From the Departments of Medicine, ** Pharmacology, and
§§ Anesthesiology, Vanderbilt University Medical
Center, Nashville, Tennessee 37232 and the ¶ Molecular Physiology
Unit, Instituto Nacional de la Nutrición Salvador Zubirán
and Instituto de Investigaciones Biomédicas UNAM, Mexico City,
Mexico
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ABSTRACT |
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The K+-Cl Potassium chloride cotransporters
(KCCs)1 were first described
as a swelling-activated K+ efflux in erythrocytes, the cell
type for which functional characterization is the most complete (1, 2).
The cotransport of K+ and Cl A major impediment to the study of K+-Cl Full-length cDNAs encoding two K+-Cl An increasing amount of data suggest further heterogeneity in the
proteins that mediate K+-Cl cDNA Cloning
mKCC3--
A BLAST (basic alignment and search tool) search (26)
of the EST data base revealed a number of mouse ESTs that were
homologous to rat KCC1 and KCC2. The cDNAs corresponding to four of
these ESTs (IMAGE clones 568084, 633794, 313521, and 698105) were
obtained from the IMAGE consortium (Research Genetics, Genome Systems
and/or the ATCC) and sequenced. These ESTs contained partial open
reading frames homologous to various segments of KCC1 and KCC2 (Fig.
1). The 3'-end of the open reading frame and the entire 3'-UTR were identified in a fifth IMAGE cDNA (clone 1314678). The tissue
distribution of mKCC3 was then assessed by RT-PCR, and widespread
expression of mKCC3 was detected with the S1/AS1 primer pair (Table
I), with particularly abundant
transcripts in kidney and heart (data not shown). Using a primer pair
(S2 and AS2 primers, Table I) spanning the gap between the IMAGE clones
313521 and 568084, a 1.2-kb fragment was amplified from C57BL/6J
kidneys and subcloned into the EcoRV site of pBluescript by
blunt-end ligation. PCR conditions for these and other gene-specific
primers were optimized using Taq 2000 and the Opti-Prime
buffer system (Stratagene). The following amplification protocol was
followed, unless specified otherwise: 30 cycles of denaturation
(92 °C, 2 min), annealing (54 °C, 1 min), and extension
(72 °C, 1 min), followed by a final extension step (72 °C, 8 min). The extreme 5'-end of mKCC3 was cloned from BALB/c mouse kidney
5'-RACE template (CLONTECH), using two antisense
primers (primers AS3 and AS4, Table I) and the AP1 adaptor primer from
CLONTECH (S3 primer). This PCR utilized AmpliTaq-Gold DNA polymerase (Perkin-Elmer) and a hot-start
amplification protocol, consisting of a 9-min enzyme activation step at
94 °C, followed by 35 cycles of 94 °C for 1 min and 68 °C for
2.5 min, and a final 10-min extension at 72 °C.
hKCC3--
A human KCC EST clone (TIGR clone 150738) was
obtained from the ATCC. Sequencing revealed that this cDNA is
derived from the human ortholog of mKCC3. Another human 5'-EST
(GenBankTM accession number F12342) exhibited strong amino
acid homology with the extreme 3' coding sequence of the other KCCs.
This EST overlaps with a large number of ESTs from the 3'-UTR of hKCC3, including the IMAGE clones 22250 and 51311 (Fig. 1). The gap between these cDNAs and the TIGR clone 150738 (Fig. 1) was bridged by RT-PCR with the primer pair S4/AS5 (Table I), using a human kidney template (CLONTECH). The PCR products for this
reaction were subcloned into pBluescript. Finally, the 5'-end of hKCC3
was cloned by sequential RT-PCR of human kidney with the primer pairs
S5/AS6 and S6/AS7. S5 and S6 were derived from IMAGE clones 1403108 and
mKCC3, respectively.
hKCC4--
Sequence analysis of another human EST cDNA (TIGR
clone 150620) indicated the existence of a fourth KCC, hKCC4. Northern
blot analysis revealed significant expression in muscle, and a single 5'-RACE cDNA was cloned from a human muscle template
(CLONTECH) using the S3/AS8 primer pair. This
5'-RACE PCR used Advantage polymerase mix
(CLONTECH) and a hot-start protocol consisting of
the following: 94 °C for 1 min, followed by 35 cycles of 94 °C
for 30 s, 62 °C for 30 s, and 68 °C for 3 min.
Screening a random-primed human muscle cDNA library
(CLONTECH) with a probe encompassing nucleotides
741-871 of hKCC4 yielded a single cDNA that extended 5' of the
start codon. Finally, two overlapping PCR fragments were amplified from
human brain template (CLONTECH) with the S7/AS9 and
S8/AS10 primer pairs (Table I), and subcloned into pCR2.1 by TA cloning (Invitrogen).
Sequence Analysis of KCC3 and KCC4 cDNAs
All cDNA clones were sequenced on both strands using
fluorescent dye terminator chemistry (Applied Biosystems). For cDNA
sequence derived exclusively from PCR, at least two cDNAs from two
separate PCR reactions were sequenced. Analyses of the nucleotide and
amino acid sequences were performed using the GeneWorks 2.5 and
MacVector 6.5 software packages (Oxford Molecular). Alignments and
other analyses also made use of the computer programs BLAST (26), DNAStar, and SMART (simple modular architecture research tool) (27).
