(Received for publication, February 23, 1995; and in revised form, May 12, 1995)
From the
By moving chloride into epithelial cells, the Na-K-Cl
cotransporter aids transcellular movement of chloride across both
secretory and absorptive epithelia. Using cDNA probes from the recently
identified elasmobranch secretory Na-K-Cl cotransporter (sNKCC1) (Xu,
J. C., Lytle, C. Zhu, T. T., Payne, J. A., Benz, E., and Forbush, B.,
III(1994) Proc. Natl. Acad. Sci. 91, 2201-2205), we have
identified the human homologue. By screening cDNA libraries of a human
colonic carcinoma line, T84 cell, we identified a sequence of 4115
bases from overlapping clones. The deduced protein is 1212 amino acids
in length, and analysis of the primary structure indicates 12
transmembrane segments. The primary structure is 74% identical to
sNKCC1, 91% identical to a mouse Na-K-Cl cotransporter (mNKCC1), 58%
identical to rabbit and rat renal Na-K-Cl cotransporters (NKCC2), and
43% identical to the thiazide-sensitive Na-Cl cotransporters from
flounder urinary bladder and rat kidney. Similar to sNKCC1 and mNKCC1,
the 5`-end of the human colonic cotransporter is rich in G+C
content. Interestingly, a triple repeat (GCG) The vectorial transport of chloride across epithelia is a
prominent mechanism in the maintenance of water and electrolyte
homeostasis. Chloride transport is involved in reabsorption of NaCl in
the thick ascending limb of the loop of Henle in mammalian kidney (1) and in secretion of NaCl in a diverse array of secretory
epithelia, including the intestine, trachea, and parotid and the avian
and elasmobranch salt
glands(2, 3, 4, 5, 6) . In
all of these tissues, the chloride entry into the epithelia cell is
mediated by a Na-K-Cl cotransport protein, which couples the
electroneutral movement of sodium, potassium, and chloride ions. In
order to carry out net salt transport, the Na-K-Cl cotransporter
functions in concert with three other membrane proteins: chloride and
potassium channels and the sodium pump. The importance of the proper
functioning of these ion transport mechanisms in chloride secretory
epithelia is exemplified by the disease states of cystic fibrosis and
secretory diarrhea, where there are defects in the regulation of ion
transport(7) . The Na-K-Cl cotransporter is characterized by
a sensitivity to the sulfamoylbenzoic acid ``loop''
diuretics, furosemide, and bumetanide and a strict ion dependence of
transport (for review, see (8) ). Among the Na-K-Cl
cotransporters that have been described in various cells and tissues,
there is substantial variation in both molecular weight and
``loop'' diuretic sensitivity(9) , suggesting the
presence of different isoforms. For example, studies using the
photosensitive bumetanide analog,
4-[ Recently, we reported the cloning,
sequencing, and expression of a cDNA encoding the basolateral Na-K-Cl
cotransporter from the shark rectal gland
(sNKCC1)
Figure 1:
Schematic diagram of selected clones
encoding the Na-K-Cl cotransporter isolated from T84 cDNA libraries.
The 3636-nucleotide open reading frame is displayed at top.
The shadedregion of the clones indicates the open
reading frame. The solidregion indicates
untranslated sequence, and the hatchedregion indicates intronic sequence. The sequence corresponding to
consensus splice acceptor and donor sites (28) are displayed underneath each intronic region, where intronic sequence is in lowercase, and coding sequence is in uppercase. Two exons are displayed as open regions A and B in clones TEF 11a and TEF
2a.
The full-length cDNA was sequenced
bidirectionally by the dideoxy chain termination method (18) 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 sequence and deduced amino acid
sequence were performed with programs from the Genetics Computer Group
software. The program TBLASTN (19) was used to search GenBank.
Identities with other sequences are given as the fraction of residues
in hNKCC1, which are found to be identical in the matched sequence.
Samples of rabbit 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 rabbit tissue Northern blot used in this study
was the one used in (16) . The membrane was completely stripped
of previously hybridized probe by incubating in 0.1
Figure 5:
Northern blot analysis of expression of
the human colonic Na-K-Cl cotransporter and related mRNAs. A,
rabbit tissues and shark rectal gland. B, human tissues.
Poly(A)
The human embryonic kidney cell
line (HEK-293) was maintained in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 10%
fetal bovine serum, penicillin (50 units/ml), and streptomycin (50
µg/ml) in a humidified incubator (5% CO
Figure 2:
Sequence
alignment of the deduced primary structure of Na-K-Cl cotransporters in
human colon (hNKCC1), mouse inner medullary collecting duct cells
(mNKCC1; Ref. 26), shark rectal gland (sNKCC1; (15) ),
and rabbit kidney (rNKCC2; splice variant A, (16) ). Lower
case letters identify residues not identical to hNKCC1. The
predicted transmembrane segments are underlined. Two putative N-linked glycosylation sites between predicted transmembrane
segments 7 and 8 are identified by a verticalbar.
