Endogenous and exogenous Na-K-Cl cotransporter expression in a
low K-resistant mutant MDCK cell line
John A.
Payne,
Christina
Ferrell, and
Chee Yeun
Chung
Department of Human Physiology, School of Medicine, University of
California, Davis, California 95616
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ABSTRACT |
A low K-resistant mutant
Madin-Darby canine kidney (MDCK) cell line, LK-C1, has been shown
previously to lack functional Na-K-Cl cotransporter (NKCC) activity,
indicating that it may be a useful NKCC "knockout" cell line for
structure-function studies. Using immunological probes, we first
characterized the defect in the endogenous NKCC protein of the LK-C1
cells and then fully restored NKCC activity in these cells by stably
expressing the human secretory NKCC1 protein (hNKCC1). The endogenous
NKCC protein of the LK-C1 cells was expressed at significantly lower
levels than in wild-type MDCK cells and was not properly glycosylated.
This latter finding indicated that the lack of functional NKCC activity
in the LK-C1 cells may be due to the inability to process the protein
to the plasma membrane. In contrast, exogenously expressed hNKCC1
protein was properly processed and fully functional at the plasma
membrane. Significantly, the exogenous hNKCC1 protein was regulated in
a manner similar to the protein in native secretory cells as it was
robustly activated by cell shrinkage, calyculin A, and low-Cl incubation. Furthermore, when the LK-C1 cells formed an epithelium on
permeable supports, the exogenous hNKCC1 protein was properly polarized
and functional at the basolateral membrane. The low levels of
endogenous NKCC protein expression, the absence of any endogenous NKCC
transport activity, and the ability to form a polarized epithelium
indicate that the LK-C1 cells offer an excellent expression system with
which to study the molecular physiology of the cation Cl cotransporters.
cation chloride cotransporter; secretory epithelium; polarity; Madin-Darby canine kidney cells; Na-K-Cl cotransporter
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INTRODUCTION |
THE CATION CHLORIDE
COTRANSPORTER gene family represents a small group of
transporters that function in intracellular [Cl] regulation,
epithelial salt transport, and cell volume control. One member of this
gene family, the Na-K-Cl cotransporter (NKCC), is represented by two
separate gene products, NKCC1 and NKCC2. Whereas NKCC2 appears to be
restricted in its distribution to the thick ascending limb cells of the
vertebrate kidney, NKCC1 exhibits a much wider tissue expression
pattern, being found in most cell types. NKCC1 expression is especially
high in secretory epithelial cells where it is under significant
regulatory control. Recent studies have been aimed at correlating
structure and function of this transport protein
(5-7). For the most part, these studies have centered
on determining the sites of ion and inhibitor binding. Fewer studies
have been performed on the structural aspects involved in modulation of
the NKCC1 protein by conditions or agents that activate the protein.
The NKCC1 protein in secretory epithelial cells as well as in duck
erythrocytes is activated by cell shrinkage, by increases in cAMP, and
by calyculin A (e.g., Refs. 8-10, 12, 15). Interestingly, all of these activators appear to
involve increased phosphorylation of the NKCC1 protein at a common set of serine and threonine residues (8). In the shark rectal
gland, three of the NKCC1 phosphorylation sites have been identified as
Thr-184, Thr-189, and Thr-202 (1, 11).
In an effort to identify a useful expression system with which to
pursue the molecular physiology of NKCC1 and the cation Cl
cotransporter proteins in general, we undertook a study to characterize
more fully a low K-resistant mutant Madin-Darby canine kidney (MDCK)
cell line originally developed by McRoberts et al. (LK-C1)
(14). The low K-resistant mutant MDCK cell line, LK-C1, has been previously demonstrated to have little "loop"
diuretic-sensitive 86Rb, 22Na, or
36Cl uptake (<2% of control) (14).
Additional studies showed that specific binding of the loop diuretic
piretanide to intact LK-C1 cells was virtually undetectable (<4% of
control) (2). These data strongly indicated that the LK-C1
cells were deficient in Na-K-Cl cotransport activity. In the present
study, we used immunological probes to characterize further the defect
of the endogenous NKCC protein of the LK-C1 cells, and then we fully
restored NKCC activity by stably expressing the human secretory NKCC1
(hNKCC1) protein in these cells. The exogenously expressed hNKCC1
protein was fully functional and regulated in a manner very similar to
the protein in native secretory cells. This study introduces the MDCK
LK-C1 cell line as a very useful expression system for
structure-function studies of the cation Cl cotransporters.
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MATERIALS AND METHODS |
Tissue culture and stable cell line production.