Chromosomal Localization and Genomic Structure of hKCC3 and
hKCC4
Chromosomal assignments for the human KCC3 and KCC4 genes were
made using a PCR-based screening approach with the National Institute
of General Medical Sciences human/rodent somatic cell hybrid mapping
panel 1 (28). The primers used for hKCC3 mapping (S9 and AS11) amplify
a 377-bp segment of the 3' noncoding region from genomic DNA, and the
hKCC4 mapping primers (S10 and AS12) amplify a 561-bp segment of the 3'
noncoding sequence. PCR reactions using DNA from the NIGMS panel were
scored for the presence or absence of the appropriately sized product
using agarose gel electrophoresis.
The chromosomal localization of hKCC3 was verified by sequencing
the chromosome 5 genomic clone pMS621, a gift of Dr. John Armour (see
"Results"). Fine mapping of hKCC4 was performed by PCR using
radiation hybrid analysis with the Stanford G3 panel (Research
Genetics) and the S10/AS12 primer pair. Reaction products generated by
PCR were alkali-denatured, applied to a nylon membrane using a dot-blot
apparatus, and subjected to Southern blotting with a
32P-labeled internal oligonucleotide probe (primer S11,
Table I). Results were analyzed by querying the Stanford radiation
hybrid map (http://www-shgc.stanford.edu/RH/).
Northern Blots and RT-PCR
RNA was extracted from mouse tissues (C57BL/6J strain) using
guanidine isothiocyanate and cesium chloride. Total RNA (10 µg/lane) was size-fractionated by electrophoresis (5% formaldehyde, 1% agarose), transferred to a nylon membrane (Stratagene), and probed sequentially with 32P-labeled randomly primed probes
corresponding to full-length glyceraldehyde-3-phosphate dehydrogenase
and nucleotides 4417-5062 of mKCC3 (3'-UTR). Human multiple-tissue
Northern blots containing 2 µg/lane poly(A)+ RNA
(CLONTECH) were hybridized to probes generated by
PCR from the 3'-UTRs of hKCC3 (nucleotides 4598-4957) and hKCC4
(nucleotides 3624-4185) and to a human In Vitro Translation of mKCC3 Protein
1.0 µg of the full-length mKCC3 cDNA (see below) was
translated in vitro using [35S]methionine and
T7 RNA polymerase-coupled rabbit reticulocyte lysate (TNTTM
T7, Promega), both with and without pancreatic microsomes, for 90 min
at 30 °C. Protein was resolved by 7% SDS-polyacrylamide gel
electrophoresis followed by autoradiography.
Expression of mKCC3 in X. laevis Oocytes
For functional expression and in vitro translation, a
full-length mKCC3 cDNA was assembled in the Xenopus
expression vector pGEMHE (29). The resulting construct contains
nucleotides 55-3812 of mKCC3. For functional comparison with mKCC3, a
full-length rabKCC1 cDNA, the gift of Dr. Bliss Forbush III, was
subcloned into pGEMHE. To prepare a template for cRNA, the rabbit KCC1
and mKCC3 cDNAs were linearized at the 3'-end using
NheI, and cRNA was transcribed in vitro using the
T7 RNA polymerase and the mMESSAGE mMACHINE kit (Ambion).
Defolliculated stage V-VI oocytes were injected with 50 nl of water or
a solution containing cRNA at a concentration of 0.5 µg/µl (25 ng/oocyte). Oocytes were incubated at 17 °C in ND96 (96 mM Na+-Cl Functional expression of rabbit KCC1 and mKCC3 was assessed by
measuring tracer 86Rb+ uptake in groups of
20-25 oocytes 3 days after water or cRNA injection. Because
K+-Cl Sequence of KCC3 and KCC4--
Full-length KCC3 and KCC4 sequences
(deposited in the GenBankTM data base under accession
numbers AF087436 (mKCC3), AF105365 (hKCC3), and AF105366 (hKCC4)) were
determined from a number of overlapping cDNA clones (Fig.
1). The complete mouse and human KCC3
cDNAs are 5132 and 5230 nucleotides long, respectively, which is
close in size to the KCC3 transcripts seen on Northern blots (Figs.
5A and 6A). Both cDNAs contain open reading
frames of 3248 nucleotides, with 85% identity, and the predicted
proteins consist of 1083 amino acids and exhibit 91% identity. The
KCC3 3'-UTRs are only 34% identical, close to the lower limit of
conservation between mouse and human orthologs (30).
The hKCC4 cDNA sequence is 4237 nucleotides in length, with a
5'-UTR of 165 bp and a 3'-UTR of 622 bp. The discrepancy between the
cDNA size and the size of the KCC4 transcripts (Fig.
6C), as well as the lack of a consensus polyadenylation site
within the 3'-UTR, suggests that the 3'-UTR sequence is incomplete.
There are three in-frame start codons between nucleotides 165 and 195 of the hKCC4 cDNA, at which translational initiation would result in proteins of 1150, 1141, or 1135 amino acids in length. However, homology to KCC1 and KCC3 begins before the third methionine (Fig. 2), which is thus an unlikely
translational start site. Comparison with the mouse KCC4
sequence2 indicates
significant conservation of the first nine codons, which also contain a
PKC site (Fig. 2), and hence translation likely occurs at the first
start codon.
