Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits

Bonnie L. Blazer-Yost1, Michael Butterworth2, Amy D. Hartman1, Gretchen E. Parker1, Carla J. Faletti1, Willem J. Els2, and Simon J. Rhodes1

1 Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202; and 2 Department of Anatomy and Cell Biology, University of Cape Town Medical School, Cape Town, South Africa


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged alpha - or beta -subunits of the epithelial Na+ channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominately subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na+ transport. The basal level of Na+ transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na+ transport was 64% greater than control in both the alpha - and beta -transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na+ transport.

sodium transport; amiloride; aldosterone; insulin; channel trafficking; adenosine 3',5'-cyclic monophosphate, signal transduction; epithelial sodium channel; green fluorescent protein


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EPITHELIAL SODIUM CHANNELS (ENaCs) are crucial for the maintenance of fluid and electrolyte homeostasis. These amiloride-sensitive channels are found in the apical membrane of polarized epithelia and form the rate-limiting step for Na+ reabsorption. At a molecular level, the primary components of the channels have been described. The basic building blocks of ENaC consist of the alpha -, beta -, and gamma -subunits (12, 13, 28).

There are a number of hormones and other endogenous factors that regulate the amiloride-sensitive Na+ channel in vivo, including aldosterone, insulin, antidiuretic hormone (ADH), insulin-like growth factor-1, and prostaglandins (3, 5, 7, 8, 10, 16-20, 25, 26, 29, 32, 34). While much has been learned about the molecular nature of ENaC, the mechanisms of channel regulation by these factors remain largely unknown.

The use of different techniques to study channel regulation has yielded discrepant answers to such fundamental questions as channel number and the biophysical changes in channel parameters in response to hormonal regulation (9). However, there is a general consensus that the number of active channels in native epithelia is relatively low. With the use of blocker-induced noise analysis, it has been demonstrated that the total number of functional channels in model high-resistance epithelia, such as the Xenopus laevis kidney (A6) cell line, toad urinary bladder, and frog skin, is typically measured as tens to, at most, hundreds of channels per cell (9, 10, 26). This low density of channels in native apical membranes poses a number of technical problems. Attempts to overcome such difficulties have given rise to the use of overexpression by transient transfection in heterologous cell lines, as well as the use of artificial membranes, or expression systems such as Xenopus oocytes. While these studies have contributed to our understanding of channel kinetics, it is difficult to use artificial systems to study channel regulation when the endogenous regulators are unknown. The overexpression levels commonly found in transient transfections may overwhelm the normal regulatory components if those regulatory components are, in fact, present in the heterologous models.

As a complementary approach to the biophysical and biochemical studies of channel regulation, and to allow the direct visualization of active ENaC in transporting epithelia, we have produced stably transfected clones of the A6 cell line that contain low levels of enhanced green fluorescent protein (EGFP)-labeled alpha - or beta -ENaC subunits. The A6 cell line is a well-characterized model of the principal cells of the distal nephron. Thus these new clones are produced in native epithelial cells that endogenously express ENaC and contain the signal transduction pathways that modulate its activity. We have confirmed that the appropriate ENaC-EGFP RNAs and proteins are expressed in the generated cell lines and have determined the subcellular localization of ENaC in these native epithelial cells using confocal microscopy. In addition, we have characterized the functional responses of the clones to hormonal stimulation and used the cell lines to examine channel trafficking in response to forskolin stimulation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. A6 cells were grown at 27°C in a modified DMEM (no. 91-5055EC; Life Technologies, Gaithersburg, MD) supplemented with 25 U/ml penicillin, 25 µg/ml streptomycin, and 10% calf serum in a humidified incubator gassed with 5% CO2. The media used for maintaining the clones contained 0.5 mg/ml Geneticin (Life Technologies). The cells were subcultured onto 24-mm Transwell tissue culture-treated inserts (Costar, Cambridge, MA), as previously reported (8), for at least 12 days to achieve confluence.

GFP expression vector construction. cDNAs encoding Xenopus ENaC alpha  (xENaCalpha ) and beta  (xENaCbeta ) channel subunits were cloned from A6 cell cDNA by polymerase chain reaction (PCR) using oligonucleotide primers based on the sequences described by Puoti et al. (31) and Pfu DNA polymerase (Stratagene, La Jolla, CA). cDNAs were first subcloned into pBluescript KS II(-) (Stratagene) using standard procedures and confirmed by DNA sequencing using the Sanger dideoxy method and Sequenase (Amersham, Piscataway, NJ). Expression vectors for xENaCalpha -EGFP and EGFP-xENaCbeta were generated by cloning PCR products into the XhoI/BamHI sites of pEGFP-N1 and the EcoRI/BamHI sites of pEGFP-C1 (Clontech, Palo Alto, CA), respectively. Oligonucleotides were 5'-gcgctcgagaggacgcctggagatatctgagac-3' (sense), 5'-cgggatcccttctacctccattctcctcatag-3' (antisense) for xENaCalpha -EGFP and 5'-cggaattctatgaagaggctgaagcgatatttcacc-3' (sense), 5'-cgggatcccgcttgtagtgtcgtttaattctc-3' (antisense) for EGFP-xENaCbeta . Plasmids were purified by two rounds of cesium chloride gradient centrifugation (4) and confirmed by DNA sequencing.

