photomicrographs of cerebellum (dotted box in A).
D shows a field containing both granular and Purkinje
neurons (arrows). Note the high level of expression in the
Purkinje neurons surrounding the granular neurons. E shows a
higher power view of Purkinje neurons and granular neurons. Scale
bars represent 100 µm (C and E) and 500 µm (B and D).
) and drug inhibition (``loop'' diuretics),
physiological studies have indicated that the K-Cl cotransporter was
likely related to the Na-K-Cl cotransporter (29, 30). The
identification of KCC1 and KCC2 now appears to confirm this hypothesis
and allows us to add a new branch to the CCC gene family.
efflux pathway in a
number of cells, where it participates in cell volume regulation and
net transepithelial salt movement. In neurons, the K-Cl cotransporter
has been suspected to function as a ``Cl pump,''
maintaining low [Cl]i and a favorable inwardly
directed Cl electrochemical gradient (for review see Ref.
40). The importance of this function of K-Cl cotransport in neurons is
clearly illustrated when one considers that the action of the
inhibitory neurotransmitters, GABA and glycine, is dependent upon an
influx of Cl through ligand-gated channels, leading to
the generation of the IPSP. Early work using microelectrodes and the
giant neuron of the Aplysia abdominal ganglion clearly
showed that ECl was less negative than the
membrane potential (Em), demonstrating that
Cl must be actively extruded in these cells (41, 42).
Although reports have suggested the involvement of a primary active
transport mechanism (i.e. Cl-ATPase; 43, 44),
Cl movement against an electrochemical gradient in animal
cells has often been attributed to electroneutral cation-chloride
cotransport mechanisms where the electrical gradient is no longer a
factor (e.g. Na-K-Cl cotransport in the thick ascending limb
of mammalian kidney). In fact, evidence supporting the role of K-Cl
cotransport as the Cl efflux pathway in neurons has been
reported in both invertebrate and vertebrate systems. The crayfish
stretch receptor neuron (16, 17), insect neurosecretory cells (22), and
vertebrate cortical and hippocampal neurons (18, 19, 20, 21) appear to extrude
Cl against an electrochemical gradient by coupling
Cl transport to the favorable outwardly directed
K+ chemical gradient, i.e. K-Cl cotransport.
These studies have indirectly characterized this transport pathway by
examining shifts in ECl (as estimated from
EGABA or EIPSP) following
alterations in [K+]o or furosemide treatment.
through the
GABAA receptor (45, 46, 47). In these neurons, elevated
[Cl]i is required (ECl > Em), and it is the Na-K-Cl cotransporter that
appears to be involved in maintaining this high
[Cl]i (for review, see Ref. 40). Under normal
physiological conditions, the ion chemical gradients are such that
Na-K-Cl cotransport operates in a net inward direction, and this
cotransporter is known to participate in maintaining high
[Cl]i in epithelial cells of
Cl-secreting tissues for the proper function of apical
Cl channels (48). Thus, it appears that electroneutral
cation-chloride cotransport processes (i.e. K-Cl and Na-K-Cl
cotransporters) play an important role in regulating
[Cl]i in neurons and determining the response
of neurons to inhibitory neurotransmitters. The use of electroneutral
transporters to maintain [Cl]i in neurons has
obvious benefits for an excitable cell.
pump'' of neurons that maintains low [Cl]i for
the proper function of ligand-gated Cl-sensitive channels
(GABAA and glycine receptors) in postsynaptic
inhibition.
To whom correspondence should be addressed. Tel.: 916-752-1359;
Fax: 916-752-5423.
1
The abbreviations used are: CCC, cation-chloride
cotransporter; KCC, K-Cl cotransporter; NKCC, Na-K-Cl cotransporter;
NCC and TSC, thiazide-sensitive Na-Cl cotransporter; IPSP, inhibitory
postsynaptic potential; GABA, -aminobutyric acid; EST, expressed
sequenced tag; TM, transmembrane segment; ECl,
equilibrium potential for Cl; [X]i,
intracellular concentration; PCR, polymerase chain reaction; RT-PCR,
reverse transcriptase PCR; bp, base pair(s); nt, nucleotide(s).