At least two KCC4 transcripts of 6-7 kb are detected by Northern blot
analysis (Fig. 6C), consistent with alternative splicing. In
comparison with other KCC4 5' cDNAs (Fig. 1), the single 5'-RACE cDNA contained a deletion of nucleotides 708-854, encoding TM-1 and TM-2 in the predicted KCC4 protein. The deleted region corresponds precisely to exon 4 of hKCC1 (16), and hence at least part of the
heterogeneity in KCC4 transcripts is the result of the alternative splicing of coding exons.
The four KCC proteins are 65-71% identical (Fig. 2). Sequence
alignments indicate that hKCC3 shares 69% identity with rat KCC2, 65%
identity with hKCC1, and 66% identity with hKCC4. The hKCC4 protein
shares 71% identity with hKCC1, 66% identity with rat KCC2, and 66%
identity with hKCC3. The identity between the KCCs and other
cation-chloride cotransporters is in the range of 27 to 33%. A
phylogenetic tree indicates that the mammalian cation-chloride
cotransporters fall into two groups, one composed of the
Na+-K+-2Cl
The seven mammalian cation-chloride cotransporters share a predicted
membrane topology. A central core of 12 TM domains is flanked by
hydrophilic amino- and carboxyl-terminal domains that are known to have
a cytoplasmic orientation (18). The major structural difference between
the KCCs and the Na+-linked cotransporters is the position
of a large glycosylated extracellular loop, which is predicted to occur
between TM-5 and TM-6 in the KCCs and between TM-7 and TM-8 in the
Na+-K+-2Cl
A comparison of the four KCCs reveals a number of intriguing
differences. KCC4 is the longest of the four because of an extension of
~60 amino acids at the extreme amino-terminal end (Fig. 2). Although
highly conserved, none of the TM domains in the four KCCs are
completely identical. Within the cytoplasmic domains the four KCCs
differ significantly in the distribution of consensus phosphorylation
sites for tyrosine kinases, protein kinase A, and protein kinase C
(PKC). A carboxyl-terminal tyrosine phosphorylation site in KCC2
(Tyr1087) is conserved in mouse and human KCC3
(Tyr1054) (Fig. 2). The KCC4 sequence predicts a total of
11 PKC sites, 7 contained within the first 90 amino acids (Fig. 2).
KCC4 has two potential protein kinase A sites, one of which
(Ser939) is a predicted substrate for both protein kinase A
and C. The KCC3 sequences predict fewer PKC sites, of which only two
are conserved in both mouse and human (Thr814 and
Ser1006).
KCC1 (3), KCC2 (4), and other members of the cation-chloride
cotransporter gene family (18) are known to be glycoproteins, and the
four KCC sequences contain three identical N-linked
glycosylation sites in the otherwise poorly conserved extracellular
loops (Fig. 2). The in vitro translation of mKCC3 results in
a protein with an apparent molecular mass of ~115 kDa, slightly lower
than the predicted core weight of 119 kDa. The addition of canine
pancreatic microsomes results in the appearance of an additional band
of higher molecular mass (Fig. 5C), which is consistent with
in vitro glycosylation.
Chromosomal Localization of hKCC3 and hKCC4--
Chromosomal
assignments for human KCC3 and KCC4 were defined using a PCR-based
somatic cell hybrid mapping strategy. The genes for KCC3 and KCC4 were
assigned to chromosomes 5 and 15, respectively. Fine mapping by
radiation hybrid analysis places the hKCC4 gene on chromosome 15q14
between the markers D15S1040 and D15S118. Further localization of KCC3
was facilitated by the finding that D5S110 (31), a chromosome 5 VNTR
(variable number of tandem repeats) minisatellite marker, is contained
within the gene. The corresponding genomic subclone, pMS621 (the gift
of Dr. John Armour), was sequenced to verify that it contained hKCC3
exons just 5' of the VNTR (Fig. 4). The
exon/intron boundaries in the hKCC1 gene (16) are conserved in this
portion of the hKCC3 gene, and the exons in pMS621 correspond to exons
14-17 of hKCC1 (Table II). The
chromosome 5 summary map generated by the Wessex Human Genetics
Institute (http://cedar.genetics.soton, ac.uk/pub/chrom5/map.html) indicates that D5S110 is on 5p15.3, between D5S678 and the
telomere.
Tissue Distribution of KCC3 and KCC4--
Northern blot analysis
was performed with probes derived from the 3'-UTRs of KCC3 and KCC4.
KCC3 probes detect 5.3-kb transcripts in a number of tissues, most
prominently in the heart and kidney (Figs.
5A and
6A). Very little KCC3
transcript is detectable in adult brain. KCC4 has a more restricted
expression pattern, with significant amounts of transcript found only
in muscle, brain, lung, heart, and kidney (Fig. 6C). At
least two different transcripts of 6-7 kb hybridize to KCC4 probes,
consistent with alternative splicing (see above).
Functional Expression of mKCC3 and Rabbit KCC1--
Microinjection
of Xenopus oocytes with cRNA in vitro transcribed
from mKCC3 and rabbit KCC1 resulted in significant increases in
86Rb+ uptake compared with water-injected
oocytes. The increased 86Rb+ uptake was evident
only when oocytes were incubated in hypotonic medium. Fig.