Both wild-type MDCK and a low K-resistant MDCK mutant were maintained
in growth medium containing Dulbecco's modified Eagle's medium
(DMEM), 10% fetal bovine serum, penicillin (50 U/ml), and streptomycin
(50 µg/ml). The low K-resistant mutant MDCK cells (MDCK LK-C1)
(14) were kindly provided by Dr. Jim McRoberts (UCLA). The
T84 cells were maintained in DMEM, 5% fetal bovine serum, Ham's F-12,
penicillin (50 U/ml), and streptomycin (50 µg/ml). All cells were
maintained in a humidified incubator with 5% CO2 at
37°C.
hNKCC1 was stably expressed in MDCK LK-C1 cells using calcium phosphate
precipitation and a previously described full-length hNKCC1 expression
construct (17). After 3 wk of growth in 900 µg/ml of
Geneticin (GIBCO), single resistant colonies were amplified and
screened by Western blot (T4) and by bumetanide-sensitive 86Rb influx (see 86Rb influx
analysis). The MDCK LK-C1 cells stably expressing hNKCC1 (MDCK LK-C1-hNKCC1) were maintained in growth media containing 900 µg/ml Geneticin.
To assess the establishment of epithelial polarity, wild-type MDCK
cells, MDCK LK-C1 cells, and MDCK LK-C1 cells stably expressing hNKCC1
were grown on permeable supports (0.4-µm Transwell inserts; Costar)
with growth media in both upper and lower wells. Epithelial monolayers
were analyzed for transepithelial resistance and potential difference
using a voltohmmeter (Millipore) at various times after reaching
confluency (1-4 days). The electrical resistance of the media and
filter without cultured cells was subtracted from the measured values
to obtain transepithelial resistance. In addition to resistance and
potential difference measurements, 5-day postconfluent epithelial
monolayers were analyzed for polarized activity of the Na-K-ATPase
(ouabain sensitive) and NKCC (bumetanide sensitive) by 86Rb
influx assay across the apical and basolateral membranes (see 86Rb influx analysis), and E-cadherin expression
was analyzed by immunofluorescence (see Protein analysis).
Protein analysis.
Membranes were prepared from cultured cells using differential
centrifugation. Briefly, cells were scraped off culture plates and
homogenized in 10-40 ml of homogenization buffer (250 mM sucrose, 10 mM Tris, 10 mM HEPES, and 1 mM EDTA, pH adjusted to 7.2 at 24°C)
containing protease inhibitors. After 10 strokes in a glass-Teflon homogenizer, the homogenate was centrifuged at 7,000 rpm for 10 min at
4°C (Sorval RC5, SS-34 rotor). The supernatant was centrifuged at
20,000 rpm for 30 min at 4°C. The final pellet was resuspended in
~100-500 µl of homogenization buffer with protease inhibitors and stored at
80°C. Protein concentration was determined using a
Micro BCA protein kit (Pierce, Rockford, IL). Deglycosylation of
membrane protein (20 µg) was performed by incubating membranes for
4 h at 37°C in a medium containing 0.5%
n-octylglucoside, 20 mM sodium phosphate buffer (pH 8.0), 50 mM EDTA, protease inhibitors, and N-glycosidase F (20 U/ml;
Boehringer Mannheim). Control samples were treated similarly, but
incubation was carried out in the absence of N-glycosidase
F. Enzymatic treatment was terminated by addition of electrophoresis
sample buffer. Membrane proteins were resolved by SDS-PAGE using a
7.5% Tricine gel system. Gels were electrophoretically transferred
from unstained gels to polyvinylidene difluoride (PVDF) membranes
(Immobilon P; Millipore, Bedford, MA) in transfer buffer (192 mM
glycine, 25 mM Tris-Cl, pH 8.3, and 15% methanol) for
5 h at 50 V
using a Bio-Rad Trans-Blot tank apparatus. PVDF-bound protein was
visualized by staining with Coomassie brilliant blue R-250. The PVDF
membrane was blocked in phosphate-buffered saline (PBS)-milk (7%
nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.4) for 1 h and
then incubated in PBS-milk with an anti-NKCC monoclonal antibody (T4 or
T10) (13) overnight at 4°C or for 2 h at 24°C.
After three 10-min washes in PBS-milk, the PVDF membrane was incubated
with secondary antibody (horseradish peroxidase-conjugated goat
anti-mouse IgG; Amersham) for 2 h at 24°C in PBS-milk. After
three washes in PBS-0.1% Tween 20, bound antibody was detected using
an enhanced chemiluminescence assay (NEN, Boston, MA).
