Am J Physiol Cell Physiol Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 280: C1607-C1615, 2001;
0363-6143/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Payne, J. A.
Right arrow Articles by Chung, C. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Payne, J. A.
Right arrow Articles by Chung, C. Y.
Vol. 280, Issue 6, C1607-C1615, June 2001

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (52K):
[in this window]
[in a new window]
  		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.



View larger version (17K):
[in this window]
[in a new window]
  		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.

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).


View larger version (30K):
[in this window]
[in a new window]
  		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.

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.


View larger version (20K):
[in this window]
[in a new window]
  		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.

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.


View larger version (27K):
[in this window]
[in a new window]
  		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).

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.


View larger version (72K):
[in this window]
[in a new window]
  		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).

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.


View larger version (30K):
[in this window]
[in a new window]
  		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).



View larger version (26K):
[in this window]
[in a new window]
  		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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Behnke, RD, and Forbush B, III. Stimulatory phosphorylation sites on the shark rectal gland Na-K-Cl cotransporter (Abstract). FASEB J 12: A1013, 1998.

2.   Giesen-Crouse, EM, and McRoberts JA. Coordinate expression of piretanide receptors and Na+, K+, Cl- cotransport activity in Madin-Darby canine kidney cell mutants. J Biol Chem 262: 17393-17397, 1987[Abstract/Free Full Text].

3.   Gillen, CM, Brill S, Payne JA, and Forbush B, III. Molecular cloning and functional expression of the K-Cl cotransport from rabbit, rat, and human: a new member of the cation-chloride cotransporter family. J Biol Chem 271: 16237-16244, 1996[Abstract/Free Full Text].

4.   Haas, M, McBrayer D, and Lytle C. [Cl-]i-dependent phosphorylation of the Na-K-Cl cotransport protein of dog tracheal epithelial cells. J Biol Chem 270: 28955-28961, 1995[Abstract/Free Full Text].

5.   Isenring, P, and Forbush B, III. Ion and bumetanide binding by the Na-K-Cl cotransporter: importance of transmembrane domains. J Biol Chem 272: 24556-24562, 1997[Abstract/Free Full Text].

6.   Isenring, P, Jacoby SC, Chang J, and Forbush B, III. Mutagenic mapping of the Na-K-Cl cotransporter for domains involved in ion transport and bumetanide binding. J Gen Physiol 112: 549-558, 1998[Abstract/Free Full Text].

7.   Isenring, P, Jacoby SC, and Forbush B, III. The role of transmembrane domain 2 in cation transport by the Na-K-Cl cotransporter. Proc Natl Acad Sci USA 95: 7179-7184, 1998[Abstract/Free Full Text].

8.   Lytle, C. Activation of the avian erythrocyte Na-K-Cl cotransporter protein by cell shrinkage, cAMP, fluoride, and calyculin-A involves phosphorylation at common sites. J Biol Chem 272: 15069-15077, 1997[Abstract/Free Full Text].

9.   Lytle, C. A volume-sensitive protein kinase regulates the Na-K-2Cl cotransporter in duck red blood cells. Am J Physiol Cell Physiol 274: C1002-C1010, 1998[Abstract/Free Full Text].

10.   Lytle, C, and Forbush B, III. Na-K-Cl cotransport in the shark rectal gland. II. Regulation in isolated tubules. Am J Physiol Cell Physiol 262: C1009-C1017, 1992[Abstract/Free Full Text].

11.   Lytle, C, and Forbush B, III. The Na-K-Cl cotransport protein of shark rectal gland. II. Regulation by direct phosphorylation. J Biol Chem 267: 25438-25443, 1992[Abstract/Free Full Text].

12.   Lytle, C, and Forbush B, III. Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl. Am J Physiol Cell Physiol 270: C437-C448, 1996[Abstract/Free Full Text].

13.   Lytle, C, Xu JC, Biemesderfer D, and Forbush B, III. Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies. Am J Physiol Cell Physiol 269: C1496-C1505, 1995[Abstract/Free Full Text].

14.   McRoberts, JA, Tran CT, and Saier MHJ Characterization of low potassium-resistant mutants of the Madin-Darby canine kidney cell line with defects in NaCl/KCl symport. J Biol Chem 258: 12320-12326, 1983[Abstract/Free Full Text].

15.   Palfrey, HC, and Pewitt EB. The ATP and Mg2+ dependence of Na-K-2Cl cotransport reflects a requirement for protein phosphorylation: studies using calyculin A. Pflügers Arch 425: 321-328, 1993[Web of Science][Medline].

16.   Payne, JA. Functional characterization of the neuronal-specific K-Cl cotransporter: implication for [K+]o regulation. Am J Physiol Cell Physiol 273: C1516-C1525, 1997[Abstract/Free Full Text].

17.   Payne, JA, Xu JC, Haas M, Lytle CY, Ward D, and Forbush B, III. Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon. J Biol Chem 270: 17977-17985, 1995[Abstract/Free Full Text].

18.   Simmons, NL. Ion transport in "tight" epithelial monolayers of MDCK cells. J Membr Biol 59: 105-114, 1981[Web of Science][Medline].

19.   Simmons, NL. Stimulation of Cl- secretion by exogenous ATP in cultured MDCK epithelial monolayers. Biochim Biophys Acta 646: 231-242, 1981[Medline].

20.   Tanimura, A, Kurihara K, Reshkin SJ, and Turner RJ. Involvement of direct phosphorylation in the regulation of the rat parotid Na-K-2Cl cotransporter. J Biol Chem 270: 25252-25258, 1995[Abstract/Free Full Text].

21.   Torchia, J, Lytle C, Pon DJ, Forbush B, III, and Sen AK. The Na-K-Cl cotransporter of avian salt gland. Phosphorylation in response to cAMP-dependent and calcium-dependent secretagogues. J Biol Chem 267: 25444-25450, 1992[Abstract/Free Full Text].

Am J Physiol Cell Physiol 280(6):C1607-C1615
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
J. R. Williams and J. A. Payne
Cation transport by the neuronal K+-Cl- cotransporter KCC2: thermodynamics and kinetics of alternate transport modes
Am J Physiol Cell Physiol, October 1, 2004; 287(4): C919 - C931.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
L. M. Maglova, W. E. Crowe, and J. M. Russell
Perinuclear localization of Na-K-Cl-cotransporter protein after human cytomegalovirus infection
Am J Physiol Cell Physiol, June 1, 2004; 286(6): C1324 - C1334.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
J. C. de Jong, P. H.G.M. Willems, L. P.W.J. van den Heuvel, N. V.A.M. Knoers, and R. J.M. Bindels
Functional Expression of the Human Thiazide-Sensitive NaCl Cotransporter in Madin-Darby Canine Kidney Cells
J. Am. Soc. Nephrol., October 1, 2003; 14(10): 2428 - 2435.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Payne, J. A.
Right arrow Articles by Chung, C. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Payne, J. A.
Right arrow Articles by Chung, C. Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online