7A shows
86Rb+ uptake in H2O, rabbit KCC1,
and mKCC3-injected oocytes, using isotonic and hypotonic conditions. In
H2O-injected oocytes 86Rb+ uptake
increased from 26.3 ± 5.33 pmol/oocyte/h under isotonic conditions to 46.8 ± 2.79 pmol/oocyte/h in hypotonic conditions (p < 0.01). In hypotonic medium, however, tracer
Rb+ uptake decreased to 4.2 ± 0.43 pmol/oocyte/h in
the absence of extracellular Cl We report here the cloning of KCC3 and KCC4, expanding the family
of mammalian cation-chloride cotransporters to a total of seven members
(Fig. 3). All of the known cation-chloride transporters belong
functionally and/or structurally in one of three groups, encompassing
two Na+-K+-2Cl The four KCC proteins share a 65-71% identity in primary structure.
Within the central hydrophobic core, TM-2 and TM-4 show the most
variation (Fig. 2); data from an extensive study of ion affinity in the
Na+-K+-2Cl The structural similarity between the four KCCs strongly suggests that
both KCC3 and KCC4 encode K+-Cl The 5.3-kb KCC3 transcript is widely expressed in both mouse and human,
and ESTs have been reported from an array of cDNA libraries.
However, in both species KCC3 transcript is most abundant in the heart,
followed by the kidney. Cultured chick cardiac cells possess a
K+-Cl KCC4 has a more restricted expression pattern than KCC1 and KCC3,
confined primarily to muscle, brain, heart, and kidney. An
insulin-stimulated K+-Cl The combined results from this and other reports indicate that the
mammalian kidney expresses KCC1 (3, 16), KCC3, and KCC4 (Figs. 5 and
6). There is strong physiological evidence for K+-Cl The human KCC3 gene has been localized to chromosome 5. Fine mapping to
5p15.3, between the anchor marker D5S678 and the telomere, was
facilitated by the identity between a segment of hKCC3 and sequence
flanking the VNTR D5S110 (31). A genomic clone containing this VNTR
also contains four exons of the hKCC3 gene (Fig. 4) homologous to exons
14-17 of the hKCC1 gene (Table II) (16). Polymorphism involving the
D5S110 VNTR is generated by variable repetition of a core 11-bp
sequence motif (31). The hKCC3 gene is not a particularly likely
candidate for genetic disorders mapped to 5p15. However, KCC3 is
expressed within the kidney, and it may play a role in basolateral ion
transport within the thick ascending limb (6, 11). KCC3 is therefore a
functional candidate gene for the remaining cases of Bartter's
syndrome not caused by mutations in BSC1/NKCC2, ROMK, or CLC-NKB (42).
In this regard, repeat expansion of a 12-bp motif within the cystatin B
promoter has been shown to cause progressive myoclonic epilepsy (43), and expansion of the D5S110 VNTR could also conceivably affect the
expression of hKCC3.
Human KCC4 has been localized to chromosome 15q14 between the markers
D15S1040 and D15S118. This section of chromosome 15 has been linked to
two subtypes of idiopathic generalized epilepsy (44, 45) and to a
neurophysiological phenotype associated with schizophrenia (46). The
markers D15S1040 and D15S118 also flank a 5-centimorgan region of
chromosome 15q14 containing the gene for peripheral neuropathy with or
without agenesis of the corpus callosum (Andermann's syndrome)
(47).
In summary, we have cloned KCC3 and KCC4, two new members of the
cation-chloride cotransporter family. The unexpected heterogeneity of
K+-Cl
cotransporters (KCCs) belong to the gene family of electroneutral
cation-chloride cotransporters, which also includes two
bumetanide-sensitive Na+-K+-2Cl
cotransporters and a thiazide-sensitive
Na+-Cl
cotransporter. We have cloned
cDNAs encoding mouse KCC3, human KCC3, and human KCC4, three new
members of this gene family. The KCC3 and KCC4 cDNAs predict
proteins of 1083 and 1150 amino acids, respectively. The KCC3 and KCC4
proteins are 65-71% identical to the previously characterized
transporters KCC1 and KCC2, with which they share a predicted membrane
topology. The four KCC proteins differ at amino acid residues within
key transmembrane domains and in the distribution of putative
phosphorylation sites within the amino- and carboxyl-terminal
cytoplasmic domains. The expression of mouse KCC3 in Xenopus
laevis oocytes reveals the expected functional characteristics of
a K+Cl
cotransporter:
Cl
-dependent uptake of
86Rb+ which is strongly activated by cell
swelling and weakly sensitive to furosemide. A direct functional
comparison of mouse KCC3 to rabbit KCC1 indicates that KCC3 has a much
greater volume sensitivity. The human KCC3 and KCC4 genes are located
on chromosomes 5p15 and 15q14, respectively. Although widely expressed,
KCC3 transcripts are the most abundant in heart and kidney, and KCC4 is
expressed in muscle, brain, lung, heart, and kidney. The unexpected
molecular heterogeneity of K+-Cl
cotransport
has implications for the physiology and pathophysiology of a number of tissues.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in erythrocytes
is interdependent, with a 1:1 stoichiometry and low affinity constants
for both ions (2). KCCs are not influenced by membrane potential, and
under most physiological conditions, they function as an efflux
pathway. K+-Cl
cotransport is activated by
hypotonicity and functions in regulatory volume decrease (1, 2).