E-cadherin, a marker for the formation of junctional complexes between
epithelial cells, was detected using immunofluorescence. Five-day
postconfluent monolayers grown on permeable supports were fixed with
4% paraformaldehyde in PBS for 10 min at 24°C. After four washes
with PBS, cells were blocked for 1 h at 24°C in 20% goat serum
in PBS (GS-PBS) and then incubated for 1 h at 24°C with a mouse
monoclonal anti-E-cadherin antibody in GS-PBS (1:100). This
anti-E-cadherin antibody recognizes an extracellular domain of the MDCK
cell protein and was kindly provided by the laboratory of Dr. James
Nelson (Stanford University). After three washes in PBS, the cells were
incubated for 2 h at 24°C with FITC-conjugated goat anti-mouse
IgG secondary antibody (1:200; Jackson Labs, West Grove, PA) in GS-PBS.
Filters were cut from their supports and mounted on clean slides using
Gel Mount (Biomedia, Foster City, CA). Cells were examined and digital
images obtained using a laser scanning confocal microscope (Zeiss 510).
86Rb influx analysis.
Flux experiments with MDCK cells were performed at 24°C with ~90%
confluent cells grown on 96-well plates or with 5-day postconfluent cells grown on Transwell permeable supports. In contrast to our earlier
expression studies with nonpolarized HEK-293 cells (16, 17), we found that bumetanide-sensitive 86Rb uptake
for MDCK cells grown on 96-well plates is dramatically reduced after
attainment of confluency. Although we have not investigated this
finding further, it is likely related to differentiation of the MDCK
cells into a polarized epithelium and the targeting of NKCC to the
basolateral membrane.
96-well plate fluxes.
Before flux measurement, cells were washed three times and preincubated
for 15 min in control media. Control media contained 135 mM NaCl, 5 mM
RbCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM
Na2HPO4, 2 mM Na2SO4, 3 mM glucose, and 15 mM HEPES titrated to pH 7.4 with
N-methyl-D-glucamine (NMDG) base and with a
final osmolarity of 290 mosmol/kgH2O. For experiments using
basal conditions, cells were then incubated for an additional 15 min in
control media in the presence of various drugs. For experiments
using low Cl to activate NKCC, cells were incubated for an
additional 40 min in low-[Cl] media. Low-[Cl] media contained 135 mM sodium methanesulfonate, 5 mM rubidium methanesulfonate, 1 mM
CaCl2, 1 mM MgCl2, 1 mM Na2HPO4, 2 mM
Na2SO4, 3 mM glucose, and 15 mM HEPES titrated
to pH 7.4 with NMDG base and with a final osmolarity of 290 mosmol/kgH2O. Any drugs that were to be tested after low-Cl
preincubation were included in the final 15 min of the low-Cl
incubation period. After these various preincubations, cells were
brought up in control media containing 2 µCi/ml 86RbCl,
0.1 mM ouabain, and ± 200 µM bumetanide. The
bumetanide-sensitive 86Rb uptake in both wild-type MDCK
cells and MDCK LK-C1 cells expressing hNKCC1 was linear for at least 8 min under either basal or low-Cl conditions, and 3- to 5-min
influx assays were routinely performed to obtain initial rates (see
Fig. 2). Influx of 86Rb was
terminated by five washes in Tris-buffered saline (pH 7.4) containing 200 µM bumetanide. Cells were solubilized in 2% SDS and
assayed for 86Rb by Cerenkov radiation and for protein by
the Micro BCA method (Pierce).
The volume sensitivity of hNKCC1 activity was determined by exposing
cells to isotonic, hypotonic, or hypertonic media osmotically adjusted
with NMDG-methanesulfonate (MSA). All solutions contained similar final
Cl concentrations (104 mM). Hypotonic media contained 95 mM NaCl, 5 mM
RbCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM
Na2HPO4, 2 mM Na2SO4, 3 mM glucose, 15 mM HEPES titrated to pH 7.4 with NMDG base, and 0.1 mM
ouabain, with a final osmolarity of 220 mosmol/kgH2O. The
isotonic media contained an additional 40 mM NMDG-MSA (290 mosmol/kgH2O), and the hypertonic media contained an
additional 95 mM NMDG-MSA (400 mosmol/kgH2O). Cells were
preincubated for 10 min in hypotonic, isotonic, or hypertonic media
before 86Rb flux analysis.
Transwell fluxes.
