Address correspondence to James A. Schafer, Department of Physiology and Biophysics, 1918 University Boulevard, Room 958 MCLM, Birmingham, AL 35294-0005. Fax: (205) 934-5787; E-mail: jschafer{at}uab.edu
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ABSTRACT |
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Key Words: retroviral transfection FLAG epitope channel number membrane trafficking short-circuit current
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INTRODUCTION |
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The Na+ channels involved in this response to ADH and cAMP are now known to be oligomeric complexes of three protein subunits, -, ß-, and
ENaC, encoded by discrete genes (Canessa et al., 1994
). All three subunits share a similar topology, consisting of two transmembrane regions, a large cysteine-rich extracellular loop, and short COOH- and NH2-terminal regions in the cytoplasm. When the three cloned subunits are expressed together in Xenopus oocytes, they form channels with characteristics comparable to those found in the apical membrane of intact epithelia such as the CCD, including: a single channel conductance of
5 pS, a high Na+ to K+ selectivity ratio, and inhibition by submicromolar concentrations of amiloride (Canessa et al., 1994
).
Interestingly, not all epithelia expressing ENaC respond to ADH or cAMP with increased Na+ transport. For example, ADH-dependent cAMP production in the rabbit CCD produces either no change or a decrease in Na+ transport (Schafer and Hawk, 1992; Schafer, 1994
, 2002
). Even in the same species, cAMP or ADH may have a different effect in different tissues, for example, stimulating Na+ transport in the rat CCD (Tomita et al., 1985
; Reif et al., 1986
), but not in the rat colon (Bridges et al., 1984
). This variability in the response to cAMP or ADH has been attributed to differences in the structure of the
ENaC subunit (Schnizler et al., 2001
), or to differences in the actions of autacoids such as prostaglandin E2 that modify the response to ADH (Schafer, 2002
).
The increase in Na+ reabsorption that occurs with ADH or cAMP in the rat CCD and other responsive epithelia represents an increase in the activity of ENaC hetero-oligomers in the apical membrane. Two general mechanisms have been put forward to explain this increased channel activity. First, modification of ENaC subunits, occurring as one of the ultimate events of ADH action, might cause an increase in the open probability (Po) or an increase in the unit conductance of the multimeric channel complexes already present in the apical membrane with no change in their numbers. Alternatively, or in addition to its effects on individual channel kinetics, ADH might cause the insertion of assembled ENaC multimers into the apical membrane in a manner analogous to the ADH-stimulated insertion of aquaporin-2 water channels in the CCD (Wade, 1985).
The former mechanism of ADH action, i.e., an increase in Po of individual Na+ channels, has been supported by studies using patch clamping and reconstituted Na+ channels. Changes in the single channel properties might be a consequence of protein kinase A (PKA) activation by cAMP, and Shimkets et al. (1998) have shown that the COOH-terminal regions of ß- and
ENaC are phosphorylated by this signaling cascade. Using inside-out patches of A6 cell apical membranes, Prat et al. (1993)
showed that application of PKA plus ATP activated Na+ channels at least in part by an increase in Po. They also showed, however, that this effect of PKA was dependent on the presence of "short" actin filaments and hypothesized that the action of PKA might be mediated by phosphorylation of G-actin rather than the channel itself. A role for actin in activating ENaC was also supported by Berdiev et al. (1996)
, who demonstrated that PKA activation of
-, ß-, and
ENaC subunits incorporated into planar lipid bilayers required the presence of short actin filaments. PKA has also been shown to increase the Po of several biochemically purified Na+ channel complexes incorporated into planar bilayers (Oh et al., 1993
; Bradford et al., 1995
; Ismailov and Benos, 1995
; Senyk et al., 1995
).