The single potential cAMP-dependent protein kinase phosphorylation site
is identified by an asterisk. Two threonines that correspond
to known phosphoacceptors in sNKCC1 are identified by solidtriangles. The regions encoded by the two exons
identified from the cDNA libraries are distinguished by the hatchedlinesabove the sequence (from Fig. 1;
exon A and B).
Figure 3:
Hypothetical model of the human colonic
Na-K-Cl cotransporter. The amino acid residues are color-coded by
hydropathic index (A) as determined by the Kyte-Doolittle
algorithm (42) or by the fractional identity of hNKCC1 to
sNKCC1 (B). Both the hydropathic index and identity are
averaged over a running window of 15 amino acids. Potential
glycosylation sites between putative transmembrane segments 7 and 8 are
indicated by branchedlines. Secondary structural
elements predicted by the PHD program (45, 46) are
shown (helices,
The hNKCC1 cDNA includes a full-length open reading frame encoding
1212 amino acids, beginning with the first ATG (GCTATGG)
downstream of a stop codon. The predicted molecular mass is 132 kDa,
which is very similar to the core polypeptide identified in N-glycosidase-F-treated membranes from T84 cells with
monoclonal antibodies developed against the shark rectal gland Na-K-Cl
cotransporter ( We noted that a number of the clones isolated from one of
the libraries contained intronic sequences with consensus splice sites
for intron-exon boundaries ((28) , Fig. 1). Many of
these clones were undoubtedly produced from incompletely processed mRNA
during library construction (see (29) ). This finding allowed
us to identify some of the exons in the hNKCC1 gene (Fig. 1).
Interestingly, one cDNA contained consensus splice sites and intronic
DNA on either side of a 96-bp exon (Fig. 1, exon B), which corresponds exactly to the position of an
alternatively spliced cassette exon in the rabbit kidney Na-K-Cl
cotransporter(16) .
The Na-K-Cl cotransporter in the T84 cell
is known to be stimulated by agents such as vasoactive intestinal
peptide that activate adenylate cyclase(2) . Unlike sNKCC1,
which has no consensus sites for phosphorylation by cAMP-dependent
protein kinase, there is a single potential cAMP-dependent protein
kinase site in hNKCC1 located within the predicted cytoplasmic
C-terminal domain (Ser
Figure 4:
Chromosomal localization of hNKCC1 gene.
The hNKCC1 gene is localized to chromosome 5 at position 5q23.3 on
metaphase spreads of human peripheral lymphocytes. Note red signal against the green R-banded
chromosomes.
Western blot analysis using monoclonal antibody T10 (see ``Materials and Methods'') revealed that HEK-293
cells stably expressing hNKCC1 produced a glycoprotein with very
similar mass to that observed in native T84 membranes,
Figure 6:
Western blot analysis of membranes from
native T84 cells, HEK-293 cells stably expressing hNKCC1, and
untransfected HEK-293 cells. Membrane protein was separated by
SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene
difluoride membrane, and probed with monoclonal antibody T10 (see ``Materials and Methods''). Membrane protein from
each cell line was treated with (+) or without(-) N-glycosidase F prior to gel electrophoresis. Molecular mass
standards are indicated in kDa.
Fig. 7presents the time course of
Figure 7:
Time course of
Figure 8:
The dependence of the initial rate of
The results of an experiment
examining the ion dependences of cotransport in cells expressing sNKCC1
or hNKCC1, as well as in untransfected HEK-293 cells are presented in Fig. 8. The affinities for sodium, rubidium, and chloride are
4-9-fold greater in cells expressing hNKCC1 compared with those
expressing sNKCC1 (in three experiments, the values (millimolar) for
hNKCC1-transfected cells were as follows: K Fig. 9displays the bumetanide
inhibition curves for HEK-293 untransfected cells and cells expressing
hNKCC1. The stably expressing cells displayed a K
Figure 9:
Bumetanide inhibition of the initial rate
of
This paper presents the cloning, functional expression, and
chromosomal localization of the basolateral isoform of the Na-K-Cl
cotransporter in the human T84 colonic carcinoma cell line. We have
previously cloned the basolateral Na-K-Cl cotransporter from the shark
rectal gland (NKCC1; (15) ) as well as the apical isoform of
this protein from the rabbit kidney (NKCC2; (16) ). Two T84
cell cDNA libraries were screened under low stringency conditions to
obtain the full-length coding region of human NKCC1 (hNKCC1) from
overlapping clones. Over the entire length, the amino acid sequence is
91% identical to a putative basolateral Na-K-Cl cotransporter from
mouse(26) , 74% identical to the shark rectal gland Na-K-Cl
cotransporter(15) , 58% identical to the rabbit and rat kidney
Na-K-Cl cotransporters(16, 17) , and 43% identical to
the flounder and rat thiazide-sensitive Na-Cl
cotransporters(17, 30) . Analysis of the primary
structure of all NKCC and thiazide-sensitive Na-Cl cotransporter
proteins cloned to date suggests that they have very similar overall
structures with large N- and C-terminal hydrophilic regions bounding a
central core hydrophobic domain containing 12 putative transmembrane
segments (Fig. 3). These cotransporter proteins display
significant homology to predicted protein sequences encoded by open
reading frames identified in sequencing projects of a cyanobacterium (Synococcus sp.; (33) ), yeast (Saccharomyces
cerevisiae, unpublished, Z36104), a nematode (Caenorhabditis
elegans, Refs. 34 and 35), and a moth (Manduca sexta,
unpublished, U17344). An expressed sequence tag from a human colorectal
tumor (unpublished, T25163) was identified from the data base, and the
nucleotide sequence is 100% identical to hNKCC1. Another expressed
sequence tag from human brain (unpublished, T16107) displays 38% amino
acid identity to hNKCC1. The N terminus is the most divergent region
among the different NKCC proteins ( Fig. 2and Fig. 3B) with numerous insertions and deletions
present. Interestingly, NKCC1 is a significantly larger protein than
NKCC2, due primarily to an additional 80 amino acids at the N terminus.