The attainment of polarity by confluent MDCK monolayers was examined by
measuring the transport activity of the Na-K-ATPase (i.e.,
ouabain-sensitive 86Rb uptake) at the apical and
basolateral membranes. Five-day postconfluent monolayers grown on
permeable supports were washed twice and incubated for 15 min in
control media (see above). Fresh control media (±0.1 mM ouabain)
containing 2 µCi 86RbCl was then added to either the
apical or basolateral compartment of the Transwell support. The
opposite compartment received fresh control media containing both 0.1 mM ouabain and 10 µM bumetanide to prevent any back flux of isotope.
After 6 min, filters were removed and submerged into a large volume of
control media containing 0.1 mM ouabain and 10 µM bumetanide. After
eight washes, filters were removed from their support and transferred
to scintillation vials and lysed with 2% SDS. Isotopic decay was
measured by Cerenkov radiation and protein by the Micro BCA method. For
the MDCK LK-C1 cells expressing hNKCC1, we also monitored NKCC activity
at the apical and basolateral membranes using a similar protocol;
however, the cells were preincubated with low-[Cl] media for 60 min
in both compartments to stimulate the cotransporter before flux
analysis. After this preincubation, 86Rb influx was
monitored as above in control media containing 0.1 mM ouabain ±10 µM
bumetanide in the cis compartment. For all Transwell fluxes,
a sample of the supernatant from the trans compartment was
monitored for isotope as an indicator of transepithelial transport of
86Rb. In all cases, the appearance of 86Rb in
the trans compartment was <0.2% of the total radioactivity in the cis compartment.
Data analysis.
Results were analyzed statistically using a t-test in which
experimental values were compared with control measurements. Error bars
are ± SE. Statistical significance was defined as
P < 0.05.
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RESULTS |
NKCC protein analysis.
Using the NKCC monoclonal antibody T4, we analyzed membranes prepared
from wild-type MDCK cells, LK-C1 cells, and T84 cells by Western blot
(Fig. 1). These antibodies were prepared
against a portion of the COOH terminus of the hNKCC1 protein and
clearly recognized a single band at ~170-kDa in membranes prepared
from the human colonic T84 cell line. Upon removal of
N-linked sugar groups by treatment with
N-glycosidase F, the T4 antibody recognized a single band at
~135 kDa corresponding to the core protein deduced from the
nucleotide sequence (132 kDa) (17). Like T84 cells, wild-type MDCK cells expressed an endogenous ~170-kDa NKCC protein that was recognized by the T4 antibody, and its mobility in the gel
increased upon deglycosylation. In contrast to these two cell types,
the LK-C1 cells expressed NKCC protein that was significantly reduced
in its apparent molecular weight as well as in quantity compared with
the wild-type MDCK cells (note different protein loads). Furthermore,
the molecular weight of the NKCC protein of LK-C1 cells was only
slightly reduced by treatment of the membranes with
N-glycosidase F. These data indicate that the LK-C1 cells produce significantly less endogenous NKCC protein compared with the
wild-type MDCK cells (5- to 10-fold less) and that the protein was not
properly glycosylated. The lack of proper glycosylation of endogenous
NKCC protein in the LK-C1 cells suggested to us that the protein likely
never made it to the membrane surface and would probably be confined
largely to the endoplasmic reticulum. Immunolocalization experiments
confirmed this finding as endogenous NKCC protein of LK-C1 cells
exhibited a distinct perinuclear staining indicative of
endoplasmic reticulum labeling (data not shown). These findings are
consistent with the hypothesis that the LK-C1 cells have a defect in
the production and processing of the endogenous NKCC protein, leading
to the lack of any functional NKCC at the plasma membrane surface.
Whereas the wild-type MDCK cells exhibited a significant
bumetanide-sensitive 86Rb uptake after low-[Cl]
incubation, the LK-C1 cells displayed no such activity (Fig.
2, A and B). The
defect in the endogenous NKCC protein of the LK-C1 cells appears to be
specific because these cells have normal transport activities for the
Na-K-ATPase and Na/H exchanger (Ref. 14 and see below).

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Fig. 1.
Western blot analysis of Na-K-Cl cotransporter (NKCC)
from membranes of Madin-Darby canine kidney (MDCK) and T84 cells. NKCC
was detected with the anti-NKCC monoclonal antibody T4 after treatment
of membranes with (+) or without ( ) N-glycosidase F for
4 h at 37°C.
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Fig. 2.
Time course of 86Rb uptake into wild-type MDCK cells
(A), MDCK LK-C1 cells (B), and MDCK LK-C1 cells
expressing human secretory NKCC1 protein (hNKCC1; C). Cells
were incubated for 40 min in low-[Cl] media (4 mM) to stimulate NKCC.
Open symbols indicate 86Rb uptake in the presence of 200 µM bumetanide. Lines were fit by eye, and samples were taken in
triplicate. Data are representative of at least 3 separate
experiments.