Although variable in magnitude, the increases in Po that occur in these patch-clamp and bilayer experiments have been taken to support the view that direct activation of channels by cAMP/PKA can contribute to the increased amiloride-sensitive Na+ transport produced by ADH. This conclusion must, however, be tempered by the limitations of the experimental systems used. As indicated by the studies in lipid bilayers and detached membrane patches (Prat et al., 1993; Berdiev et al., 1996
), the activity of ENaC channels depends on the presence of components of the cytoskeleton. In a detached patch or in a reconstituted system in which normal interactions of ENaC subunits with the cytoskeleton are disrupted, activation by PKA may involve changes in the interaction between residual or exogenous actin and ENaC that do not occur in the intact cell. It is also impossible to quantify the density of channels in the membrane of the native cells from the numbers measured in detached patches. When multiple channels are present in a patch, not only is it technically very difficult to determine their precise number, but the channels may also not be uniformly distributed, resulting in the "hot spots" of channel activity as described by Marunaka and Eaton (1991)
in vasotocin-treated A6 cells.
Both patch-clamp and biochemical approaches have been used in support of the alternative hypothesis that ADH increases Na+ transport by the insertion of additional channels into the apical membrane. Marunaka and Eaton (1991) used patch clamping in A6 cell monolayers to show that both vasotocin and cAMP increased the number of Na+ channels per patch with no change in single channel kinetics. Kleyman et al. (1994)
addressed the effect of ADH on the total pool of Na+ channels in A6 apical membranes using an antiidiotypic antibody (antianti-amiloride) that has been shown to recognize
ENaC (Kieber-Emmons et al., 1995
). In these experiments, apical membrane proteins in intact A6 cells, with and without ADH treatment, were radio iodinated and the Na+ channels were subsequently immunoprecipitated from extracted proteins using the antiidiotypic antibody. Kleyman et al. (1994)
reported there was approximately a doubling of the immunoprecipitated Na+ channels after treatment with the hormone, which they concluded would account for the increase in Na+ transport produced by ADH. In another approach to quantifying Na+ channel density in the apical membrane, Snyder (2000)
used transient transfection to introduce human ENaC subunits, which had been modified to allow fluorescence labeling, into cultured epithelial cells of thyroid origin. He demonstrated that cAMP stimulation produced an acute increase in the fluorescent labeling of ENaC in the apical membrane that paralleled the increase in Na+ transport.
The measurement of the surface density of ENaC subunits in the intact epithelium has definite advantages. Not only are the subunits in their native environment, but the use of epithelial monolayers with >106 cells inherently provides the statistical averaging that is lost when examining individual patches or reconstituted transporters. However, although the two surface-labeling studies discussed above undeniably show an increase in the density of ENaC subunits in the apical membrane of A6 cells with ADH or cAMP, both are limited in their ability to relate the change in the surface density of the label quantitatively to the change in Na+ transport. In the experiments of Kleyman et al. (1994), the identity and the number of antigenic sites per ENaC subunit, as well as the affinity of the antiidiotypic antibody for these sites, are unknown. Similarly, in Snyder (2000)
fluorescence could not be related quantitatively to channel density, and the cells examined by confocal microscopy were not representative of the average labeling of the whole epithelium, because not all cells expressed ENaC subunits in this transient transfection system.
These limitations were overcome in the surface-labeling experiments described by Firsov et al. (1996). These investigators modified rat
-, ß-, and
ENaC by introducing the octapeptide FLAG epitope (DYKDDDDY) into the early (NH2-terminal) region of the extracellular loop of each. When these subunits were expressed in Xenopus oocytes, the binding of 125I-labeled monoclonal antibody to FLAG allowed these investigators to determine the surface density of the ENaC subunits on a femtomole per oocyte basis. The macroscopic amiloride-sensitive Na+ current and the surface density of ENaC subunits measured in the same oocyte were linearly related over a wide range with a slope of 1.1 µA/fmol. Although Firsov et al. (1996)
did not examine the effect of cAMP,1 they found that a mutation in the ß subunit associated with Liddle's syndrome resulted in an increase in the surface density of ENaC subunits and an increase in amiloride-sensitive current.