K+-Cl
cotransport is sensitive to the loop
diuretics bumetanide and furosemide, albeit with much lower drug
affinities than Na+-K+-2Cl
cotransport (3, 4). A number of other important characteristics serve
to distinguish K+-Cl
cotransport from
Na+-K+-2Cl
cotransport, in
particular the response to protein phosphorylation and
dephosphorylation (1).
cotransport has been the lack of specific high affinity inhibitors.
Convincing evidence for K+-Cl
cotransport has
nonetheless been reported for a number of tissues, including epithelia
(5, 6), heart (7, 8), skeletal muscle (9), and brain (4, 10). In
addition to cell volume regulation, non-erythroid
K+-Cl
cotransport has been implicated in
trans-epithelial salt absorption (5, 11), renal K+
secretion (12, 13), and the regulation of both intra- and extracellular
K+-Cl
(4, 9, 10, 14).
cotransporters, KCC1 and KCC2, were recently reported (3, 4, 15-17).
Both proteins are homologous to the other electroneutral
cation-chloride cotransporters, the bumetanide-sensitive
Na+-K+-2Cl
cotransporters BSC1
and BSC2 (also known as NKCC2 and NKCC1, respectively), and the
thiazide-sensitive Na+-Cl
cotransporter TSC
(also known as NCC) (18, 19). Heterologous expression of KCC1 in
HEK-293 cells results in Cl
-dependent,
furosemide-sensitive uptake of 86Rb+ that is
activated by cell swelling (3, 16). KCC1 transcript is abundant in both
mouse and human erythroleukemia cell lines, indicating that KCC1 is the
major erythroid K+-Cl
cotransporter (17). The
widespread expression of KCC1 also suggests a significant role in
non-erythroid K+-Cl
cotransport (3, 16, 20).
In contrast to KCC1, the expression of KCC2 is restricted to neurons
within the central nervous system (15, 21). Along with other neuronal
pathways for chloride (21, 22), KCC2 plays an important role in the
regulation of the transmembrane chloride gradient and thus affects the
neuronal response to stimuli such as
-aminobutyric acid (14,
23).
cotransport. At
the functional level, KCC1 and KCC2 differ in K+ affinities
(3, 4) (Km > 25 and 5.2 mM,
respectively), and a dramatically higher K+ affinity
(Km 0.9 mM) has also been reported for
insulin-stimulated K+-Cl
cotransport in a
mouse muscle cell line (9). Erythroid K+-Cl
cotransport is heterogeneous with respect to inhibition by the K+-channel blocker quinidine, with a significant
quinidine-insensitive fraction of
Cl
-dependent K+ flux (24). At the
molecular level, cDNA probes that include human KCC1 coding
sequences detect a number of transcripts, indicative of either
alternative splicing or the expression of closely related gene
products. Thus, a rat 3'-UTR probe detects transcripts of 3.8 and 4.4 kb in size (3), and hKCC1 probes from within the open reading frame
also detect transcripts of 5.5 (20, 25) and 6-7 kb (3) in various
human tissues and cell lines. Using the mouse and human EST data bases
as a starting point, we have cloned cDNAs corresponding to two of
these new KCC isoforms, denoted KCC3 and KCC4. Functional expression in
Xenopus laevis oocytes confirms that mouse KCC3 is a
furosemide-sensitive K+-Cl
cotransporter,
with much greater volume sensitivity than rabbit KCC1. Although highly
homologous, KCCs 1-4 differ in potentially important transmembrane
(TM) segments and in the distribution of putative cytoplasmic
phosphorylation sites. Northern blot analysis with specific 3'-UTR
probes indicates that the KCCs have distinct but overlapping expression
patterns. The identification of all four KCCs thus reveals significant
differences in structure, function, and expression.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Oligonucleotide primers for PCR
-actin probe. Hybridization
for all blots was performed overnight at 42 °C in 4× SSCP, 40%
formamide, 4× Denhardt's solution, 0.5% SDS, and 200 µg of salmon
sperm DNA, and membranes were washed twice for 10 min at room
temperature in 2× SSCP, 0.1% SDS and twice for 1 h at 65 °C
in 0.1× SSCP, 0.1% SDS. Exposure times varied as noted in the legends
of Figs. 4 and 5.
, 2 mM
K+-Cl
, 1.8 mM
Ca2+-Cl
, 1.0 mM
Mg2+-Cl
, and 5 mM Hepes/Tris, pH
7.4), supplemented with 2.5 mM sodium pyruvate and 5 mg/100
ml gentamicin, for 3 days.
cotransport is activated by cell
swelling, both hypotonic and isotonic conditions were used.
86Rb+ uptake was measured after a 30-min
incubation period in either hypotonic or isotonic Na+-free
and Cl
-free medium. Hypotonic medium (~125 mosmol/kg)
contained 48 mM N-methyl-D-glucamine
gluconate, 2 mM K+ gluconate, 4.0 mM Ca2+ gluconate, 1.0 mM
Mg2+ gluconate, and 5 mM Hepes/Tris, pH 7.4. To
generate the isotonic conditions (~220 mosmol/kg) sucrose was added
to a concentration of 100 mM. Ouabain was added to both
solutions at a concentration of 1 mM. This incubation
period was followed by a 60-min uptake period using
Na+-free uptake medium, both hypotonic and isotonic.