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Interestingly, the inability of the LK-C1 cells to process endogenous
NKCC protein properly did not impair their ability to process
exogenously expressed NKCC protein (Figs. 2C and
3). The hNKCC1 protein stably
expressed in LK-C1 cells was properly glycosylated, as determined by
Western blot analysis (Fig. 3), and 86Rb influx
measurements with these stable cells confirmed the surface expression
of functional exogenous hNKCC1 protein (Fig. 2C). It should
be pointed out that in contrast to the T4 antibody used in Fig. 1,
which reacts with most vertebrate NKCC proteins, the mouse monoclonal
T10 antibody used in Fig. 3 is specific for the hNKCC1 protein
(13). This specificity was confirmed by the lack of any
endogenous NKCC protein recognition by T10 in the control vector-transfected LK-C1 cells (Fig. 3).

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Fig. 3.
Western blot analysis of hNKCC1 from membranes of T84
cells and MDCK LK-C1 cells stably transfected with vector alone or with
hNKCC1. hNKCC1 was detected with the anti-NKCC monoclonal antibody
specific for the hNKCC1 protein, T10. Membranes were treated with (+)
or without ( ) N-glycosidase F for 4 h at 37°C.
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NKCC regulation.
The activity of the NKCC1 protein is known to be modulated by reagents
and conditions that alter its phosphorylation state. In general,
conditions that promote the phosphorylation of NKCC1 activate the protein (11, 12). We examined the
modulation of both the endogenous NKCC protein of MDCK
wild-type cells and the exogenously expressed hNKCC1 protein by
reagents known to affect the phosphorylation state and activity of
NKCC1 in secretory cells (Fig. 4,
A-E). We tested these reagents under both basal conditions and after stimulation by low-[Cl] incubation. Figure 4A illustrates that under basal conditions, exogenously
expressed hNKCC1 protein was essentially inactive because little
bumetanide-sensitive 86Rb uptake could be detected. In
direct contrast, the endogenous NKCC protein of MDCK wild-type cells
exhibited a small but significant basal flux activity that was
~10-fold higher than the exogenously expressed hNKCC1 protein in
LK-C1 cells (Fig. 4D). We hypothesized that this higher
basal flux activity of the endogenous NKCC protein was likely the
result of a higher basal phosphorylation state of the protein. In
support of this hypothesis are data from both kinase and phosphatase
inhibitors (Fig. 4, A and D). Calyculin A, a
protein phosphatase inhibitor known to lead to the increased phosphorylation and activation of NKCC1 (12), markedly
stimulated exogenously expressed hNKCC1 but only increased the activity
of the endogenous NKCC protein approximately twofold. Furthermore, the
various kinase inhibitors tested, including staurosporine and
N-ethylmaleimide (NEM), had no significant effect on the
basal activity of exogenous hNKCC1, but these reagents inhibited the basal activity of the endogenous NKCC protein of MDCK wild-type cells.

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Fig. 4.
Regulation of exogenous hNKCC1 in MDCK LK-C1 cells
(A-C) and endogenous NKCC in MDCK wild-type cells
(D-E). Bumetanide-sensitive 86Rb influx was
monitored after treatment with various reagents (LK-C1 cells,
A and B; MDCK wild-type cells, D and
E) or after changes in cell volume (LK-C1 cells,
C). The effect of the various reagents was monitored either
under basal conditions (A and D) or after
incubation in low-[Cl] media (4 mM) for 40 min (B and
E). Values are means ± SE of >3 experiments.
*Significance from control values (P < 0.05;
t-test). NEM, N-ethylmaleimide.
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Both the exogenously expressed hNKCC1 and endogenous NKCC proteins were
activated by low-[Cl] incubation, which presumably reduces
intracellular [Cl] ([Cl]i) and leads to phosphorylation and activation of the NKCC protein (Fig. 4, B and
E). In both cell lines, staurosporine and NEM inhibited
virtually all of the NKCC activity stimulated by low-[Cl] incubation.