Our objective in the present studies was to use FLAG antibody surface labeling to quantify any change in the surface density of ENaC in the apical membrane of mammalian epithelial cells with cAMP treatment and to relate it to transepithelial Na+ transport measured in the same epithelia. We used retroviral transfection to develop a line of MDCK cells expressing "flagged" rat ENaC subunits, i.e., subunits labeled in the extracellular loop-domain with FLAG as described by Firsov et al. (1996). We measured the density of total ENaC subunits on the apical membrane surface and compared it with the amiloride-sensitive short-circuit current (Isc) measured in the same cells in the presence and absence of cAMP stimulation. Our results indicate that the increase in Isc produced by cAMP can be accounted for entirely by a proportional increase in the surface density of ENaC.
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MATERIALS AND METHODS |
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Cultures of MDCK cells were expanded in T-75 flasks in a humidified incubator at 37°C in the presence of 4% CO2. The medium was Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) supplemented with 50 mM HEPES (pH 7.4), 1% Pen/Strep-fungizone, and 10% FBS. The medium was exchanged as needed, usually daily, and the cells were split upon confluence. For the both electrophysiological and surface labeling experiments, the cells were seeded on inserts with permeable membranes: either 24-mm cyclopores (Catalog No. 35-3090; Falcon) or 24-mm transwells (Catalog No. 3412; Costar) at a density of 400,000 cells/insert, after which they were grown in the modified DMEM without selection antibiotics (see below).
Other Tissues
In some of the immunoblotting and RT-PCR studies described below, we also used protein and total RNA extracted from fresh samples of dog and rat renal cortex and inner medulla. These tissues were obtained from cadarveric animals that had been used in other studies in our own laboratory or in other laboratories at this university. All such studies were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham.
Retroviral Transfection of Parental MDCK Cells
Three plasmids containing rat -, ß-, and
ENaC subunits, into each of which the FLAG epitope had been introduced in the early portion of the extracellular loop as described by Firsov et al. (1996)
, were provided by Dr. B. Rossier (University of Lausanne, Lausanne, Switzerland). These flagged subunits are designated hereafter by a subscript F, e.g.,
FENaC. These subunit cDNAs were provided in mammalian plasmids (a modification of pSD65), and new restriction sites appropriate for subcloning into the retroviral vectors (5' XhoI and 3' NotI) were added to each by PCR. The resulting cDNA constructs were bidirectionally sequenced in order to verify that the ENaC subunits had not been modified, and they were then subcloned into bicistronic retroviral vectors, which were generously provided by Dr. R. Boucher (University of North Carolina, Chapel Hill, NC). The three vectors, LXPIN, LXPIH, and LXPIP, contained antibiotic resistance genes for, respectively, neomycin (G418), hygromycin, and puromycin, in one cloning position, with the
F-, ßF-, or
FENaC subunit in the other. Sequencing from the 5' and 3' termini verified the orientation and nucleotide fidelity of the subcloned ENaC subunits. Using the protocol of Comstock et al. (1997)
, PA317 packaging cells (American Type Culture Collection) were transfected with the retroviral vectors to produce three retrovirions: L
F PIN, LßF PIH, and L
F PIP, each containing the indicated flagged ENaC subunit and the respective antibiotic resistance gene.
Parental MDCK cells were first infected with one of the three retrovirions, and then selected with the corresponding antibiotic, followed by transfection and selection cycles with the other two virions. Selection media included (in combinations appropriate to the history of infection): G418 (800 µg/ml), hygromycin (300 µg/ml), and puromycin (5 µg/ml) DMEM. At the end of three rounds of infection and selection, colonies of cells that had been infected with all three subunits and exhibited the corresponding antibiotic resistances were isolated and expanded for screening while maintaining selection pressure with all three antibiotics.
Clones of the triply transfected MDCK cells (referred to as FßF
F MDCK cells) were subsequently grown on permeable supports. After 45 d in culture, expression of the transfected ENaC subunits was increased by overnight "induction" with 1 µM dexamethasone plus 2 mM Na+ butyrate in the culture medium as described by Stutts et al. (1995)
. Functional expression of the transfected ENaC subunits was monitored by the amiloride-sensitive transepithelial voltage (VT) using an EVOM (World Precision Instruments). The clone with the most robust transepithelial amiloridesensitive voltage was selected. RT-PCR was used to verify the presence of flagged (but not wild-type) ENaC subunits in the transfected cells, and to confirm the absence of wild-type ENaC subunit cDNA in the parental cell line. The suitability of the primers used for this purpose was confirmed by using RNA extracted from fresh dog kidney as a positive control. Expression of all three subunits in the
FßF
F MDCK cells was also confirmed by immunoblotting as described below.