A6 cell transfection. A6 cells were seeded onto plastic petri dishes and grown to ~20% confluence. Transfection with the EGFP expression vectors was accomplished by incubating the cells with DNA (4 µg/100-mm2 dish) and Lipofectamine (Life Technologies) in serum-free medium overnight. The following day, the mixture was replaced with normal serum-containing medium and the cells were grown to confluence. The cells were trypsinized and replated on petri dishes at a concentration equal to 10% confluent density in the presence of a toxic concentration of Geneticin (2 mg/ml). Under these conditions, it was possible to identify clones arising from single cells that had incorporated ENaC-EGFP expression vector containing a neomycin resistance gene. When individual clones had grown to colonies of 100-200 cells, they were trypsinized using cloning cylinders (Bel-Art Products, Pequannock, NJ), seeded onto 25-cm2 flasks, and maintained in 0.5 mg/ml Geneticin. The individual clones were maintained as normal A6 cells (see above) but were kept under selective pressure with the addition of 0.5 mg/ml Geneticin to the culture medium. Cells seeded onto Transwell inserts were fed with normal A6 medium.

RNA isolation. Total RNA was extracted from the parent A6 and stable clonal lines using Tri-Reagent (Molecular Research Center, Cincinnati, OH) and bromochloropropane as a chloroform substitute. The RNA was further purified by isopropanol precipitation, and integrity was checked by electrophoresis on 1% agarose-formaldehyde gels.

RT-PCR analysis. cDNA was synthesized using Superscript II reverse transcriptase (Life Technologies) and random hexamer or gene-specific 3' primers. Primer pairs for PCR were 5'-ggtgagcaagggcgaggagctg-3'/5'-ctcgtccatgccgagagtgatcc-3' or 5'-ggtgagcaagggcgaggagctg-3'/5'-cccttcagctcgatgcggttcacc-3' for EGFP and 5'-ttgaacaagatggattgcacgcag-3'/5'-cgccttgagcctggcgaacagttc-3' for neomycin phosphotransferase. Thermal cycling parameters were 95°C for 10 s, 57°C for 10 s, and 72°C for 45 s for 30 cycles. Reaction products were analyzed on 1.5% agarose Tris-borate gels and visualized by staining with ethidium bromide.

PAGE and Western analysis. A6 cell homogenates were prepared from cells grown on Transwell filters. The apical medium of confluent cells was aspirated and replaced with fresh serum-free medium twice. The cells were scraped from the nucleopore filter with a rubber policeman, pelleted by centrifugation (1,000 g), resuspended in serum-free medium, and repelleted. The pellet was solubilized in sodium dodecyl sulfate (SDS) lysis buffer (50 mM Tris, pH 6.8, 2% SDS, 120 mM dithiothreitol, and 10% glycerol; 0.5 ml/Transwell) using 20 strokes of a tight-fitting, hand-held Dounce homogenizer. The homogenate was centrifuged in a microcentrifuge, and the supernatant was stored at -20°C until polyacrylamide gel electrophoresis (PAGE) analysis. The solubilized proteins were separated by one-dimensional PAGE using a 4-20% Tris-glycine gel (Novex, San Diego, CA). After electrophoresis, the separated proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF)-blotting membranes, and the membranes were stained with Coomassie brilliant blue R to visualize the protein bands. The PVDF-immobilized, Coomassie-stained protein blots were blocked with 1% bovine serum albumin to prevent nonspecific binding and probed with an anti-GFP polyclonal antibody (1:1,000 dilution; Clontech Laboratories), followed by binding of a secondary goat-anti rabbit horseradish peroxidase IgG (1:40,000 dilution; Cappel Laboratories; Durham, NC). The specific binding was detected using a peroxidase-based chemiluminescent detection method (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions.

Electrophysiology. Short-circuit current (SCC) techniques were used to determine net ion flux across high-resistance, confluent epithelia (27). Cells grown on Transwell inserts were placed in modified Ussing chambers and monitored as previously described (8). During the electrophysiological experiments, the cells were bathed in serum-free medium maintained at 27°C, with gentle circulation provided by a 5% CO2-95% O2 gas lift. The monolayers were monitored until a steady baseline current was obtained (typically 0.5-2 h). Hormones were added to the serosal bathing medium. At the end of each experiment, amiloride (10-5 M) was added to the apical bathing medium to determine the proportion of the current that was due to Na+ flux mediated by ENaC. In both the parent cell line and the clonal lines, >90% of the current was amiloride sensitive. Transepithelial resistance was determined every 2 min via a brief 2-mV pulse. All cellular monolayers (parent line as well as clones) had resistances >1,000 Omega  · cm2.

Confocal microscopy. A6 cells were cultured on Millipore millicell HA permeable supports in DMEM for at least 14 days to produce confluent monolayers. For live cell imaging, tissues on the filter were washed in cold DMEM, whereafter the apical membranes were labeled for 30 min on ice with FM 4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)-hexatrienyl) pyridinium dibromide] (Molecular Probes, Leiden, The Netherlands) at 16 µM (6). After plasma membrane labeling, excess fluophore was removed with two cold DMEM washes to reduce background fluorescence. Next, an ~0.75-cm2 area of filter was punched out of the supports, and the cells were transferred to a special chamber where tissues were mounted apical surface down for imaging. Cells were bathed continuously in DMEM during imaging.