The tyrosine kinase inhibitor genistein was a less potent inhibitor of
NKCC activity in each cell line. Calyculin A caused a 100% increase in
hNKCC1 activity above that observed with low-[Cl] incubation alone,
whereas it had no effect on the endogenous NKCC activity. Forskolin had
little effect on the exogenous or endogenous NKCC activity after
low-[Cl] incubation (Fig. 4, B and E). Cell
shrinkage, which is known to activate NKCC in most cells, significantly
activated the exogenously expressed hNKCC1 (Fig. 4C). Cell
swelling had no effect on the basal exogenous hNKCC1 activity. These
experiments demonstrated that exogenously expressed hNKCC1 protein was
properly processed and functional at the plasma membrane of the LK-C1
cells. Furthermore, the exogenous hNKCC1 protein was regulated by
reagents and conditions in a manner similar to that of NKCC in most
other cells, including endogenous MDCK wild-type cells, i.e., it was activated by agents and conditions that promote its phosphorylation (phosphatase inhibitors, low-[Cl] incubation, hypertonicity) and inactivated by agents that promote its dephosphorylation (kinase inhibitors). The exogenously expressed hNKCC1 protein, however, was
significantly different from the endogenous NKCC protein in that it
exhibited no basal flux activity, and this finding can be explained by
a lower basal phosphorylation state of the exogenous hNKCC1 protein.
Establishment of a polarized epithelium with functional tight
junctions.
The LK-C1 cells represent a very useful expression system for the
cation Cl cotransporters since there is no apparent background loop diuretic-sensitive 86Rb flux activity. Furthermore,
the hNKCC1 protein appeared to be appropriately regulated when
exogenously expressed in the LK-C1 cells. An additional feature that
may be useful in cation Cl cotransporter expression experiments is the
ability of the LK-C1 cells to form a polarized epithelium. It is well
known that the wild-type MDCK cells are capable of differentiating into
a secretory-like epithelium if allowed to form confluent monolayers on
permeable supports (19). Thus we tested the ability of the
LK-C1 cells to form tight epithelial monolayers when grown on Transwell
supports. We first examined the development of transepithelial
resistance and transepithelial potential difference after the
attainment of confluency on permeable supports. As shown in Fig.
5, the wild-type MDCK cells formed a
tight epithelium 2 days after reaching confluency, with relatively high
transepithelial resistance (>1,000 ohm · cm2) and
potential difference (>20 mV). The LK-C1 cells, both control and
hNKCC1-expressing cells, exhibited significantly lower transepithelial resistances (~400 ohm · cm2) and potential
differences (~9 mV) 2-4 days after reaching confluency, but
these values still indicated that functionally intact tight junctions
had formed.

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Fig. 5.
Time course of changes in transepithelial resistance and
potential difference of wild-type MDCK cells, MDCK LK-C1 cells, and
MDCK LK-C1 cells expressing hNKCC1. Cells were grown on permeable
supports (Transwell; 0.4 µm) and resistance and potential difference
measurements were made using a voltohmmeter (Millipore).
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To investigate the formation of epithelial junctional complexes of the
LK-C1 cells further, we examined E-cadherin staining of 5-day
postconfluent monolayers. E-cadherin is a marker for the development of
adherens junctions between epithelial cells and is expressed upon
development of a polarized epithelial monolayer. Figure
6 displays both en face (x-y
plane) and cross-sectional (z plane) views through the
epithelia of the MDCK cell lines, including wild-type MDCK cells, LK-C1
cells, and LK-C1 cells expressing hNKCC1. Each cell line exhibited
E-cadherin expression along the lateral surfaces of the epithelial
cells, demonstrating the formation of junctional complexes between
them.

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Fig. 6.
Confocal laser scanning images
taken en face (A-C, top) or cross-sectional
(A-C, bottom) of E-cadherin in wild-type MDCK cells
(A), MDCK LK-C1 cells (B), or MDCK LK-C1 cells
(C) expressing hNKCC1. Cells were grown 5 days
postconfluence on permeable supports and then fixed with 4%
paraformaldehyde in PBS for 10 min at 24°C. E-cadherin was visualized
with a mouse monoclonal antibody (see MATERIALS AND
METHODS). Apical (Ap) and basolateral (Bs) membranes are
designated (bottom).
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We next examined the polarity of the cell lines by monitoring the
transport activity of the Na-K-ATPase at both the apical and
basolateral membranes. Five days after reaching confluency, no apical
Na-K-ATPase transport activity could be detected in any of the three
cell lines. All three cell lines, however, did express significant
Na-K-ATPase transport activity at the basolateral membrane (Fig.