Electrophysiological Measurements
Short-circuit currents (Isc) were measured by mounting 12-mm diameter cell culture inserts (Millicell PCF; Millipore) in Ussing-type chambers (Jims Instruments) enclosed in a circulating water-jacket maintained at 37°C and equipped with bubble-lifts driven by the 95% O2/5% CO2 gassing. The half-chambers were connected via agar bridges to potential-sensing and current-passing Ag-AgCl electrodes. All electrodes were connected to a voltage clamp (model VCC600; Physiological Instruments). Isc was continuously recorded on a strip-chart recorder.
In addition to the traditional Ussing-type chambers, Isc was also measured in the 24-mm diameter cyclopore and transwell culture inserts that were used to conduct binding experiments. For these experiments, we built a special "multiinsert apparatus" that allowed rapid sequential recording of Isc and RT, as well as the open-circuit VT. The inserts were held in a manifold constructed from a Lucite sheet with 24 equally spaced openings. The lower portions of the inserts protruded below the manifold into the basolateral medium, which was contained in an inexpensive plastic container and was gassed with a mixture of 95% O2/5% CO2. This container was placed in a larger plastic container that served as a water bath whose temperature was maintained at 37°C by an immersion heater and temperature regulator. The basolateral currentreceiving electrode was a stainless-steel sheet, whereas the basolateral voltagesensing electrode, an Ag-AgCl2 electrode, was connected to the basolateral medium by an agar bridge. These basolateral electrodes served as a common electrode pair for all 24 inserts mounted in the Lucite manifold. The apical electrodes consisted of a small stainless-steel disc for current passing and a central agar bridge connected to an Ag-AgCl2 electrode. The apical electrode pair was encased in a housing made from a rubber stopper, which rested on the top rim of the cell culture inserts with the current-passing electrode and the agar bridge extending into the apical solution. Using this housing, the apical electrode pair could be moved rapidly from insert to insert for sequential electrophysiological measurements with the VCC600 voltage-clamp. A MacLab model 8e (ADInstruments) interface and a Macintosh computer were used to control the clamp and perform a rapid sequence of short-circuit, open-circuit, and current-passing recordings, and the data were stored on the computer.
Two solutions were used in the Ussing chambers, as well as in the multiinsert apparatus: unmodified DMEM or a "chloride-free" solution containing (in mM): 140 Na+ aminobenzenesulfonate, 24 Na+ bicarbonate 6 Ca2+ gluconate, 3 Mg2+ gluconate, 5 K+ gluconate, and 10 D-glucose.
Surface Labeling of Flagged ENaC Subunits
Anti-FLAG antibody (Sigma-Aldrich; referred to below as M2) was radiolabeled with 125I in the UAB radiolabeling core facility at the Comprehensive Cancer Center using Iodo-Beads (Pierce Chemical Co.) per the manufacturer's instructions. Labeled M2 antibody was initially purified over an anion-exchange column, eluted, and subsequently placed in a 10,000 MWC Slide-A-Lyzer dialysis cassette (Pierce Chemical Co.) and dialyzed for at least 24 h against PBS. The amount of labeled antibody was determined by a microprotein assay (# 23235; Pierce Chemical Co.) and was diluted to the desired specific activity by the addition of unlabeled M2 antibody. The specific activity was typically 1.8 µCi/µg antibody, but it was modified as needed to give sample count rates that ranged from at least 20 times background to >10,000 cpm, depending on the antibody concentration used.