The wide interspecies sequence divergence at the N terminus suggests
that much of this region is not directly involved in carrying out ion
translocation. Since the Na-K-Cl cotransporter is known to be regulated
by various factors in a cell-specific fashion (9) , the
divergence at the N terminus might reflect differences in modulation by
phosphorylation and other regulatory processes. Despite great
variation in the N-terminal hydrophilic region of the NKCC proteins,
all of these proteins contain a short 11-amino acid segment (for
sNKCC1, TFGHNT The 5`-end of hNKCC1 is very rich
in cytosine and guanine nucleotides. This appears to be a
characteristic of the secretory isoform of NKCC since it is also
observed in sNKCC1 and mNKCC1 but not in the rabbit or rat renal
absorptive isoforms, NKCC2. We have noted that within this
(G+C)-rich region of hNKCC1 is a triple repeat (GCG) The central core hydrophobic domain of hNKCC1,
containing the 12 putative transmembrane (TM) segments, displays high
identity to NKCC2 (79%) consistent with the importance of the
transmembrane segments in ion translocation. Six of the 12 putative TM
segments of hNKCC1 are greater than 90% identical to sNKCC1, and
putative TM segments 1, 3, 6, and 10 show complete conservation of all
residues between these two secretory cotransporters. One of the
putative TM segments, which displays lower identity among the different
NKCC and thiazide-sensitive Na-Cl cotransporter proteins is TM2
(50-80%). We have previously reported that putative TM2 in rNKCC2
is encoded largely by an alternatively spliced 96-bp exon, resulting in
three separate renal NKCC2 splice variants that are structurally
different only over the 31 amino acids encoded by this
exon(16) . Although we have no evidence of alternative splicing
in the human colonic Na-K-Cl cotransporter, we have identified a
similar 96-bp exon encoding the identical region of hNKCC1 (exon B in Fig. 1and Fig. 2). We have hypothesized that the
portion of TM2 encoded by this exon may form part of the ion
translocation pocket and thus may confer differences in ion affinities
among the NKCC proteins(16) . It may be significant, therefore,
that sNKCC1 and hNKCC1 stably expressed in HEK-293 cells display very
different affinities for the three cotransported ions ( (15) and see Fig. 8). In contrast to the variability
of the N-terminal hydrophilic region, the C terminus displays
remarkable homology among the NKCC proteins ( Fig. 2and Fig. 3B). Outside of a small region where there is
considerable divergence and a 6-bp deletion in comparison with sNKCC1
(residues 946-975), hNKCC1 aligns perfectly with sNKCC1 and is
greater than 82% identical to it. A very well conserved region within
the C-terminal domain (hNKCC1; Gln Using Northern blot analysis to examine the tissue expression of
hNKCC1, we have found that a 7.2-7.5-kb transcript is found in
most tissues examined (Fig. 5), similar to the result previously
obtained in shark rectal gland(15) . This result suggests that
in addition to its special role in salt-secreting epithelia, NKCC1 may
represent the ``housekeeping'' form of the Na-K-Cl
cotransporter. A prominent 6.3-kb message was detected in human
skeletal and heart muscle; based on its strength of hybridization, this
transcript is most likely a muscle-specific splice variant of NKCC1. An
additional transcript was weakly detected by the hNKCC1 probe in human
kidney (Fig. 5B) and in rabbit kidney at The ion affinities obtained from similar The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
U30246[GenBank Link].
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
occurs within
the 5`-coding region and contributes to a large alanine repeat
(Ala
). Two sites for N-linked glycosylation are
predicted on an extracellular loop between putative transmembrane
segments 7 and 8. A single potential site for phosphorylation by
protein kinase A is present in the predicted cytoplasmic C-terminal
domain. Northern blot analysis revealed a 7.4-7.5-kilobase
transcript in T84 cells and shark rectal gland and a
7.2-kilobase
transcript in mammalian colon, kidney, lung, and stomach. Metaphase
spreads from lymphocytes were probed with biotin-labeled cDNA and
avidin fluorescein (the cotransporter gene was localized to human
chromosome 5 at position 5q23.3). Human embryonic kidney cells stably
transfected with the full-length cDNA expressed a
170-kDa protein
recognized by anti-cotransporter antibodies. Following treatment with N-glycosidase F, the molecular mass of the expressed protein
was similar to that predicted for the core protein from the cDNA
sequence (132-kDa) and identical to that of deglycosylated T84
cotransporter (
135-kDa). The stably transfected cells exhibited a
15-fold greater bumetanide-sensitive
Rb influx than
control cells, and this flux required external sodium and chloride.