7). Interestingly, the LK-C1 cells
expressing hNKCC1 exhibited a dramatically reduced Na-K-ATPase
transport activity. The expression of Na-K-ATPase exclusively at the
basolateral surface is consistent with the successful establishment of
epithelial polarity of all three cell lines. We also investigated the
polarity of hNKCC1 in stably transfected LK-C1 cells. We observed
little hNKCC1 activity at either the apical or basolateral membrane
under basal unstimulated conditions (data not shown). This latter
finding is consistent with the data presented in Fig. 4 in which the
expressed hNKCC1 protein appeared to be relatively inactive under basal conditions. In contrast, when the LK-C1 cells expressing hNKCC1 were
stimulated by incubation in low-[Cl] media, there was a robust activation of hNKCC1 exclusively at the basolateral membrane (Fig. 8). No hNKCC1 activity could be measured
at the apical membrane following the low-[Cl] incubation. These data
indicate that, like the endogenous Na-K-ATPase, exogenously expressed
hNKCC1 is properly localized at the basolateral membrane of the
polarized LK-C1 cells and that hNKCC1 retains its regulation by changes
in [Cl]i.

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Fig. 7.
Polarization of the Na-K-ATPase in wild-type MDCK cells, MDCK LK-C1
cells, or MDCK LK-C1 cells expressing hNKCC1. Under basal conditions,
86Rb influx was monitored in 5-day postconfluent cells in
the absence and presence of 0.1 mM ouabain at both the apical
(A) and basolateral (B) membranes. Values are
means ± SE of 3 experiments for MDCK wild-type and MDCK LK-C1
cells and 6 experiments for MDCK LK-C1 cells expressing hNKCC1.
*Significance from corresponding values of MDCK wild-type cells
(P < 0.05; t-test).
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Fig. 8.
Polarization of exogenous hNKCC1 in MDCK LK-C1 cells.
Bumetanide-sensitive 86Rb influx was monitored in 5-day
postconfluent cells in the absence and presence of 10 µM bumetanide
at both the apical (A) and basolateral (B)
membranes. Exogenous hNKCC1 was stimulated by a 40-min incubation in
low-[Cl] media.
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DISCUSSION |
McRoberts et al. (14) developed a number of MDCK cell
lines that were capable of growing under low extracellular [K]
conditions following treatment of the parent cell line with the
mutagen
N-methyl-N'-nitro-N-nitrosoguanidine. These MDCK mutants were shown to have specific defects in the Na-K-Cl
cotransport system, since they expressed reduced levels of
furosemide-sensitive 22Na, 86Rb, and
36Cl influxes as well as reduced
3[H]piretanide binding but exhibited normal transport
activities for the Na/H exchanger and the Na-K-ATPase (2,
14). Among the three mutant cell lines identified and
characterized by McRoberts et al. (14), the LK-C1 cells
were unique in that they expressed no detectable NKCC transport
activity and did not exhibit any significant loop diuretic binding
(2). In the absence of any functional endogenous NKCC
activity, the LK-C1 cell line offers a unique system with which to
examine the molecular physiology of the cation Cl cotransporters
because it represents an important expression system for
structure-function studies of this gene family. In the present report,
we characterized both endogenous and exogenous NKCC expression in the
MDCK LK-C1 cell line.
Using Western blot analysis, we found that the LK-C1 cells do express
NKCC protein but at significantly lower levels than the wild-type cells
(Fig. 1). Furthermore, the apparent molecular weight of the endogenous
cotransporter protein was only slightly greater than the core
deglycosylated mammalian NKCC1 protein (~132 kDa), indicating that
the NKCC protein of the LK-C1 cells was not properly glycosylated and
unlikely to be processed to the plasma membrane. This is fully
consistent with the lack of specific loop diuretic binding observed by
Giesen-Crouse and McRoberts (2). Immunocytochemistry
confirmed that the endogenous NKCC protein of the LK-C1 cells exhibits
a perinuclear staining, indicative of a protein localization in the
endoplasmic reticulum. These data suggest that some defect exists in
the processing of the endogenous protein, preventing its targeting to
the plasma membrane. The lack of any functional NKCC protein at the
plasma membrane of the LK-C1 cells was confirmed by the absence of any
bumetanide-sensitive 86Rb influx (Fig. 2). Even though the
LK-C1 cells have a defect in the processing of the endogenous NKCC
protein, when we stably expressed the hNKCC1 protein in the LK-C1
cells, we were able to restore Na-K-Cl cotransporter activity. Thus the
defect in the processing of the endogenous NKCC protein does not appear to impede the functional expression of the exogenous hNKCC1 protein. Both Western blot analysis and bumetanide-sensitive 86Rb
influx assay confirmed that the exogenous hNKCC1 protein was properly
glycosylated and processed to the plasma membrane (Figs. 2 and 3).