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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H. Fujise, K. Higa, T. Kanemaru, M. Fukuda, N. C. Adragna, and P. K. Lauf GSH depletion, K-Cl cotransport, and regulatory volume decrease in high-K/high-GSH dog red blood cells Am J Physiol Cell Physiol, December 1, 2001; 281(6): C2003 - C2009. [Abstract] [Full Text] [PDF]
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W. Kelsch, S. Hormuzdi, E. Straube, A. Lewen, H. Monyer, and U. Misgeld Insulin-Like Growth Factor 1 and a Cytosolic Tyrosine Kinase Activate Chloride Outward Transport during Maturation of Hippocampal Neurons J. Neurosci., November 1, 2001; 21(21): 8339 - 8347. [Abstract] [Full Text] [PDF]
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G. Gamba Alternative splicing and diversity of renal transporters Am J Physiol Renal Physiol, November 1, 2001; 281(5): F781 - F794. [Abstract] [Full Text] [PDF]
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M. L. Jennings and M. F. Adame Direct estimate of 1:1 stoichiometry of K+-Cl{-} cotransport in rabbit erythrocytes Am J Physiol Cell Physiol, September 1, 2001; 281(3): C825 - C832. [Abstract] [Full Text] [PDF]
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A. Mercado, P. de los Heros, N. Vazquez, P. Meade, D. B. Mount, and G. Gamba Functional and molecular characterization of the K-Cl cotransporter of Xenopus laevis oocytes Am J Physiol Cell Physiol, August 1, 2001; 281(2): C670 - C680. [Abstract] [Full Text] [PDF]
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Z. J. Zhou A Critical Role of the Strychnine-Sensitive Glycinergic System in Spontaneous Retinal Waves of the Developing Rabbit J. Neurosci., July 15, 2001; 21(14): 5158 - 5168. [Abstract] [Full Text] [PDF]
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T. S. Perrot-Sinal, A. M. Davis, K. A. Gregerson, J. P. Y. Kao, and M. M. McCarthy Estradiol Enhances Excitatory Gammabutyric Acid-Mediated Calcium Signaling in Neonatal Hypothalamic Neurons Endocrinology, June 1, 2001; 142(6): 2238 - 2243. [Abstract] [Full Text] [PDF]
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M. F. Karadsheh and E. Delpire Neuronal Restrictive Silencing Element Is Found in the KCC2 Gene: Molecular Basis for KCC2-Specific Expression in Neurons J Neurophysiol, February 1, 2001; 85(2): 995 - 997. [Abstract] [Full Text] [PDF]
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E. Delpire Cation-Chloride Cotransporters in Neuronal Communication Physiology, December 1, 2000; 15(6): 309 - 312. [Abstract] [Full Text] [PDF]
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K. Strange, T. D. Singer, R. Morrison, and E. Delpire Dependence of KCC2 K-Cl cotransporter activity on a conserved carboxy terminus tyrosine residue Am J Physiol Cell Physiol, September 1, 2000; 279(3): C860 - C867. [Abstract] [Full Text] [PDF]
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V. C. Kotak and D. H. Sanes Long-Lasting Inhibitory Synaptic Depression is Age- and Calcium-Dependent J. Neurosci., August 1, 2000; 20(15): 5820 - 5826. [Abstract] [Full Text] [PDF]
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Y. Kakazu, S. Uchida, T. Nakagawa, N. Akaike, and J. Nabekura Reversibility and Cation Selectivity of the K+-Cl- Cotransport in Rat Central Neurons J Neurophysiol, July 1, 2000; 84(1): 281 - 288. [Abstract] [Full Text] [PDF]
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A. Monroy, C. Plata, S. C. Hebert, and G. Gamba Characterization of the thiazide-sensitive Na+-Cl- cotransporter: a new model for ions and diuretics interaction Am J Physiol Renal Physiol, July 1, 2000; 279(1): F161 - F169. [Abstract] [Full Text] [PDF]
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T. Q. Vu, J. A. Payne, and D. R. Copenhagen Localization and Developmental Expression Patterns of the Neuronal K-Cl Cotransporter (KCC2) in the Rat Retina J. Neurosci., February 15, 2000; 20(4): 1414 - 1423. [Abstract] [Full Text] [PDF]