Flux kinetics were consistent with an electroneutral cotransport of
1Na:1K:2Cl. Preincubation in chloride-free media was necessary to
activate fully the expressed cotransporter, suggesting a
[Cl]-dependent regulatory mechanism.
H]benzoyl-5-sulfamoyl-3-(3-thenyloxy) benzoic
acid, have demonstrated the specific labeling of a
150-kDa protein
in membranes from dog kidney(10) , mouse kidney(11) ,
and duck red blood cells(12) , whereas in shark rectal gland
membranes, a
195-kDa protein is observed(13) . Secretory
epithelia, such as the rectal gland and parotid gland typically show a
10-fold lower affinity for bumetanide than absorptive epithelia, such
as the thick ascending limb of the loop of Henle of mammalian kidney
and flounder intestine(8) . Additionally, in absorptive and
secretory epithelia, the Na-K-Cl cotransporter displays distinct
differences in its polarized membrane distribution. In secretory
epithelia, the Na-K-Cl cotransporter is an exclusively basolateral
membrane protein, whereas in absorptive epithelia it is localized to
the apical membrane(14) .
(
)(15) . In addition, we (16) and others (17) have identified the apical Na-K-Cl
cotransporter from mammalian kidney (NKCC2). We proposed that NKCC1 and
NKCC2 represented distinct isoforms, since they display only 61% amino
acid identity and are encoded by mRNAs of different sizes and tissue
specificities(15, 16) . In the present report, we have
utilized homology to sNKCC1 to obtain the cDNA encoding the basolateral
Na-K-Cl cotransporter (hNKCC1) from a human intestinal cell line, the
T84 cell. The human Na-K-Cl cotransporter displays remarkable identity
to sNKCC1 within the predicted transmembrane segments (88%), yet when
examined in the same expression system, hNKCC1 exhibits considerably
different ion affinities compared with sNKCC1.
Cloning and Sequence Analysis
A human colonic
(T84 epithelial cell line) cDNA library in Uni-Zap XR was obtained from
Stratagene (La Jolla, CA). This library was screened with two P-labeled cDNA probes derived from the shark rectal gland
Na-K-Cl cotransporter (nucleotides -298 to 1235 and 1236 to 3411;
see (15) ). Using low stringency hybridization (34 °C in
50% formamide, 5
SSPE, 5
Denhardt's solution,
0.1% SDS, and 100 µg/ml fish sperm DNA) and high stringency wash
conditions (55 °C in 0.5
SSC and 0.1% SDS), a single
positive clone (TEF 1-1) was identified, carried through two
rounds of plaque purification, and successfully excised into Bluescript
SK
with the helper phage R408. A second T84 cDNA
library in
obtained from Dr. John R. Riordan
(Hospital for Sick Children; Toronto, Ontario, Canada) was screened
with a 5`-end 0.7-kb XbaI-XbaI fragment of TEF
1-1. Using similar screening procedures as described above, 23
positive cDNAs were obtained in Bluescript SK
(see Fig. 1). All cDNAs were characterized by Southern blot analysis
and partial sequencing.
Northern Blot Analysis
T84 cells were obtained
from Dr. James L. Madara (Brigham and Women's Hospital, Boston,
MA) and cultured as described previously(20) . Total RNA was
isolated from fresh rabbit tissues, spiny dogfish (Squalus
acanthias) rectal gland, and T84 cells by the guanidine
thiocyanate method(21) . Poly(A) RNA was
purified from total RNA using magnetic beads (PolyATtract, Promega
Corp.).
SSC and
0.5% SDS at 75 °C, and confirmation of probe removal was determined
by film exposure (48 h at -70 °C). A blot of human tissue
mRNA was obtained commercially (Clonetech, human MTN blot,
7760-1, lot 52805). The blots were prehybridized 4 h at 45 °C
in 50% formamide, 2
SSPE, 2
Denhardt's, 1% SDS,
200 µg/ml yeast RNA, 100 µg/ml fish sperm DNA and then
hybridized for 24 h in fresh hybridization solution containing 10
cpm/ml
P-labeled cRNA probe. Antisense cRNA probes
were produced as run-off transcripts (MAXIscript, Ambion Inc)
(nucleotides 650-1102 for Fig. 5a and nucleotides
796-1926 for Fig. 5b) from cDNA clones. The blots were
subjected to a final wash of 20 min at 65 °C in 0.1
SSC and
0.5% SDS and analyzed by autoradiography at -70 °C with an
intensifying screen.