Hypotonic uptake medium (~125 mosmol/kg) contained 48 mM
N-methyl-D-glucamine-Cl
, 2 mM K+-Cl
, 1.8 mM
Ca2+-Cl
2, 1 mM
Mg2+-Cl
2, and 5 mM
Hepes/Tris, pH 7.4. The isotonic conditions (~220 mosmol/kg) included
100 mM sucrose. Both uptake solutions contained 5.0 µCi/ml of 86Rb+ (New England Nuclear) and 1 mM ouabain. To determine the
Cl
-dependent fraction of
86Rb+ uptake, paired groups of oocytes were
incubated in uptake medium without Cl
(substituted with
gluconate). When indicated, 2 mM furosemide was added to
both the incubation and uptake media. All uptakes were performed at
30 °C, after which oocytes were washed five times in ice-cold uptake
solution without isotope to remove tracer activity in the extracellular
fluid. After the oocytes were dissolved in 10% SDS, tracer activity
was determined by
-scintillation counting.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of KCC3 and KCC4
cDNAs. A full-length cDNA is shown for each isoform.
Coding sequences are boxed, and solid lines
represent the 5'- and 3'-UTR. The relative positions of partial
cDNAs, derived from EST cDNA clones, RT-PCR, 5'-RACE RT-PCR,
and library screening, are shown below each full-length
sequence. The IMAGE or TIGR clone numbers are displayed below EST
cDNAs. The figure is drawn to the scale indicated.
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Fig. 2.
Protein alignments of the four mammalian
K+-Cl
cotransporters. The deduced amino acid sequences of mKCC3,
hKCC3, and hKCC4 are aligned together along with hKCC1 and rat KCC2.
Identical segments are boxed in gray, and
putative TM segments are underlined in black.
Consensus sites for N-linked glycosylation are
boxed in green within the predicted extracellular
loop between TM-5 and TM-6. Consensus phosphorylation sites for PKC are
boxed in red, protein kinase A sites are in
yellow, and tyrosine kinases are boxed in
blue.
cotransporters and the
Na+-Cl
cotransporter, and the other
encompassing the four K+-Cl
cotransporters.
As indicated by direct sequence alignments, the four KCCs form two
subgroups, KCC1 paired with KCC4 and KCC3 paired with KCC2 (Fig.
3).
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Fig. 3.
Phylogenetic relationships between mammalian
cation-chloride cotransporters. The six human cation-chloride
cotransporters are compared with rat KCC2 in a phylogenetic tree
generated with the DNAStar program.
cotransporters and the
Na+-Cl
cotransporter (18). Homology is most
marked in the TM domains, the intracellular loops, and the
cytoplasmic carboxyl terminus (Fig. 2).
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Fig. 4.
Structure of the pMS621 genomic
subclone. The pMS621 clone contains exons 14-17 of the hKCC3
gene, which flank the D5S110 VNTR. The polymorphic tandem repeat, 1.8 kb in length in the pMS621 clone, can vary between 0.5 and 10 kb (31).
Arrows and GenBankTM/EBI accession
numbers indicate the previously reported DNA sequence
flanking the repeat.
Exon/intron boundaries of hKCC3 exons 14-17
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Fig. 5.
A, Northern blot analysis of mKCC3 (48-h
exposure). The transcript is abundant in kidney and heart and
undetectable in brain. B, the same blot was stripped and
reprobed with glyceraldehyde-3-phosphate dehydrogenase (12-h exposure).
C, In vitro translation of mKCC3 protein.
35S-labeled mKCC3 polypeptide was translated from cDNA
by rabbit reticulocyte lysate, both with (+) and without ( )
pancreatic microsomes, resolved by 7% SDS-polyacrylamide gel
electrophoresis, and visualized by autoradiography (12-h
exposure).
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Fig. 6.
A, Northern blot analysis of hKCC3.
Human multiple-tissue Northern blots (CLONTECH)
were probed with a cDNA probe from the 3'-UTR of hKCC3 (4-day
exposure). A 5.3-kb transcript is seen in multiple tissues.
B, the same Northern blots reprobed with a human -actin
probe (12-h exposure). C, Northern blot analysis of hKCC4,
using a 3'-UTR probe (2-day exposure). Only one multiple-tissue blot
was positive. Variable expression of two 6-7 kb transcripts is
detected in muscle, heart, kidney, lung, and brain.
(p < 0.001) and to 17.9 ± 1.6 pmol/oocyte/h in the presence of 2 mM furosemide, suggesting the presence of an endogenous
K+-Cl
cotransporter in Xenopus
oocytes. In rabbit KCC1-injected oocytes, 86Rb+
uptake increased from 8.8 ± 0. 5 pmol/oocyte/h in isotonic medium to 200 ± 13.8 in hypotonic medium (p < 0.001).