Equilibrium binding assays were performed on monolayers of FßF
F MDCK cells grown on either cyclopore or transwell 24-mm cell culture inserts. After experimental measurements and treatments, the inserts were placed in an ice bath and briefly rinsed with ice-cold PBS, and then incubated for 30 min with ice-cold blocking solution (PBS + 5% FBS). The apical blocking solution was aspirated and replaced with 500 µl of ice-cold blocking solution containing the desired concentration of 125I-labeled M2 antibody and incubated for 1 h. To determine the nonspecific binding, M2 antibody was added to paired inserts together with a 100-fold excess (by weight) of FLAG peptide (Research Genetics). After a 1-h incubation, excess label was aspirated and monolayers were washed four times with 1 ml of ice-cold blocking solution. Label remaining bound to the apical surface of the monolayers was then removed by "acid-stripping" as per Wiley and Cunningham (1982)
. Briefly, 750 µl of ice-cold acid wash solution (0.5 M NaCl, 0.2 M Na+ acetate, pH 2.4) was aliquoted onto the apical membrane of monolayers, incubated on ice for 5 min, and then harvested for gamma counting. Two acid strips were performed per monolayer to confirm that
80% of the bound label was removed. The counts from both acid strips were combined for data analysis. For each experimental point, triplicate inserts were labeled with 125I-labeled M2 antibody in the absence of competing peptide and a paired triplicate in its presence. Specifically, bound counts were taken to be the difference in average counts obtained in the presence and absence of peptide.
Western Blotting and Immunoprecipitation of ENaC Subunits
For each Western blot, protein was harvested from two confluent 24-mm monolayers of FßF
F or parental MDCK cells. Monolayers were washed extensively with ice-cold PBS, removed from the filter inserts with cell scrapers, and briefly centrifuged. After aspirating the PBS, 600 µl of ice-cold lysis buffer (10 mM triethanolamine, 250 mM sucrose, pH 7.6, plus PMSF [1 mg/10 ml] and leupeptin [1 µg/ml]) was added to the pellet, and the cells were homogenized with a glass mortar and pestle. SDS was then added (1% final concentration) to the homogenate, which was mixed with a 21-ga needle and syringe. The sample was transferred to a 1.5-ml tube and sheered with a 30-ga needle 34 times before centrifuging 15 min at 14,000 g. Approximately 90% of the supernatant was then recovered and concentrated using a Microcon 30 (Millipore) as per the manufacturer's instructions. Protein determinations were made with a MicroBCA Kit (Pierce Chemical Co.). Laemmli buffer containing mercaptoethanol was added to 10-µg samples of protein, which were briefly boiled and run on 10% polyacrylamide gels. Protein was electrophoretically transferred to a nitrocellulose membrane (Nitrobind; Osmonics) and processed for immunoblotting.
Antibodies specific to three rat ENaC subunits (Masilamani et al., 1999) were provided by Dr. M. Knepper (National Institutes of Health, Bethesda, MD). The membrane blots were blocked with Blotto for 1 h at room temperature and incubated overnight at 4°C with primary ENaC antibodies at the appropriate dilutions (anti-
ENaC L766 1:2,500, anti-ßENaC L558 1:1,500, anti-
ENaC L550 1:2,000). They were then washed three times with T-TBS (TBS/0.05% Tween-20), incubated for 1 h at room temperature with secondary antibody diluted 1:5000 in Blotto, and washed again with T-TBS. Blots were visualized with the ECL detection system (Amersham Pharmacia Biotech) per the manufacturer's instructions. Autoradiograms of immunoblots were digitized on an Epson transparency scanner (Expression Model 1600). Densitometric comparisons of bands on the same blot were made using the NIH Image program and calibrated gel analysis subroutine (version 1.62; National Institutes of Health).
To verify that the expressed ENaC subunits were indeed flagged, F, ßF, and
F subunits were immunoprecipitated from protein harvested from two confluent 24-mm diameter monolayers of induced
FßF
F MDCK cells using M2 antibody (5 µl) and a protein-G immunoprecipitation kit (1719386; Roche) according to the manufacturer's instructions. After immunoprecipitation, samples were boiled briefly in Laemmli buffer and processed for Western blotting as described above. In other experiments, the subunit-specific antibodies were used to immunoprecipitate the proteins, and the immunoblots were probed with the M2 antibody.
Statistics and Nonlinear Regression of Binding Saturation Plots
StatView for Macintosh (version 4.5.1, SAS Institute Inc.) was used for standard statistical calculations. Numerical results are presented as averages ±SEM. ANOVA with Bonferroni-Dunn post-hoc testing was used for comparisons of paired and nonpaired averages as appropriate, with significance ascribed at P < 0.05.