Laser scanning confocal microscopy (LSCM) was performed with an inverted Leica TSC-NT confocal imaging system equipped with a ×63 Plan APO 1.2 NA water immersion objective that was used for all imaging. For the dual imaging of EGFP and FM 4-64, fluorescent images were collected by exciting the fluorophores with a krypton-argon laser at 478 nm (EGFP) and 568 nm (FM 4-64). Emissions from EGFP and FM 4-64 were detected with band-pass filter sets of 495-525 nm and 690-730 nm, respectively. A sequential scanning program was used to collect z-optical sections at 0.3-µm intervals, with an optimal scan time that varied between 8 and 32 s and with the use of 4-16 line averages, depending on the intensity of the fluorescence and the degree of photobleaching. The images detected by excitation with the 478-nm line were directed to the green channel, while those from excitation with the 568-nm line were directed to the red channel. An overlay of collected images at each depth was performed to produce the final double-labeled images (Leica TSC-NT software). Because the ENaC-EGFP signal most often emitted at a lower level than the FM 4-64 signal, the emitted EGFP signal was optimized by increasing the photomultiplier gain until levels in both channels appeared approximately equal. Laser power was kept below 50% to minimize photo damage, with optimal pinhole size automatically determined for the objective. Under these conditions there was minimal autofluorescence from the preparation and no bleed-through between emitted fluorophore channels.

FM 4-64, which was used to specifically label cell membranes in the live cell studies, did not provide optimal intensity to unequivocally localize EGFP-tagged subunits within the apical membrane. Localization of the tagged subunits at the apical membrane was improved by using fixed monolayers of cells and labeling the apical membrane with wheat germ agglutinin (WGA). Cells were incubated on ice with rhodamine-conjugated WGA (2 µg/ml; Molecular Probes) prepared in a buffered Ringer solution containing 108 mM NaCl, 3 mM KCl, 2.5 mM NaHCO3, 1 mM K2HPO4, 10 mM glucose, 1 mM CaCl2, and 0.8 mM MgCl2 at a pH of 7.6. After incubation, cells were washed twice with ice-cold Ringer solution for 5 min and fixed on ice in 1% paraformaldehyde (Sigma Aldrich, St. Louis, MO) prepared in the Ringer solution for 30 min. Finally, cells were washed in Ringer solution twice, and filters were mounted on slides. Red and green channels used to visualize FM 4-64 and EGFP in the studies on live cells were similarly suitable to image the rhodamine/EGFP combination.

To examine trafficking in response to forskolin stimulation, we reconstituted serial optical sections to form three-dimensional (3-D) images using the Leica TCS-NT software. Images were oriented with coordinates (xy 0°, yz 0°, xz 90°) to visualize the epithelial monolayer from the side. Because the EGFP signal was relatively weak, sliced orthogonal sections were not taken, but the entire imaged EGFP signal within all cells was viewed from the side. In this configuration the FM 4-64 fluorescent signal was far stronger than the EGFP levels, and the red signal was consequently omitted from reconstituted 3-D images to better observe internal EGFP. It is therefore important to note that in figures illustrating EGFP trafficking, no membrane label is shown and images are orientated with the apical membrane toward the top. Because of autofluorescent signals produced by the chamber coverslip, the first optical section was typically acquired 1 µm from the apical membrane.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression vectors for xENaCalpha -EGFP and EGFP-xENaCbeta were generated by cloning the respective Xenopus ENaC cDNAs into cytomegalovirus promoter-driven plasmids. One vector expressed the ENaCalpha -subunit protein fused to the amino terminus of the enhanced form of EGFP (Fig. 1A). The second vector generated a fusion protein where the beta -ENaC subunit was fused to the carboxy terminus of EGFP (Fig. 1A). This latter fusion protein design was guided by the observation that mutations in the carboxy terminus of the human beta -ENaC subunit cause Liddle's syndrome (33). We therefore wanted to avoid the possibility of compromising this region of the beta -subunit protein. Likewise, Bonny et al. (11) have shown that the amino terminus, particularly the first 535 amino acids of the alpha -subunit, may be important for targeting to the plasma membrane. Therefore, the EGFP was added to the carboxy terminus of the alpha -subunit to avoid potential alteration in the targeting domain.


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Fig. 1.   Molecular characterization of stable A6 kidney cell lines expressing Xenopus (x) epithelial Na+ channel (ENaC) subunit-enhanced green fluorescent protein (EGFP) fusion proteins. A: schematic of the expression vector plasmids (p). CMV, cytomegalovirus promoter. B: RT-PCR analysis of ENaC subunit-EGFP fusion protein gene expression. PCR was used to amplify green fluorescent protein (EGFP)-containing mRNA sequences from the stable cell lines. Products were separated on 1.5% agarose gels and visualized by staining with ethidium bromide. Negative controls using either cDNA generated from nontransfected A6 cells or alpha 5 cDNA that was not treated with reverse transcriptase (no RT) did not produce products. RT-PCR analysis of the neomycin phosphotransferase (NPT) indicated similar levels of expression in the stable cell lines. The integrity of total RNA from the ENaC subunit-EGFP stable cell lines was confirmed by denaturing agarose gel electrophoresis (RNA). The 28S and 18S ribosomal RNA can be seen in these preparations. C: Western analysis of the ENaC subunit-EGFP stable cell lines. Protein was isolated from the indicated clones or the parent A6 cells, separated by SDS-PAGE (4-20% gels), transferred to polyvinylidene difluoride membranes, and stained with Coomassie brilliant blue (bottom) or probed with anti-GFP antibody and visualized by chemiluminescence (top). The position of the molecular weight standards (in kDa) are provided at left. The arrows indicate a calculated molecular weight of 120 kDa.