-selected RNA (5-10 µg in A,
2 µg in B) was hybridized with antisense cRNA probes
transcribed in vitro from hNKCC1 (upperpanels). The blots were reprobed with a
-actin cDNA
probe as a control for RNA integrity (lower panels); note that
an additional band in some lanes of the
-actin control panel
reflects a muscle-specific actin transcript,
1.8-kb.
Chromosomal Localization
Fluorescence in situ hybridization was performed as described previously(22) .
Metaphase spreads were prepared from human peripheral blood lymphocytes
after methotrexate synchronization and bromodeoxyuridine incorporation.
Probes were assigned to R bands generated by bromodeoxyuridine
incorporation and subsequent photolysis(23) . Briefly, TEF
1-1 cDNA was labeled with 11-biotin dUTP by nick translation. The
probe DNA (60 ng) was coprecipitated with 3 µg of human Cot 1 DNA
plus 6 µg of salmon sperm DNA. After resuspension in 12 µl of
hybridization solution (50% formamide, 2 SSC, 10% dextran
sulfate) the DNA was denatured at 75 °C for 10 min. Denatured DNA
was applied directly to denatured metaphase spreads (50% formamide and
2
SSC for 2 min at 70 °C). The slides were incubated for 12
h at 37 °C, washed 3 times in 42% formamide and 2
SSC at 42
°C, washed 3 additional times at low stringency (1
SSC, 40
°C), blocked with bovine serum albumin (3% in 4
SSC for 30
min at 37 °C), and stained with Hoechst 33258 (2.5 mg/ml) for 15
min. After washing briefly with distilled water, they were placed under
a UV lamp at a distance of 10 cm for 1 h; they were subsequently
incubated for an additional hour in 2
SSC at 42 °C.
Avidin-DCS-fluorescein isothiocynate (Vector Laboratories) in 1% bovine
serum albumin and 4
SSC was used to detect probe sequences in
which 11-biotin dUTP had been incorporated, and, after incubation for
30 min at 37 °C, the slides were washed 3 times in 4
SSC
and 1% Tween 20 for 5 min at 42 °C. Propidium iodide (200 ng/ml) in
antifade (Dabco) was used to counterstain chromosomes. A CCD camera
(PM512, Photometrics, Tucson, AZ) was used to visualize fluorescent
signals; gray scale images were obtained sequentially for fluorescein
and propidium iodide with precision filter sets (C. Zeiss), and the
images were pseudocolored and merged.
Stable Expression in a Mammalian Cell Line
A
full-length construct of the sequence for the human colonic Na-K-Cl
cotransporter was prepared from the two cDNAs, TEF 11a and TEF
1-1 (see Fig. 1). Much of the 5`-untranslated region and
all of the intronic DNA were removed from TEF 11a by subcloning a PstI-PstI fragment into Bluescript KS (nucleotides -24 to 831). The small subcloned fragment of
TEF 11a and the cDNA of TEF 1-1 were ligated at a common NcoI site and subcloned into the mammalian expression vector
pJB20 at EcoRI and KpnI restriction sites of the
polylinker(24) . Transcription of the insert is under the
control of the cytomegalovirus promoter, and the vector contains a
neomycin (G418) resistance gene.
at 37 °C).
The full-length construct was transfected into HEK-293 cells by calcium
phosphate precipitation(25) . Nearly confluent cells were split
1:6 the day before transfection and were 50-70% confluent in a
10-cm dish immediately prior to transfection with 20 µg of DNA in
140 mM NaCl, 25 mM HEPES, 0.75 mM
Na
HPO
, 125 mM CaCl
, pH
7.05. 48 h after transfection, cells were selected for neomycin
resistance by growth in G418 (900 µg/ml), and after 3 weeks, single
resistant colonies were amplified and screened by
Rb
influx measurements and Western blotting.
Enzymatic Modification of
Protein
CHAPS-solubilized membrane protein (50 µg) was
deglycosylated by incubation (4 h at 37 °C) with 20 units/ml of N-glycosidase F (Boehringer Mannheim) in a 0.05-ml solution
containing 4% CHAPS, 0.15 M sodium phosphate buffer, pH 7.8,
2.5 mM EDTA, and protease inhibitors. Enzymatic treatment was
terminated by the addition of electrophoresis sample buffer.
HEK-293
cells were subcultured into 96-well plates (1:5 split) and grown to
confluency (2-3 days). Prior to the flux assay, the cells were
preincubated for 1 h in a low chloride hypotonic medium (1:2 dilution
of isotonic low chloride medium but lacking ouabain) and then briefly
washed in a low chloride isotonic medium containing 135 mM sodium gluconate, 5 mM potassium gluconate, 1 mM CaClRb Influx Assay
, 1 mM MgCl
, 1 mM NaHPO
, 2 mM Na
SO
, 15
mM HEPES, pH 7.4, and 0.1 mM ouabain. The initial
rate of
Rb influx was determined in quadruplicate wells in
the presence or absence of 200 µM bumetanide. Uptake was
determined in isotonic medium in which
Rb (1 µCi/ml)
replaced potassium. Influx was terminated after 0.5 min by the addition
of an ice-cold, high potassium saline followed by five rinses in
phosphate-buffered saline. Cellular extracts (80 µl of 2% SDS) were
assayed for
Rb by Cherenkov radiation, for protein with
the Micro BCA protein kit (Pierce) and for Na-K-Cl cotransporter
production by Western blot analysis(13) . Reported K
and K
values were obtained from nonlinear least squares fits to
the data using one- or two-site (for chloride) models. Western blot
analysis made use of a monoclonal antibody (T10) produced against a
glutathione S-transferase fusion protein containing the
hydrophilic C terminus of hNKCC1 (amino acids 902-1212).