Kaleidagraph for Macintosh (version 3.5.1; Synergy Software) was used for nonlinear least squares fitting of specific binding data to the Michaelis-Menten relationship. Quality of fit is indicated by the correlation coefficient (r) and associated P value.
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RESULTS |
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To verify that the ENaC subunits expressed in transfected MDCK cells contained the FLAG epitope, two series of immunoprecipitation experiments were performed. First, proteins were immunoprecipitated from FßF
F MDCK cell lysates with the M2 antibody. The immunoprecipitated proteins were visualized on subsequent Western blots using the same subunit-specific antibodies as above. Bands of the appropriate molecular weights (Fig. 3 A) confirmed the presence of the FLAG in each of the ENaC subunits, but with the presence of a doublet for the ß subunit. As further confirmation, a second series of immunoprecipitation experiments was performed in which each ENaC subunit was initially immunoprecipitated from
FßF
F MDCK cell lysates using the subunit-specific anti-ENaC antibodies. Immunoprecipitated proteins were processed for Western blotting and visualized with the M2 antibody shown in Fig. 3 B. Using this approach, the doublet for ßF subunit was absent, although, as noted above, the presence of this band was variable. Together, the results (Figs. 2, A and B, and 3, A and B) demonstrate that all three flagged ENaC subunits are expressed in the
FßF
F MDCK cells.
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Fig. 4 presents the results of seven experiments with FßF
F MDCK cells in DMEM in which specific binding was measured as a function of the 125I-labeled M2 antibody concentration. The specific binding showed saturation with increasing antibody concentration as would be expected for a typical receptor ligand interaction; in this case, the binding of 125I-labeled M2 antibody to the FLAG present in the ectodomain of the ENaC subunits in the apical membrane. Because less than 1% of the M2 antibody added to the apical medium was bound to the membrane surface at equilibrium, we fit the data in Fig. 4 according to the Michaelis-Menten equation. Nonlinear least squares fitting gave estimates of k0.5 and Bmax of 7.9 ± 1.9 nM and 7.4 ± 0.7 fmol/cm2, respectively.2 The k0.5 thus obtained is not significantly different from the k0.5 of
3 nM reported by Firsov et al. (1996)
using the same FLAG-M2 antibody pairing in the Xenopus oocyte expression system.
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Finally, we conducted four experiments in which we measured the specific binding of 125I-labeled M2 antibody applied to the basolateral surface of the FßF
F MDCK monolayers in the presence and absence of FLAG peptide. There was no significant specific binding, however, it would have been difficult to discern specific binding at the lower levels observed on the apical surface (i.e., <2 fmol/cm2) because of the high levels of nonspecific binding when the antibody was added to basolateral side. We attributed these higher nonspecific counts to antibody binding to the membrane support because we found similar levels of nonspecific binding to membrane inserts in the absence of cells. The absence of significant ENaC activity in the apical membrane was also supported by the absence of any change in VT or Isc when amiloride was added to the basolateral membrane (see below).
Electrophysiological Characteristics
Parental MDCK cells exhibited very little evidence of amiloride-sensitive Na+ transport. In three experiments with parental MDCK cells that were not induced, the average baseline open-circuit VT and Isc in DMEM were, respectively, -1.2 ± 0.3 mV and 0.8 ± 0.1 µA/cm2. In five experiments with parental cells that had been induced overnight with butyrate and dexamethasone, baseline VT and Isc were, respectively, -7.1 ± 2.1 mV and 3.7 ± 0.3 µA/cm2. After the latter cells were treated with 20 µM 8-CPT-cAMP plus 200 µM IBMX, Isc decreased by only 1.8 ± 0.3 µA/cm2 upon addition of 20 µM amiloride to the apical solution. In many other groups of parental cells, treated with both induction and cAMP, there was no change whatsoever in Isc upon the addition of amiloride to the apical solution. Thus, while there may be a small amiloride-sensitive Isc in some groups of parental cells after overnight induction, it was small in comparison with the transfected cells.