A6 cells were transfected and selected as single-cell clones using the conferred resistance to Geneticin (neomycin). Multiple clones were selected expressing each of the ENaC-EGFP fusion proteins. Here we present the analysis of two of the beta  clones and one of the alpha  clones.

To confirm expression of the ENaC subunit-EGFP vectors in the clonal cell lines, we analyzed both RNA and protein products from the introduced genes. By Northern analysis, ENaC subunit-EGFP mRNAs were barely detectable (data not shown). With RT-PCR analysis, fusion protein transcripts were detected in the derived cell lines but not in the parent A6 cell line or in controls (Fig. 1B). Parallel experiments indicated that the generated cell lines expressed similar levels of the neomycin phosphotransferase mRNA, which allows resistance to the selection antibiotic (Fig. 1B). On the basis of multiple, independent repeats of this analysis, we estimate that all of the ENaC subunit-EGFP mRNAs are expressed at low abundance, with highest levels in the alpha 5 line and lowest levels in the beta 3 line.

To confirm the presence of an EGFP-labeled ENaC protein, we grew the individual clones to confluence on permeable Transwell supports, and the proteins were isolated, separated by SDS-PAGE, immobilized on PVDF membranes, stained with Coomassie, and probed with an anti-GFP polyclonal antibody (Fig. 1C). A distinct 115- to 125-kDa band was illuminated by the Western detection system in the lanes containing clonally derived proteins while the parent A6 cell line does not contain a similar epitope (Fig. 1C, top). This is the predicted molecular weight for the ENaC-EGFP chimeric proteins. The Coomassie-stained membrane that was used for the Western detection is shown in Fig. 1C, bottom. The arrow denotes a molecular weight of ~120 kDa on each panel. On the basis of the Coomassie staining, the EGFP-labeled ENaC is not an abundant protein in any of the clonal cell lines.

The ENaC-EGFP chimeric proteins are expressed as functional Na+ transporters in the clonal lines. The basal level of ion transport exhibited by the parent A6 cell line compared with the alpha  and beta  clones is shown in Fig. 2. The SSC technique measures net ion transport. Although there are statistical differences among the individual transfected lines, all displayed ion transport levels higher than those of the parent line. A second alpha  clone (alpha 2) was also examined in a more limited series of studies. This alpha 2 line had basal transport rates that were similar to those of the alpha 5 clone (data not shown). A cell line created as a stable transfectant with the use of only the neomycin empty vector had basal currents that were the same as the control A6 cells (data not shown). Thus all the ENaC-transfected lines show higher basal transport rates than the parent line.


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Fig. 2.   Basal levels of ion transport in A6 cells and ENaC-EGFP-transfected A6 cell clones. Ion transport across confluent monolayers of normal and transfected A6 cells was measured as short-circuit currents (SCC). Values are means ± SE; n = no. of monolayers measured.

LSCM allowed us to clearly visualize ENaC-EGFP subunits at high resolution in living cells. FM 4-64, a membrane dye, does not permeate cell membranes and fluoresces strongly only when bound to plasma membranes. Accordingly, the label does not bind to intracellular membranes or organelles (6). This property allowed us to orientate and determine the intracellular distribution of the EGFP-tagged subunits in living cells.

Representative confocal microscope images of A6 cells transfected with the ENaC-EGFP subunits in Fig. 3 illustrate the localization of the individual EGFP subunits. Green fluorescence was not detected in nontransfected cells (data not shown). The intracellular localization of EGFP signal in live, unstimulated cells is illustrated in Fig. 3, A and B. The optical sections of clone alpha 5 (Fig. 3A) show clusters of EGFP that are more abundant in the upper portions of the cell than in the deeper intracellular compartments. In the first optical sections there appeared to be some green fluorescence colocalized with the red membrane marker to form yellow areas (Fig. 3A). A similar distribution was observed with the beta  clones (Fig. 3B). In Fig. 3C, the xz section taken from fixed A6 cells shows localization of EGFP and rhodamine-conjugated WGA-labeled membrane. A portion of the ENaC subunits were located in the apical membrane where they could participate in the basal transport of Na+. The bulk of the subunits in these unstimulated cells were, however, localized in the subapical regions of the cells. We have noticed that the levels of expression of GFP-ENaC subunits can be variable between cloned cells.