The observed uptake was Cl
-dependent and
furosemide-sensitive. 86Rb+ uptake in
mKCC3-injected oocytes increased from 15.3 ± 1.4 pmol/oocyte/h when oocytes were incubated in isotonic conditions (~220
mosmol/liter) to 2,552 ± 126 pmol/oocyte/h when incubated in
hypotonic medium (~125 mosmol/liter). Most of the
86Rb+ uptake observed in mKCC3-injected oocytes
was Cl
-dependent, because uptake in mKCC3
oocytes in Cl
-free medium was 65 ± 5.8 pmol/oocyte/h. The activation of mKCC3 by cell swelling is
significantly higher than that observed for H2O-injected
oocytes or for rabbit KCC1-injected oocytes. Fig. 7B shows
that the Cl
-dependent fraction of
86Rb+ uptake was 1.9 ± 0.1-fold higher
than the isotonic base line in control oocytes, 38.6 ± 2.8-fold
higher in rabbit KCC1-injected oocytes, and 226 ± 11.5-fold
higher than base line in oocytes injected with mKCC3.
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Fig. 7.
Functional expression of mKCC3 and rabKCC1 in
X. laevis oocytes. A,
86Rb+ uptake in oocytes injected with
H20, rabKCC1 or mKCC3. Uptakes were performed under both
isotonic (200 mosmol/kg) and hypotonic (100 mosmol/kg) conditions in
the presence (open bars) or absence (gray bars)
of extracellular chloride and in the presence of 2 mM
furosemide (black bars). There is a significant difference
in the vertical scale between the three graphs.
B, relative activation by hypotonicity of
K+-Cl cotransport expressed as a multiple of
the respective uptakes in isotonic conditions. The endogenous
K+-Cl
cotransporter (H20)
exhibits only a 2-fold activation, compared with the 39-fold activation
of rabbit KCC1 (rabKCC1) and a 226-fold activation of
mKCC3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cotransporters,
one Na+-Cl
cotransporter, and four
K+-Cl
cotransporters. A phylogenetic
comparison of protein sequences indicates that the four
K+-Cl
cotransporters evolved separately from
the Na+-dependent transporters, forming a
distinct subfamily (Fig. 3). This separation is also evident at the
genomic level, since the exon/intron structure of the BSC/NKCC genes
(32, 33) is similar to that of the TSC/NCC gene (34), but differs
significantly from that of the KCC1 gene (16). Partial
characterizations of the mouse KCC2 gene (GenBankTM
accession number AJ011033) and the human KCC3 gene (Table II) indicate
that the KCCs have a conserved genomic structure.
cotransporters
implicates TM-2 in cation transport (35) and TM-4 in anion transport
(36). The cytoplasmic carboxyl termini contain segments of substantial
homology interspersed with a number of more variable regions. The
extreme carboxyl terminus is identical in the four KCCs. Indeed, this
homology was crucial in identifying ESTs that contained the 3'-end of
hKCC3 (see "Materials and Methods"). Presumably some of the
conserved cytoplasmic segments are involved in protein-protein
interactions; however, none of the KCCs contain recognizable protein
signaling domains (27). The predicted amino and carboxyl termini differ
significantly in the distribution of putative phosphorylation sites
(see Fig. 2 and "Results"). KCC4, in particular, is rich in
potential PKC sites. The KCC2 and KCC3 sequences predict a tyrosine
phosphorylation site near the extreme carboxyl terminus. This consensus
phosphorylation site is altered in KCC1 and KCC4 by variation in
amino acids flanking the conserved tyrosine (Fig. 2).
cotransporters. The functional expression of both KCC1 (3, 16) and KCC2
(4) has already been reported, using heterologous expression in HEK-293
cells. Using modification of a previously published protocol (37), we
have utilized X. laevis oocytes for the functional
expression of rabbit KCC1 and mKCC3. The significant identity between
hKCC4 and the other KCCs suggests that it too encodes a
K+-Cl
cotransporter; however, functional
expression of this cDNA has not yet been examined.
Xenopus oocytes have an endogenous bumetanide-sensitive Na+-K+-2Cl
cotransporter that is
activated by hypertonicity and inhibited by hypotonicity (37). The
activity of this cotransporter was abolished in the present study by
the use of Na+-free conditions. The lack of extracellular
Na+ also exposes an endogenous
K+-Cl
cotransporter that is only weakly
activated by cell swelling (Fig. 7A), permitting a clear
distinction between oocytes injected with KCC cRNA and controls
injected with water. Oocytes injected with cRNA generated from the
rabbit KCC1 and mKCC3 cDNAs express Cl
-dependent uptake of
86Rb+, which is partially sensitive to
furosemide. A furosemide concentration of 2 mM inhibited
the K+-Cl
cotransport activity of rabbit KCC1
and mKCC3 by 81 and 69%, respectively. Rabbit KCC1 and mKCC3 are both
activated by cell swelling but differ markedly in the degree of
activation over base-line isotonic conditions. Thus, rabbit KCC1 is
activated 35-fold by hypotonic conditions, compared with the 226-fold
activation of mKCC3 (Fig. 7B). Functional expression in
HEK-293 cells suggests that rat KCC2 is insensitive to cell swelling
(4). However, this KCC has not yet been compared with KCC1 and KCC3
using the Xenopus expression system. Although direct
phosphorylation of KCC proteins has not been demonstrated, it is known
that protein phosphorylation and dephosphorylation play important roles
in the regulation of red cell K+-Cl
cotransport (1, 38). KCC1 and KCC3 differ in the distribution of
putative phosphorylation sites for a number of protein kinases (Fig.