Table I documents the effects of 20 µM amiloride on the open-circuit VT and RT that develop across confluent monolayers of FßF
F MDCK cells after overnight induction with both dexamethasone and butyrate. Addition of 20 µM amiloride to the apical medium significantly reduced VT from -27.7 ± 1.2 mV to -6.4 ± 0.3 mV (n = 95, P < 0.001), and significantly elevated RT from 2.2 ± 0.1 to 2.8 ± 0.1 k
·cm2 (n = 90, P < 0.001). We also examined the dose response of the transfected cells to amiloride. Based on the percent inhibition of Isc, the ki for amiloride in the absence and presence of cAMP treatment (20 µM 8-CPT-cAMP plus 200 µM IBMX) was, respectively, 0.48 ± 0.14 and 0.48 ± 0.18 µM. Addition of 10100 µM amiloride to the basolateral membrane had no significant effect on either the basal VT, or on Isc in the presence or absence of cAMP treatment. These observations demonstrate the presence of functional amiloride-sensitive Na+ channels in the apical membranes of the
FßF
F MDCK cells.3
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Correlation between ENaC Surface Labeling and Isc
We used two different approaches to produce cell cultures that exhibited a wide range of ENaC expression and, thus, of surface binding. In both sets of experiments, the chloride-free medium was used. First, we prepared mixtures of varying proportions of parental (wild-type) to FßF
F MDCK cells at the time of seeding onto cell culture inserts while keeping the total number of cells constant. For each experiment, a tray of six cell culture inserts was seeded for each mixture of cells. This arrangement typically resulted in a total of four trays, or 24 inserts for each experiment. After reaching confluence, the cells were induced with butyrate and dexamethasone and the following day the cell culture inserts were placed in the multiinsert apparatus for rapid, consecutive measurement of Isc in each of the 24 inserts. The amiloride-sensitive short-circuit current (AS-Isc) was calculated as the mean paired difference between Isc measured just before and that measured within 3 min after adding 20 µM amiloride to the apical solution. There was considerable variability in AS-Isc measured for a given cell mixture, which probably reflects a variable rate of growth of parental compared with
FßF
F MDCK cells among the different preparations of cells used to plate the inserts. Nevertheless, this approach provided inserts with the wide range of AS-Isc that was desired.
In the second protocol, we varied the expression of ENaC among the four trays used for each experiment by varying the induction method. In each experiment, we seeded FßF
F MDCK cells onto cell culture inserts resulting in four trays, or 24 inserts, per experiment. The evening before the experiment, one tray was not induced, the second tray was induced with 2 mM butyrate only, the third with 1 µM dexamethasone only, and the fourth tray of cells was induced with the standard combination of dexamethasone and butyrate. As in the previous protocol, Isc was measured in the presence and absence of amiloride using the multi-insert apparatus. In nine experiments conducted in chloride-free medium, the average AS-Isc values in uninduced MDCK cells, and in cells induced with butyrate alone, dexamethasone alone, or both agents were, respectively, 2.7 ± 0.7, 8.1 ± 1.3, 11.8 ± 0.7, and 16.7 ± 1.6 µA/cm2 (each mean significantly different from the other three by ANOVA).
In each of the two sets of experiments described above, the specific binding 125I-labeled M2 antibody was measured at an antibody concentration of 16 nM. This intermediate concentration was chosen because the ratio of specific to nonspecific binding (i.e., the signal-to-noise ratio) decreased at higher antibody concentrations as specific binding sites became saturated. Specific binding was measured in the same inserts as Isc, and it was plotted against the AS-Isc as shown in Fig. 6. Linear regression demonstrated a significant correlation between specific binding and AS-Isc (n = 45, r = 0.82, P < 0.001). The slope of this relationship was 0.12 ± 0.03 fmol/µA, and the intercept (0.62 ± 0.33 fmol/cm2) was not significantly different from zero.