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N. C. Adragna, R. E. White, S. N. Orlov, and P. K. Lauf K-Cl cotransport in vascular smooth muscle and erythrocytes: possible implication in vasodilation Am J Physiol Cell Physiol, February 1, 2000; 278(2): C381 - C390. [Abstract] [Full Text] [PDF]
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 J. Gibson, A. Cossins, and J. Ellory Oxygen-sensitive membrane transporters in vertebrate red cells J. Exp. Biol., January 5, 2000; 203(9): 1395 - 1407. [Abstract] [PDF]
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J. M. Russell Sodium-Potassium-Chloride Cotransport Physiol Rev, January 1, 2000; 80(1): 211 - 276. [Abstract] [Full Text] [PDF]
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J. E. Race, F. N. Makhlouf, P. J. Logue, F. H. Wilson, P. B. Dunham, and E. J. Holtzman Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter Am J Physiol Cell Physiol, December 1, 1999; 277(6): C1210 - C1219. [Abstract] [Full Text] [PDF]
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W. Su, B. E. Shmukler, M. N. Chernova, A. K. Stuart-Tilley, L. de Franceschi, C. Brugnara, and S. L. Alper Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation Am J Physiol Cell Physiol, November 1, 1999; 277(5): C899 - C912. [Abstract] [Full Text] [PDF]
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I. Ehrlich, S. Lohrke, and E. Friauf Shift from depolarizing to hyperpolarizing glycine action in rat auditory neurones is due to age-dependent Cl- regulation J. Physiol., October 1, 1999; 520(1): 121 - 137. [Abstract] [Full Text] [PDF]
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M. Flagella, L. L. Clarke, M. L. Miller, L. C. Erway, R. A. Giannella, A. Andringa, L. R. Gawenis, J. Kramer, J. J. Duffy, T. Doetschman, et al. Mice Lacking the Basolateral Na-K-2Cl Cotransporter Have Impaired Epithelial Chloride Secretion and Are Profoundly Deaf J. Biol. Chem., September 17, 1999; 274(38): 26946 - 26955. [Abstract] [Full Text] [PDF]
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W. Jarolimek, A. Lewen, and U. Misgeld A Furosemide-Sensitive K+-Cl- Cotransporter Counteracts Intracellular Cl- Accumulation and Depletion in Cultured Rat Midbrain Neurons J. Neurosci., June 15, 1999; 19(12): 4695 - 4704. [Abstract] [Full Text] [PDF]
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D. B. Mount, A. Mercado, L. Song, J. Xu, A. L. George Jr., E. Delpire, and G. Gamba Cloning and Characterization of KCC3 and KCC4, New Members of the Cation-Chloride Cotransporter Gene Family J. Biol. Chem., June 4, 1999; 274(23): 16355 - 16362. [Abstract] [Full Text] [PDF]
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J. R. Williams, J. W. Sharp, V. G. Kumari, M. Wilson, and J. A. Payne The Neuron-specific K-Cl Cotransporter, KCC2. ANTIBODY DEVELOPMENT AND INITIAL CHARACTERIZATION OF THE PROTEIN J. Biol. Chem., April 30, 1999; 274(18): 12656 - 12664. [Abstract] [Full Text] [PDF]
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Y. Kakazu, N. Akaike, S. Komiyama, and J. Nabekura Regulation of Intracellular Chloride by Cotransporters in Developing Lateral Superior Olive Neurons J. Neurosci., April 15, 1999; 19(8): 2843 - 2851. [Abstract] [Full Text] [PDF]
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K. Hiki, R. J. D'Andrea, J. Furze, J. Crawford, E. Woollatt, G. R. Sutherland, M. A. Vadas, and J. R. Gamble Cloning, Characterization, and Chromosomal Location of a Novel Human K+-Cl- Cotransporter J. Biol. Chem., April 9, 1999; 274(15): 10661 - 10667. [Abstract] [Full Text] [PDF]
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 S. Haapa, S. Suomalainen, S. Eerikäinen, M. Airaksinen, L. Paulin, and H. Savilahti An Efficient DNA Sequencing Strategy Based on the Bacteriophage Mu in Vitro DNA Transposition Reaction Genome Res., March 1, 1999; 9(3): 308 - 315. [Abstract] [Full Text]
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S. N. Orlov, N. C. Adragna, V. A. Adarichev, and P. Hamet Genetic and biochemical determinants of abnormal monovalent ion transport in primary hypertension Am J Physiol Cell Physiol, March 1, 1999; 276(3): C511 - C536. [Abstract] [Full Text] [PDF]