(
)
Cloning and Sequencing of hNKCC1
Clones encoding
the bumetanide-sensitive Na-K-Cl cotransporter in human colon (the T84
cell) were initially obtained by screening a cDNA library under low
stringency conditions with nonoverlapping fragments of the shark rectal
gland Na-K-Cl cotransporter (see ``Materials and
Methods'' and (15) ). The first library screening produced
a cDNA (TEF 1-1; Fig. 1) with homology to the rectal gland
Na-K-Cl cotransporter with a large open reading frame and poly(A) tail
but that lacked the 5`-end of the coding region. A second library was
screened with a 5`-end fragment of TEF 1-1 (0.7-kb; see ``Materials and Methods''). 23 cDNAs were
plaque-purified and excised into Bluescript SK. Two
independent cDNAs (TEF 11a and 20a) extended the 5`-end of TEF
1-1, providing the complete coding region of the human colonic
Na-K-Cl cotransporter (Fig. 1). An overlapping nucleotide
sequence of 4115-bp was identified, hNKCC1. The 5`-end of hNKCC1 is
exceedingly rich in guanine and cytosine nucleotides with the first 900
bases containing 74% (G+C) content. We have reported previously
the presence of a (G+C)-rich region at the 5`-end of
sNKCC1(15) . Interestingly, within this (G+C)-rich region
of hNKCC1 is a triple repeat (GCG)
, which occurs within the
coding region and contributes to a large alanine repeat
(Ala
- Ala
; Fig. 2). This same
triple repeat is observed in the putative basolateral Na-K-Cl
cotransporter from mouse inner medullary collecting duct cells,
mNKCC1(26) .
(
)Since alanine is assigned
a positive hydropathic index (i.e. hydrophobic residue) by
Kyte and Doolittle(42) , the large alanine repeat encoded by
the GCG trinucleotide repeat is responsible for the very hydrophobic
region (residues color-coded red), which appears within the
otherwise hydrophilic N-terminal domain of Fig. 3A.
; wavy lines,
). No attempt has been made to
pair
structural elements.
135-kDa)(27) . The full-length amino acid
sequence of hNKCC1 is 93% identical to mNKCC1 and 73% identical to
sNKCC1 (Fig. 2). The most divergent region is the N terminus,
which includes a number of insertions and deletions when compared with
mNKCC1 and sNKCC1. Outside of this area, hNKCC1 aligns perfectly with
mNKCC1.
Structure of hNKCC1
Similar to previously
identified cation-chloride cotransport
proteins(15, 16, 17, 26, 30) ,
hydropathy analysis of hNKCC1 predicts a large central hydrophobic
region bounded by N- and C-terminal hydrophilic domains (Fig. 3A). As displayed in Fig. 3, 12
membrane-spanning helices are predicted in our model of hNKCC1.
Based on homology with sNKCC1, we predict that both terminal domains
reside within the cytoplasm. The polypeptide sequence of hNKCC1 has
five potential N-linked glycosylation sites. Two of these
sites are located in a predicted extracellular hydrophilic region of
the protein between putative transmembrane segments 7 and 8 ( Fig. 2and Fig. 3). In comparing the primary structure of
hNKCC1 to related proteins, we note that among the loops connecting
predicted transmembrane domains, those that are predicted to be
intracellular are considerably better conserved than are those that are
predicted to be extracellular (e.g. sNKCC1, Fig. 3B).
; Fig. 2). This residue
corresponds to one of three potential cAMP-dependent protein kinase
phosphorylation sites in the rabbit kidney Na-K-Cl cotransporter
(rNKCC2, Ser
; (16) ). In addition, hNKCC1
contains two threonines (Thr
and Thr
; Fig. 2), which correspond to known phosphoacceptors in sNKCC1
(Thr
and Thr
; (15) ). The fact
that the region around both of these threonine residues in hNKCC and
sNKCC1 is very well conserved suggests that these residues are also
phosphacceptors in hNKCC1 (Fig. 3B).