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Fig. 3.   A: confocal microscopy photomicrographs showing double fluorescent images of optical sections taken from EGFP-ENaC alpha 5 cloned cells cultured for 14 days. A schematic illustrating the sectioned depths below the apical surface is presented beneath each image (not drawn to scale). Although only the apical membrane was labeled with FM 4-64, it is likely that some of the label penetrated past the tight junctions and labeled some of the lateral plasma membranes. The signal was absent in the deepest optical sections. B: representative images of EGFP-ENaC beta 1 and beta 3 clones in live cells cultured for 21 days. Each image was acquired 2 µm beneath the apical membrane, and the membrane was labeled with FM 4-64. C: EGFP localization in fixed cells. The apical membranes of paraformaldehyde-fixed cells of the EGFP-ENaC alpha 5 clonal line were labeled with rhodamine-conjugated wheat germ agglutinin. Top image shows apical surface labeling, while the corresponding xz orthogonal section (bottom) shows the membrane (red) with colocalized ENaC (yellow) and subapical ENaC (green). The yellow line (top) indicates the area of the xz section. Scale bar, 5 µm.

We and others have previously shown that the A6 cell line responds to hormonal stimulation by insulin, aldosterone, or ADH with an increase in amiloride-sensitive Na+ transport (3, 5, 7, 8, 10, 16-20, 25, 26, 29, 32, 34). Here the functional responses to insulin, aldosterone, and forskolin were examined in the clonal lines and compared with responses in the parent line. Forskolin, which constitutively activates adenylate cyclase, was used to increase cAMP, thereby mimicking the action of ADH.

The magnitude of the amiloride-sensitive ion transport response to either insulin (Fig. 4) or aldosterone (Fig. 5) in the transfected cell lines was very similar to that of the corresponding response in the parent line. In response to forskolin, however, the alpha  and beta  clonal lines exhibited maximal responses that were 64% greater than those of the parent line. These maximal values were not maintained for more than a few minutes in the clonal lines (Fig. 6). LSCM was used to determine any changes in the subcellular localization of EGFP-ENaC in response to forskolin stimulation. Both alpha  and beta  clones exhibited a movement of labeled channel subunits toward the apical membrane in response to stimulation (Fig. 7).


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Fig. 4.   Effect of insulin on normal and ENaC-EGFP-transfected A6 cell lines. Rates of transport were measured as SCC. Insulin (30 nM) was added at time 0 to the serosal face of the epithelia, indicated by filled symbols. The epithelia indicated by open symbols are matched controls. Amiloride (10-5 M) was added to the apical face of the control and insulin-treated parent line (A) 120 min after insulin was added. Amiloride (10-5 M) was added to the apical face of the control and insulin-treated ENaC-EGFP alpha 5 (B), beta 1 (C), and beta 3 (D) clones 30 min after insulin was added. Values are means ± SE at each time point; n = no. of monolayers measured.



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Fig. 5.   Effect of aldosterone on normal and ENaC-EGFP-transfected A6 cell lines. Rates of transport were measured as SCC. Aldosterone (10-6 M) was added at time 0 to the serosal face of the epithelia, indicated by filled symbols. The epithelia indicated by open symbols are matched controls. Amiloride (10-5 M) was added to the apical face of the control and aldosterone-treated parent line (A) 300 min after aldosterone was added. Amiloride (10-5 M) was added to the apical face of the control and aldosterone-treated ENaC-EGFP alpha 5 (B), beta 1 (C), and beta 3 (D) clones 300 min after aldosterone was added. Values are means ± SE at each time point; n = no. of monolayers measured.



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Fig. 6.   Effect of forskolin on normal and ENaC-EGFP-transfected A6 cell lines. Rates of transport were measured as SCC. Forskolin (5 µM) was added at time 0 to the serosal face of the epithelia, indicated by filled symbols. The epithelia indicated by open symbols are matched controls. Amiloride (10-5 M) was added to the apical face of the control and forskolin-treated ENaC-EGFP clones 30 min after forskolin was added. Values are means (for ease of comparison, SE bars have been omitted); n = 10 monolayers measured for the parent line, n = 11 for alpha 5, and n = 6 for beta 3. In each of the cell lines, forskolin caused a statistical increase (P < 0.05) in SCC compared with nontreated controls.



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Fig. 7.   EGFP-ENaC trafficking in the alpha 5 (top) and beta 3 (bottom) clones in response to forskolin. The xz images are orientated cell side on with the apical surface uppermost. Forskolin was added at time 0, and the times of imaging are 5, 15, and 35 min, respectively. No FM 4-64 signal is shown (see MATERIALS AND METHODS), and the green signal comprises all the EGFP in the field of view. Scale bars, 5 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have generated stable epithelial cell lines expressing functional, fluorescently labeled ENaC subunits. Each clonal cell line expresses low levels of mRNAs encoding either xENaCalpha -EGFP or EGFP-xENaCbeta . Correspondingly, novel proteins that cross-react with an anti-GFP antibody were detected in all three clones. These antigens exhibited molecular weights consistent with the ENaC-EGFP chimeric proteins. We have chosen to produce stable transfectants primarily because this allows the selection of clonal derivatives that express the transfected proteins at low levels that are consistent with the endogenous levels of ENaC in native epithelial cells. Comparing the Coomassie-stained protein bands in the molecular weight range of the EGFP-labeled ENaC, it is clear that these labeled proteins represent a minor component of the total cellular protein.