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C. M. Gillen and B. Forbush III Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells Am J Physiol Cell Physiol, February 1, 1999; 276(2): C328 - C336. [Abstract] [Full Text] [PDF]
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D. W. Hochman, R. D'Ambrosio, D. Janigro, and P. A. Schwartzkroin Extracellular Chloride and the Maintenance of Spontaneous Epileptiform Activity in Rat Hippocampal Slices J Neurophysiol, January 1, 1999; 81(1): 49 - 59. [Abstract] [Full Text] [PDF]
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H. Liapis, M. Nag, and D. M. Kaji K-Cl cotransporter expression in the human kidney Am J Physiol Cell Physiol, December 1, 1998; 275(6): C1432 - C1437. [Abstract] [Full Text] [PDF]
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E. J. Holtzman, S. Kumar, C. A. Faaland, F. Warner, P. J. Logue, S. J. Erickson, G. Ricken, J. Waldman, S. Kumar, and P. B. Dunham Cloning, characterization, and gene organization of K-Cl cotransporter from pig and human kidney and C. elegans Am J Physiol Renal Physiol, October 1, 1998; 275(4): F550 - F564. [Abstract] [Full Text] [PDF]
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P. K Lauf K+-Cl- cotransport: 'to be or not to be' oxygen sensitive J. Physiol., August 15, 1998; 511(1): 1 - 1. [Full Text] [PDF]
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P. Isenring, S. C. Jacoby, and B. Forbush III The role of transmembrane domain 2 in cation transport by the Na-K-Cl cotransporter PNAS, June 9, 1998; 95(12): 7179 - 7184. [Abstract] [Full Text] [PDF]
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P. Isenring, S. C. Jacoby, J. A. Payne, and B. Forbush III Comparison of Na-K-Cl Cotransporters. NKCC1, NKCC2, AND THE HEK CELL Na-K-Cl COTRANSPORTER J. Biol. Chem., May 1, 1998; 273(18): 11295 - 11301. [Abstract] [Full Text] [PDF]
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J. A. Payne Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation Am J Physiol Cell Physiol, November 1, 1997; 273(5): C1516 - C1525. [Abstract] [Full Text] [PDF]
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P. Isenring and B. Forbush III Ion and Bumetanide Binding by the Na-K-Cl Cotransporter. IMPORTANCE OF TRANSMEMBRANE DOMAINS J. Biol. Chem., September 26, 1997; 272(39): 24556 - 24562. [Abstract] [Full Text] [PDF]
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C. M. Gillen, S. Brill, J. A. Payne, and B. Forbush III Molecular Cloning and Functional Expression of the K-Cl Cotransporter from Rabbit, Rat, and Human. A NEW MEMBER OF THE CATION-CHLORIDE COTRANSPORTER FAMILY J. Biol. Chem., July 5, 1996; 271(27): 16237 - 16244. [Abstract] [Full Text] [PDF]
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L. Caron, F. Rousseau, E. Gagnon, and P. Isenring Cloning and Functional Characterization of a Cation-Cl- Cotransporter-interacting Protein J. Biol. Chem., October 6, 2000; 275(41): 32027 - 32036. [Abstract] [Full Text] [PDF]
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A. Mercado, L. Song, N. Vazquez, D. B. Mount, and G. Gamba Functional Comparison of the K+-Cl- Cotransporters KCC1 and KCC4 J. Biol. Chem., September 22, 2000; 275(39): 30326 - 30334. [Abstract] [Full Text] [PDF]
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P. Sangan, S. R. Brill, S. Sangan, B. Forbush III, and H. J. Binder Basolateral K-Cl Cotransporter Regulates Colonic Potassium Absorption in Potassium Depletion J. Biol. Chem., September 29, 2000; 275(40): 30813 - 30816. [Abstract] [Full Text] [PDF]
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M. Di Fulvio, T. M. Lincoln, P. K. Lauf, and N. C. Adragna Protein Kinase G Regulates Potassium Chloride Cotransporter-3 Expression in Primary Cultures of Rat Vascular Smooth Muscle Cells J. Biol. Chem., June 8, 2001; 276(24): 21046 - 21052. [Abstract] [Full Text] [PDF]
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N. Vazquez, A. Monroy, E. Dorantes, R. A. Munoz-Clares, and G. Gamba Functional differences between flounder and rat thiazide-sensitive Na-Cl cotransporter Am J Physiol Renal Physiol, April 1, 2002; 282(4): F599 - F607. [Abstract] [Full Text] [PDF]
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