Chromosomal Localization of the hNKCC1 Gene
Using
the TEF 1-1 cDNA, we localized the hNKCC1 gene to human
chromosome 5 at position 5q23.3 by fluorescence in situ hybridization on R-banded chromosomes (Fig. 4). Five
separate metaphase spreads were investigated. Two had distinct signals
on both homologues of chromosome 5, with signals on all four
chromatids. Three others had distinct signals on only one homologue
with discrete signals on each chromatid. Evidence of hybridization at
other chromosomal regions with TEF 1-1 was not observed. Delpire et al.(1994) have recently mapped mNKCC1 to mouse chromosome
18 in a region that they reported to be syntenic with human chromosome
5q31-33. Interestingly, the murine renal absorptive isoform,
mNKCC2, has recently been cloned, and the gene localized to mouse
chromosome 2(43) . These studies clearly distinguish NKCC1 and
NKCC2 as separate gene products.
Tissue Northern Blot Analysis
The level of
expression of hNKCC1 and related transcripts in T84 cells, shark rectal
gland, and several rabbit and human tissues was examined by Northern
blot analysis (Fig. 5). Using antisense cRNA probes, a
7.2-7.5-kb mRNA was expressed at high levels in T84 cells,
rectal gland, rabbit stomach, rabbit kidney, and rabbit large intestine (Fig. 5A) and in all of the human tissues (Fig. 5B). A prominent message is also found at 6.3 kb
in human heart and skeletal muscle, and a signal is detected in human
kidney at 5.1 kb (Fig. 5B). After longer development of
the blot shown in Fig. 5A, transcripts were detected at
7.2 kb in rabbit brain, lung, and small intestine and at
5.1
kb in rabbit kidney (data not shown).
Functional Expression of hNKCC1 in HEK-293
Cells
The function of the protein encoded by hNKCC1 was examined
by isolating a stably expressing cell line (HEK-293) following
transfection with a full-length construct in the mammalian expression
vector, pJB20 (see ``Materials and Methods''). Of
the 40 cells lines that were screened, 37 exhibited various levels of
increased bumetanide-sensitive Rb influx above
untransfected control cells (data not shown). The cell line that
displayed the largest bumetanide-sensitive
Rb influx was
used in subsequent studies to characterize the functional expression of
hNKCC1.
170-kDa (Fig. 6). We also immunodetected a similar sized band in
untransfected HEK-293 cells but at a much reduced level (note different
protein loads). We presume that this
170-kDa band in untransfected
HEK-293 cells is the endogenous Na-K-Cl cotransport protein we have
previously identified in these cells by bumetanide-sensitive
Rb influx analysis(15) . Two bands were
immunodetected in the lane containing membranes from the stable cell
line (Fig. 6). The lower molecular weight band is indicative of
incomplete glycosylation of the expressed protein, a probable result of
the large amount of protein being produced. We presume that the higher
molecular weight band is the functional, glycosylated cotransporter
delivered to the plasma membrane, since removal of N-linked
oligosaccharides with N-glycosidase F resulted in a single
smaller molecular weight band indistinguishable from that of the native
deglycosylated T84 cotransporter (
135-kDa; Fig. 6).
Rb uptake in
HEK-293 untransfected cells and HEK-293 cells stably expressing hNKCC1.
The stably expressing cells exhibited a linear
Rb uptake
for about 30 s, and the initial rate of bumetanide-sensitive
Rb influx was
15-fold greater in cells expressing
hNKCC1 than control untransfected cells. As we have noted previously in
expression studies of the shark rectal gland
cotransporter(15) , full activity of the expressed Na-K-Cl
cotransporter from human colon required a preincubation in low chloride
media prior to the
Rb influx assay (Fig. 7). This
supports the hypothesis that intracellular chloride is an important
regulator of cotransporter activity in secretory
epithelia(15, 31) . The expressed T84 cotransport
protein displayed definitive characteristics of a Na-K-Cl cotransport
mechanism. As displayed in Fig. 8(bottomrow), these include
Rb influx required the
simultaneous presence of sodium and chloride in the flux medium, and
Rb influx was almost completely inhibited by bumetanide
(200 µM). Additionally, the
Rb influx
kinetics are consistent with a single transport site for sodium and
rubidium (hyperbolic curves) and two sites for chloride (sigmoidal
curve, see legend of Fig. 8).
Rb uptake into
untransfected HEK-293 cells and HEK-293 cells stably expressing hNKCC1
following a 1-h preincubation in chloride-free (closed symbol)
or control media (open symbol). Crosssymbols indicate
Rb uptake in the presence of 200 µM bumetanide. Lines are drawn by eye. Samples were taken in
triplicate. Data are representative of three separate
experiments.
Rb influx on external [Na], [Cl], and
[Rb] for untransfected HEK-293 cells and HEK-293 cells stably
expressing either sNKCC1 (HEK 15; Ref. 15) or hNKCC1. Cations were
replaced with N-methyl glucamine and chloride with gluconate. Curves represent the best fits of the data to a model of
activation at single sodium and rubidium sites and at two identical
chloride sites; K values for this representative experiment
are displayed in each panel (summary data are presented in the
text).