The exogenously expressed hNKCC1 in the MDCK LK-C1 cells was regulated
in a manner similar to that observed in secretory tissues as well as
that in MDCK wild-type cells (Fig. 4). It has been shown for a number
of secretory epithelia and avian red blood cells that activation of the
Na-K-Cl cotransporter involves direct phosphorylation of the protein
(4, 8, 11, 20, 21). Thus the phosphorylation state of the
protein reflects a competition between concurrent kinase and
phosphatase activities (9). The exogenously expressed
hNKCC1 protein and the endogenous NKCC protein of MDCK wild-type cells
were both activated by the phosphatase inhibitor calyculin A, and their
activities could be inhibited by the kinase inhibitors staurosporine
and NEM. Furthermore, both NKCC proteins were activated by a low-[Cl]
incubation (Fig. 4, B and E). Interestingly, the
endogenous NKCC protein of MDCK wild-type cells displayed a significant
basal bumetanide-sensitive 86Rb influx activity that was
not observed with the exogenous hNKCC1 protein expressed in LK-C1
cells. Because the kinase inhibitors staurosporine and NEM inhibited
this basal activity of the endogenous NKCC protein, we conclude that
the basal phosphorylation state of the endogenous NKCC protein must be
greater than that of the exogenously expressed hNKCC1 protein. We
observed greater stimulation of exogenously expressed hNKCC1 under
low-[Cl] conditions than with cell shrinkage. This finding is likely
related to the fact that after cell shrinkage, [Cl]i
increases, and this significantly inhibits full activation of the cotransporter.
The use of an MDCK cell line to express hNKCC1 has the benefit that
this cell line can potentially form a functional epithelium upon
differentiation. MDCK cells have been reported to be represented by two
strains, a low-resistance type and a high-resistance type (18). The wild-type MDCK cells used in the present study
were clearly of the high-resistance strain. We found that the MDCK LK-C1 cell line, however, does not form as tight an epithelium as the
wild-type MDCK cells, but it clearly forms a polarized epithelium. When
grown 5 days postconfluence on permeable supports, the MDCK LK-C1 cells
expressed junctional complexes between cells as revealed by E-cadherin
staining. Furthermore, the cells were appropriately polarized with
functional Na-K-ATPase transport activity observed exclusively at the
basolateral membrane. Interestingly, we observed that the basal
Na-K-ATPase transport activity in the MDCK LK-C1 cells expressing
hNKCC1 was significantly reduced from control LK-C1 cells (Fig. 7).
This reduced pump activity is perplexing given that it is normal in the
MDCK LK-C1 cells. It is possible that overexpression of hNKCC1 with the
potent cytomegalovirus promoter used in our construct caused reduced
expression of certain endogenous proteins like the Na-K-ATPase;
however, we never observed any reduction in growth of the stable cells.
In addition to the proper polarization of the Na-K-ATPase, we found
that the exogenously expressed hNKCC1 protein exhibited functional
activity exclusively at the basolateral membrane following activation
by low-[Cl] incubation.
In conclusion, the MDCK LK-C1 cells exhibited many characteristics that
are favorable for expression studies aimed at examining structure-function of the Na-K-Cl cotransporter. First, these cells
express little endogenous NKCC protein, and the NKCC protein that is
expressed is nonfunctional (i.e., not localized at the plasma
membrane). Second, exogenously expressed hNKCC1 protein is regulated in
a manner similar to that of secretory epithelia. Third, the cells are
capable of forming a polarized epithelium that would permit independent
manipulation of the apical and basolateral membranes. Because virtually
all of the loop diuretic-sensitive 86Rb transport activity
in the MDCK LK-C1 cells was mediated via the exogenously expressed
hNKCC1 protein, these cells offer an excellent expression system for
the cation Cl cotransporters and are especially useful for those
cotransporters that appear to exhibit very low transport activities
(e.g., K-Cl cotransporter KCC1) (3).
 |
ACKNOWLEDGEMENTS |
The authors are grateful to Jeff Williams for excellent technical
assistance and to Drs. Peter Cala, Chris Lytle, Bliss Forbush III, and
Martha O'Donnell for helpful discussions and readings of the
manuscript. The authors are indebted to Dr. Jim McRoberts (UCLA) for
providing the low K-resistant MDCK cell lines and to Dr. Bliss Forbush
III (Yale University) in whose lab this work was first started.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
DK-47661 (to B. Forbush) and National Institute of Neurological Disorders and Stroke NS-36296 (to J. A. Payne), by the University of California Davis Health Systems Research Funds (to J. A. Payne), and by a grant from the American Heart Association, Western
States Affiliate (to J. A. Payne).
Address for reprint requests and other correspondence: J. A. Payne, MED: Human Physiology, One Shields Ave., Univ. of California, Davis, CA 95616-8644 (E-mail: japayne{at}ucdavis.edu).
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.
Received 12 July 2000; accepted in final form 28 December 2000.
 |
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