The expression of individual alpha - or beta -ENaC subunits in the clonal lines produce notably higher baseline levels of Na+ transport. The increased basal current in the EGFP-alpha 5 clone is consistent with several recent studies. Bonny et al. (11), using a Xenopus oocyte expression system, reached the novel conclusion that the alpha -subunit, particularly the first 535 residues of the protein, form an assembly/targeting domain. In addition, several in vivo studies (3, 20, 29) showed that, in response to long-term aldosterone treatment, it is the alpha -subunit that is selectively increased in rat renal collecting duct. Thus the moderate overexpression of the alpha -subunit would be expected to increase Na+ transport as we have observed in these studies.

A similar increase in the basal current in the presence of excess EGFP beta -subunit was an unexpected, but consistent, finding as indicated by the analysis of two separate clones. Whether the increased Na+ transport arises from a mechanism similar to that proposed for the alpha -subunit, indicating a redundancy in the targeting/assembly function of the individual subunits, or whether the increased basal current is generated via entirely separate mechanisms in the alpha  and beta  clones is unknown.

It is interesting to speculate on the redundancy in ENaC subunit function. Originally the alpha -subunit was identified as the pore-forming subunit because of its ability to confer amiloride-sensitive transport to oocytes (12, 28). More recent analyses (29), also performed in oocytes, suggest that relatively normal channels can be formed from beta gamma -subunits or alpha L525stopbeta gamma , where the alpha -subunit is truncated before the second transmembrane domain. Thus the oocyte studies indicate that channels can be formed, assembled, and targeted correctly in the absence of one of the subunits, albeit with different time courses.

Various ENaC stoichiometries have been proposed, and several models of the channel pore have been constructed (2, 11, 13, 14, 21, 22, 35); it has been suggested that the diversity in channel function and regulation may be due to variation in subunit composition (2, 24). However, no consensus has been reached as to the exact nature of the channel in native epithelial cells. Our data suggest that an excess of either alpha - or beta -subunit can cause an increase in channel expression and/or activity. Whether this is due to a redundancy in subunit function, a change in subunit assembly and targeting, or an alteration in channel turnover or kinetics is unknown. It is clear that our understanding of the ENaC is evolving and that the nature of the channel subunit interactions and regulation remains largely unknown.

The results from LSCM indicate that under the conditions of our experiments, ENaC-EGFP fluorescence is sufficiently strong to allow dynamic imaging of the subunits. Hence, the system described in this study could be an effective tool for the noninvasive and, importantly, direct visualization of intracellular trafficking of the channel subunits in native epithelia. As demonstrated by the forskolin studies, these data may be generated in parallel with electrophysiological studies. The results also indicate that under conditions of our experiments, there are EGFP-labeled channels located at or near the apical membrane, where they are likely to participate in Na+ transport. This is consistent with the findings of Moyer et al. (30), who demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR), which also is an apically expressed protein in transporting epithelia, was localized predominately in the apical portion of the CFTR-GFP stably transfected Madin Darby canine kidney type I cells.

The relative paucity of ENaC expressed in the apical membrane as indicated by the colocalization of EGFP-ENaC and the apical plasma membrane marker is in complete agreement with the electrophysiological data. The basal currents measured in the clonal cell lines predict an average of >100 functional channels per cell (9). The majority of the expressed EGFP-ENaC is located below the apical plasma membrane. In this regard it is worth noting that studies in native epithelia using a variety of techniques have revealed the presence of quantities of ENaC that far exceed the functional number of channels predicted by the Na+-reabsorptive capacity of the tissue (9, 23). The large excess of channels may represent synthesis, storage, and/or degradative pathways. Thus, unlike the oocyte expression system where virtually all of the channels are inserted into the plasma membrane, the EGFP-ENaC synthesized in the A6 cells appears to be regulated by the physiological control mechanisms of the epithelial cells. An elucidation of these regulatory mechanisms and their modulation by hormonal stimuli represents an important area of investigation for our understanding of ENaC function.

In response to insulin or aldosterone, the clonal lines show increases in transcellular Na+ transport that are similar to the responses in the parent line. This finding is particularly evident in the case of insulin stimulation and is consistent with our previous observation that the increase in insulin-stimulated transport is an arithmetic increase (7-10 µA/cm2) that is independent of the starting current (15). The most likely explanation for these findings is that the channel regulators stimulated in response to hormonal activation form the rate-limiting component of the response.

These data are also consistent with previous findings showing that maximal responses to either insulin or aldosterone can be immediately followed by an additive response to the alternate hormone (32). Thus, in this context, the channels per se do not appear to be the limiting factor to the overall capacity for increased Na+ transport.

During the short time course shown in Fig. 5, aldosterone-stimulated Na+ transport in renal tissue does not depend on the synthesis of new channels subunits but, rather, on the synthesis of "early" aldosterone-induced proteins that are separate from the channel subunits (3, 20). Compared with the control tissue, the aldosterone-induced increase in Na+ transport is similar in the parent and clonal A6 cell lines. In the parent line, the control tissue is maintained at a steady, low basal level, while aldosterone causes an increase in Na+ transport. In the clonal lines, however, the high basal current is not maintained in the long-term voltage-clamped tissues incubated in serum-free medium, and the differences between control and aldosterone-treated cells appear to be due to an absence of falloff in the basal current. In the clonal lines, the falloff in basal current could be due to a number of factors, including turnover of a serum-specific factor that is required to maintain higher basal current or feedback inhibition of ENaC, which would be more pronounced at the higher transport levels (1, 16, 25).