= 19.6 ±
4.9, K
= 2.68
± 0.72, K
=
26.5 ± 1.3); and for sNKCC1-transfected cells K
= 165 ±
34, K
= 14.3
± 8.0, K
=
101 ± 24; see also (15) ). These findings are consistent
with much lower ion affinities observed for cotransport in the shark
rectal gland (40) compared with mammalian
systems(32, 44) . Ion affinities for sodium and
chloride in the untransfected cells (K
= 22.7 ±
9.5, K
= 12.8
± 7.0, K
=
41.5 ± 5.8; see also (15) ) were similar to those for
hNKCC1-transfected cells.
for bumetanide of 0.16 ± 0.05 µM that
was not significantly different than that observed for the endogenous
cotransport protein of untransfected HEK-293 cells, 0.09 ± 0.03
µM (t test; p > 0.05). The bumetanide
affinities of untransfected HEK-293 cells and of cells expressing
hNKCC1 are greater than that for sNKCC1 expressed in HEK-293 cells
(0.57 µM, (15) ). The bumetanide affinity of
expressed hNKCC1 is 10-fold higher than that reported by
Dharmsathaphorn et al.(2) for T84 cells; this
discrepancy can be attributed to a difference in experimental
protocol.
(
)
Rb influx into untransfected HEK cells and HEK-293
cells stably expressing hNKCC1. Cells were incubated in chloride-free
medium for 1 h and then prior to the flux assay; cells were allowed to
bind bumetanide at the indicated concentration for 20 min in the
presence of 72 mM chloride. Results are fit by a model of
bumetanide inhibition at a single site. Summary data of separate
experiments are as follows (mean ± S.E.): untransfected HEK-293
cells, K = 0.09 µM ± 0.02, V
= 8.34 nmol/mg of protein/min ±
0.98; and HEK-293 cells stably expressing hNKCC1, K =
0.16 µM ± 0.03, V
=
154 nmol/mg of protein/min ± 16.
IDAVP) that is very well conserved (note
the high identity red region of the N terminus in Fig. 3B). Within this short peptide, Ile
in sNKCC1 is the only variant residue, being replaced by
methionine in hNKCC1, mNKCC1, and NKCC2. Interestingly, the strictly
conserved Thr
is known to be phosphorylated in sNKCC1 by
stimuli that promote secretion through activation of cAMP-dependent
protein kinase(36) . Since Thr
and the region
around it are so highly conserved, it is likely that this
phosphorylation site is important for activation of both secretory and
absorptive NKCC isoforms. Our evidence support the hypothesis that the
response to cAMP in the rectal gland is an indirect result of changes
in cell [Cl] and not a direct effect of cAMP-dependent
protein kinase on the cotransporter(31, 37) . This
hypothesis is consistent with the finding that no region of sNKCC1
conforms to the consensus motif of cAMP-dependent protein
kinase(15) . Human NKCC1 contains a single strong
cAMP-dependent protein kinase consensus site at Ser
(KKES
), and it is possible that this cAMP-dependent
protein kinase site plays a role in regulation; recent work with dog
tracheal epithelial cells has provided evidence for the direct
activation of the Na-K-Cl cotransporter via a cAMP-dependent protein
kinase-dependent pathway in addition to activation via a reduction in
cell [Cl](38) .
that occurs within the coding region of the protein. A growing
number of human genes are known to contain five or more consecutive
triple repeats and several human genetic diseases, including fragile X
syndrome and myotonic dystrophy, are associated with mutations that
result in a tremendous expansion of the trinucleotide
repeat(39) .
-Thr
)
displays between 90 and 92% identity among the different NKCC isoforms
( Fig. 2and Fig. 3B). It seems likely that this
may encode a membrane-associated region involved in ion translocation.
5.1-kb
after longer development (data not shown). We and others have reported
that this
5.1-kb transcript encodes the absorptive or apical
isoform of the Na-K-Cl cotransporter which is found only in renal
tissue and is found there at high levels(16, 17) .
Rb influx
studies on HEK-293 cells stably expressing sNKCC1 or hNKCC1 are
considerably different from one another. For example, an almost 10-fold
difference in sodium affinity is observed between the two NKCC proteins (Fig. 8). Previous work in this laboratory has provided evidence
that both anions and cations are occluded within a transport
``pocket'' on the Na-K-Cl cotransporter(41) . It is
reasonable to propose that this transport ``pocket'' is
present within the TM segments and that this region of the protein
largely determines the ion affinities of the cotransporter. TM segments
that are most divergent between sNKCC1 and hNKCC1 may be responsible
for the ion affinity differences; these include TM2 (80% identity), TM4
(75%), TM5 (75%), and TM11 (80%). In this regard, we have proposed that
the alternatively spliced exons encoding most of TM2 of rNKCC2 result
in ion affinity differences for the three kidney splice
variants(16) .
10
mol/s for dog kidney, (47) ), the latter method
substantially underestimates the affinity for bumetanide.
We thank Laura Roman for technical advice and many
helpful discussions, Mike Caplan, Peter Igarashi, Chris Gillen, and
Rachel Behnke for reading the manuscript, and Grace Jones for excellent
technical assistance. We also thank John Riordan for providing a cDNA
library and James Madara for providing the T84 cells. Automated
sequencing was performed by the Yale University/Keck Foundation Nucleic
Acid Facility.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.