2), which may account in part for their differential sensitivity to
cell swelling.
cotransporter capable of both inward
and outward K+-Cl
transport (7). The role of
this transport pathway in cardiac physiology is not yet clear. However,
1-adrenoreceptor stimulation of isolated rat hearts
stimulates a K+ efflux pathway that is partially sensitive
to loop diuretics (39). The cell swelling induced by cardiac ischemia
also stimulates a significant efflux of K+ that is mediated
predominantly by K+-Cl
cotransport. This
efflux of K+ may play an important role in the genesis of
arrhythmias following myocardial ischemia (8).
cotransport system
with a high affinity for extracellular K+ has been
described in mouse skeletal muscle cells (9); however, the
physiological roles of K+-Cl
cotransport in
this tissue have not been extensively studied. KCC4 cDNAs were also
cloned by RT-PCR from human brain (Fig. 1), in which a single
transcript of ~6 kb has been detected by Northern analysis. Like
KCC2, KCC4 may function in neuronal Cl
homeostasis, with
secondary effects on the response to
-aminobutyric acid and other
stimuli (10, 14, 21, 23).
cotransport in several nephron
segments, including proximal tubule (5, 40), thick ascending limb (6,
11), distal convoluted tubule (12), and cortical collecting duct (13).
The expression of three KCCs in the kidney suggests the possibility
that there is an overlap in their intrarenal distribution. Moreover,
one or more KCCs may target to the apical membrane of renal tubular cells, particularly within the cortical nephron (12, 13). The
intrarenal distribution of hKCC1 was recently studied by in situ hybridization with a coding sequence probe (20). Although the
probe chosen cross-hybridizes with KCC3, it is evident from this study
that KCCs are expressed along the entire nephron, including within
glomeruli (20). The role of K+-Cl
cotransport
within the glomerulus is unknown. However, glomerular mesangial cells
express BSC2/NKCC1 in vivo and exhibit hormone-sensitive Na+-K+-2Cl
cotransport (18).
There is also preliminary evidence that cation-chloride cotransporters
help set intracellular chloride activity in mesangial cells
(41).
cotransport raises a number of
important issues for future study. First, the overlapping expression
patterns of the four KCCs suggests the possibility that certain cell
types will express more than one isoform, and indeed this appears to be
the case in some human cell lines (25). Second, the significant
homology within cytoplasmic domains has implications for the generation
of isoform-specific antibodies. Third, the structural and functional
comparison of the KCCs suggests that they differ in a number of
important characteristics, including the response to changes in
cellular volume (Fig. 7). Fourth, the full characterization of the
physiological role(s) of individual KCCs will likely require sequential
targeted disruption of the mouse KCC genes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Steven C. Hebert for encouragement and generous support, Drs. Bliss Forbush III and John Armour for clones, and Karen Sloan-Brown and Craig Short for technical assistance.
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Note Added in Proof |
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While this manuscript was in press, Hiki et al. published the characterization of human KCC3 (Hiki, K., D'Andrea, R. J., Furze, J., Crawford, J., Woollatt, E., Sutherland, G. R., Vadas, M. A., and Gamble, J. R. (1999) J. Biol. Chem. 274, 10661-10667). This KCC3 cDNA is an alternative splice form of the KCC4 cDNA reported here. In deference to the earlier publication, we have reversed the numbering of our GenBankTM/EBI submissions and will henceforth refer to the KCC on chromosome 15q14 as KCC3 and the KCC on chromosome 5p15 as KCC4.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants K-11 DK02103 (to D. B. M.) and R-29 HL-49251 (to E. D.) and by grants from the Mexican Council of Science and Technology (CONACYT, M3840) and the Howard Hughes Medical Institute (75197-553601) (to G. G).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF087436 (mKCC3), AF105365 (hKCC3), and AF105366 (hKCC4).
§ To whom correspondence should be addressed: Div. of Nephrology, Vanderbilt University Medical Ctr., MCN S-3223, Nashville, TN 37232. Tel.: 615-343-2853; Fax: 615-343-7156; E-mail: david.mount{at}mcmail.vanderbilt.edu.
Supported by a scholarship from Dirección Genaral del
Personal Académico of the National University of Mexico.
Established investigator of the American Heart Association.
2 D. B. Mount and E. Delpire, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
KCC, K+-Cl cotransporter;
BSC or NKCC, bumetanide-sensitive Na+-K+-2Cl
cotransporter;
TSC or NCC, thiazide-sensitive
Na+-Cl
cotransporter;
h, human
(e.g. hKCC);
m, mouse (e.g. mKCC);
86Rb+, rubidium;
EST, expressed sequence tag;
bp, base pair(s);
kb, kilobase (or kilobase pairs);
IMAGE, integrated
molecular analysis of genomes and their expression;
TIGR, The Institute
for Genome Research;
UTR, untranslated region;
RT-PCR, reverse
transcriptase polymerase chain reaction;
RACE, rapid amplification of
conserved ends;
VNTR, variable number of tandem repeats;
TM, transmembrane segment;
PKC, protein kinase C.
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REFERENCES |
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