In contrast to insulin- and aldosterone-stimulated transport, forskolin-stimulated transport is enhanced by the presence of additional subunits. Interestingly, the increased forskolin-stimulated transport in the clonal lines is not long lasting, and the current returns to the level of the stimulated parent cell line in 15-20 min. The question of whether cAMP-mediated regulation of ENaC involves channel activation or channel recruitment is controversial (34). With the use of LSCM it was possible to demonstrate that the labeled channels were trafficked to the apical membrane in response to forskolin. Because the electrophysiological data indicate that the labeled subunits are participating in ENaC-mediated Na+ transport, these data provide an independent evaluation of the mechanism of cAMP-induced increases in Na+ transport. However, the falloff in transport in the clonal lines during the 30-min time course is not accompanied by an internalization of labeled subunits. Thus the mechanism of regulation may be complex and may involve both channel insertion as well as changes in channel kinetics.

The regulation of ENaC will, undoubtedly, be under the control of regulatory elements that are specific to the epithelial cells that contain ENaC. The ENaC-EGFP subunit A6 clones will, therefore, provide valuable tools for the characterization of regulatory elements as well as the exact nature of responses to hormonal stimulation.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Faculty Development Office of Indiana University-Purdue University Indianapolis (B. L. Blazer-Yost), National Science Foundation (S. J. Rhodes), United States Department of Agriculture (S. J. Rhodes), National Research Foundation of South Africa (W. J. Els), and Kidney Foundation of Southern Africa (W. J. Els).


    FOOTNOTES

Address for reprint requests and other correspondence: B. L. Blazer-Yost, Dept. of Biology, SL358, 723 West Michigan St., Indiana Univ.-Purdue Univ. Indianapolis, Indianapolis, IN 46202 (E-mail: bblazer{at}iupui.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 13 April 2000; accepted in final form 15 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abriel, H, and Horisberger J-D. Feedback inhibition of rat amiloride-sensitive epithelial sodium channels expressed in Xenopus laevis oocytes. J Physiol (Lond) 516: 31-43, 1999[Abstract/Free Full Text].

2.   Adams, CM, Snyder PM, and Welsh MJ. Interactions between subunits of the human epithelial sodium channel. J Biol Chem 272: 27295-27300, 1997[Abstract/Free Full Text].

3.   Asher, C, Wald H, Rossier B, and Garty H. Aldosterone-induced increase in the abundance of Na+ channel subunits. Am J Physiol Cell Physiol 271: C605-C611, 1996[Abstract/Free Full Text].

4.   Ausubel, FM, Brent R, Kingston RE, Moore DD, Seldman JD, Smith JA, and Struhl K. Current Protocols in Molecular Biology. New York: Greene and Wiley, 1994.

5.   Barbry, P, and Lazdunski M. Structure and regulation of the amiloride-sensitive epithelial sodium channel. In: Ion Channels, edited by Narahashi T.. New York: Plenum, 1996, vol. 4, p. 115-167.

6.   Betz, WJ, Mao F, and Smith CB. Imaging exocytosis and endocytosis. Curr Opin Neurobiol 6: 365-371, 1996[ISI][Medline].

7.   Blazer-Yost, BL, and Cox M. Insulin-like growth factors 1 stimulates renal epithelial Na+ transport. Am J Physiol Cell Physiol 255: C413-C417, 1988[Abstract/Free Full Text].

8.   Blazer-Yost, BL, Fesseha Y, and Cox M. Aldosterone-mediated Na+ transport in renal epithelia: time-course of induction of a potential regulatory component of the conductive Na+ channel. Biochem Int 26: 887-897, 1992[ISI][Medline].

9.   Blazer-Yost, BL, and Helman SI. The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities. Am J Physiol Cell Physiol 272: C761-C769, 1997[Abstract/Free Full Text].

10.   Blazer-Yost, BL, Liu X, and Helman SI. Hormonal regulation of ENaCs: insulin and aldosterone. Am J Physiol Cell Physiol 274: C1373-C1379, 1998[Abstract/Free Full Text].

11.   Bonny, O, Chraibi A, Loffing J, Jaeger NF, Grunder S, Horisberger J-D, and Rossier BC. Functional expression of a pseudohypoaldosteronism type I mutated Na+ channel lacking the pore-forming region of its alpha  subunit. J Clin Invest 104: 967-974, 1999[Abstract/Free Full Text].

12.   Canessa, CM, Horisberger J-D, and Rossier BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361: 467-470, 1993[ISI][Medline].

13.   Canessa, CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger J-D, and Rossier BC. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463-466, 1994[ISI][Medline].

14.   Coscoy, S, Lingueglia E, Lazdunski M, and Barbry P. The Phe-Met-Arg-Phe-amide-activated sodium channel is a tetramer. J Biol Chem 273: 8317-8322, 1998[Abstract/Free Full Text].

15.   Coupaye-Gerard, B, Kim HJ, Singh A, and Blazer-Yost BL. Differential effects of Brefeldin A on hormonally regulated Na+ transport in a model renal epithelial cell line. Biochim Biophys Acta 1190: 449-456, 1994[ISI][Medline].

16.   Eaton, DC, Becchetti A, Heping M, and Ling BN. Renal sodium channels: regulation and single channel properties. Kidney Int 48: 941-949, 1995[ISI][Medline].

17.   Els, WJ, and Helman SI. Vasopressin, theophylline, PGE2, and indomethacin on active Na transport in frog skin: studies with microelectrodes. Am J Physiol Renal Fluid Electrolyte Physiol 241: F279-F288, 1981[ISI][Medline].

18.   Els, WJ, and Helman SI. Regulation of epithelial sodium channel densities by vasopressin signaling. Cell Signal 1: 533-539, 1989[ISI][Medline].

19.   Els, WJ, and Helman SI. Dual role of prostaglandins (PGE2) in regulation of channel density and open probability of epithelial Na+ channels in frog skin (R. pipiens). J Membr Biol 155: 75-87, 1997[ISI][Medline].

20.   Escoubet, B, Coureau C, Bonvalet J-P, and Farman N. Noncoordinate regulation of epithelial Na channel and Na pump subunit mRNAs in kidney and colon by aldosterone. Am J Physiol Cell Physiol 272: C1482-C1491, 1997[Abstract/Free Full Text].

21.   Eskandari, S, Snyder PM, Kreman M, Zampighi GA, Welsh MJ, and Wright EM. Number of subunits comprising the epithelial sodium channel. J Biol Chem 274: 27281-27286, 1999[Abstract/Free Full Text].

22.   Firsov, D, Gautschi I, Merillat A-M, Rossier BC, and Schild L. The heterotetrameric architecture of the epithelial sodium channel (ENaC). EMBO J 17: 344-352, 1998[Abstract/Free Full Text].

23.   Firsov, D, Schild L, Gautschi I, Merillat A-M, Schneeberger E, and Rossier BC. Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach. Proc Natl Acad Sci USA 93: 15370-15375, 1996[Abstract/Free Full Text].

24.   Fyfe, GK, and Canessa CM. Subunit composition determines the single channel kinetics of the epithelial sodium channel. J Gen Physiol 112: 423-432, 1998[Abstract/Free Full Text].

25.   Garty, H, and Palmer L. Epithelial sodium channels - function, structure and regulation. Physiol Rev 77: 359-396, 1997[Abstract/Free Full Text].

26.   Helman, SI, Liu X, Baldwin K, Blazer-Yost BL, and Els WJ. Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia. Am J Physiol Cell Physiol 274: C947-C957, 1998[Abstract/Free Full Text].

27.   Koefoed-Johnsen, V, and Ussing HH. The nature of the frog skin potential. Acta Physiol Scand 42: 298-308, 1958[ISI].

28.   Lingueglia, E, Voilley N, Waldmann R, Lazdunski M, and Barbry P. Expression cloning of an epithelial amiloride-sensitive Na+ channel: a new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett 318: 95-99, 1993[ISI][Medline].

29.   Masilamani, S, Kim G-H, Mitchell C, Wade JB, and Knepper MA. Aldosterone-mediated regulation of ENaC alpha , beta , and gamma  subunit proteins in rat kidney. J Clin Invest 104: R19-R23, 1999[Abstract/Free Full Text].

30.   Moyer, BD, Loffing J, Schwiebert EM, Loffing-Cueni D, Halpin PA, Karlson KH, Ismailov II, Guggino WB, Langford GM, and Stanton BA. Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in Madin-Darby canine kidney cells. J Biol Chem 273: 21759-21768, 1998[Abstract/Free Full Text].

31.   Puoti, A, May A, Canessa CM, Horisberger-D J, Schild L, and Rossier BC. The highly selective low-conductance epithelial Na channel of Xenopus laevis A6 kidney cells. Am J Physiol Cell Physiol 269: C188-C197, 1995[Abstract/Free Full Text].

32.   Record, RD, Johnson M, Lee SY, and Blazer-Yost BL. Aldosterone and insulin stimulate amiloride-sensitive sodium transport in A6 cells by additive mechanisms. Am J Physiol Cell Physiol 271: C1079-C1084, 1996[Abstract/Free Full Text].

33.   Shimkets, RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, Schambelan M, Gill JR, Ulick S, Milora RV, Findling JW, Canessa CM, Rossier BC, and Lifton RP. Liddle's syndrome: heritable human hypertension caused by mutations in the beta  subunit of the epithelial sodium channel. Cell 79: 407-414, 1994[ISI][Medline].

34.   Smith, PR. cAMP-mediated regulation of amiloride-sensitive sodium channels: channel activation or channel recruitment? In: Current Topics in Membranes. Amiloride-Sensitive Sodium Channels. Physiology and Functional Diversity, edited by Benos DJ.. New York: Academic, 1999, vol. 47, p. 133-154.

35.   Synder, PM, Cheng C, Prince LS, Rogers JC, and Welsh MJ. Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits. J Biol Chem 273: 681-684, 1998[Abstract/Free Full Text].


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