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To correlate the cAMP-stimulated increase in the surface density of ENaC subunits with the increase in amiloride-sensitive Isc, additional experiments were conducted in which AS-Isc measurements in the absence and presence of cAMP stimulation were performed contemporaneously with binding studies using inserts seeded at the same time and grown under the same conditions. In this series of experiments, a single concentration of M2 antibody (1.7 nM) was used for the binding assay because of the greater sensitivity (larger ratio of specific to nonspecific counts) at antibody concentrations at or below k0.5. In the seven experiments summarized in Table II, the amiloride-sensitive Isc increased from 11.2 ± 1.3 to 18.1 ± 1.3 µA/cm2 after cAMP treatment, and the specific binding of M2 antibody increased from 0.623 ± 0.126 to 1.159 ± 0.183 fmol/cm2 (n = 9) after addition of cAMP plus IBMX.
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DISCUSSION |
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ENaC Protein Expression and Amiloride-sensitive Na+ Transport in Transfected MDCK Cells
The parental line of MDCK cells that was used for retroviral transfection had no significant expression of any of the three ENaC subunits at the mRNA level and no expression of the endogenous (dog) ßENaC protein (Fig. 1), whereas the triply transfected FßF
F MDCK cells expressed all three exogenous rat ENaC proteins (Fig. 2). The immunoprecipitation experiments in Fig. 3 further demonstrate that the FLAG epitope was present in all three subunits. The
FßF
F MDCK cells also exhibited a significantly higher VT than observed in the parental cells, and that VT was inhibited >75% by amiloride (Table I) added to the apical but not to the basolateral membrane. These results demonstrated that the ENaC subunits expressed in the
FßF
F MDCK cells were assembled and trafficked preferentially to the apical membrane, and that they were mediating amiloride-sensitive Na+ transport.
The results of experiments in which Isc was measured in FßF
F MDCK cells was also consistent with previous studies in MDCK cell lines that have high endogenous rates of amiloride-sensitive reabsorption as well as similar cultured lines, such as A6 cells (Bindels et al., 1988
; Chalfant et al., 1993
; Kleyman et al., 1994
; Blazer-Yost and Helman, 1997
; Morris et al., 1998
). In particular, in DMEM the time course of the transport response to cAMP (Fig. 5 A) was the same as that observed in these previous studies. There was an initial rapid increase in Isc followed by a decline, which has been attributed to stimulation of Cl- secretion (under the voltage-clamp conditions) by cAMP, then a lesser secondary maximum, which has been attributed to stimulation of Na+ transport via ENaC and which subsequently decays (Chalfant et al., 1993
; Letz and Korbmacher, 1997
; Morris et al., 1998
). The initial transient was absent in a chloride-free medium, and Isc peaked 1015 min after cAMP treatment, followed by a slow decay, remaining elevated above baseline even 30 min after the addition. It is tempting to speculate that this more sustained current reflects the loss of an inhibitory influence of CFTR-mediated Cl- transport on Na+ transport (Schwiebert et al., 1995
); however, Stutts et al. (1995)
showed that the inhibitory effect of CFTR on ENaC channels in MDCK cells transfected with both transporters occurred even in a chloride-free medium. Regardless of the mechanism involved in the effect of Cl-, we used the chloride-free medium for most of the experiments in this study in order to avoid the complications presented by the more labile Isc in DMEM and to minimize the possible contribution of Cl- conductance changes on Isc.
Comparison of the Effects of cAMP on Isc and ENaC Subunit Density in the Apical Membrane
Labeling of the apical membrane of the FßF
F MDCK cells with the M2 antibody was shown to be saturable with an affinity in the nanomolar range. The k0.5 ranged from 3 to 8 nM (Figs. 4 and 7), which was comparable to the k0.5 of
3 nM reported by Firsov et al. (1996)
for M2 antibody binding to Xenopus oocytes transfected with the same flagged ENaC subunits. Most of the 125I-labeled M2 antibody binding could be displaced competitively either by FLAG or unlabeled antibody, but 2030% of the counts remained even in the presence of a 100-fold excess (by weight) of FLAG. These residual counts were attributed to nonspecific binding to the cells and the culture inserts, and in all cases the specific binding was calculated as the paired difference between binding in the presence and absence of FLAG. Specific binding of 125I-labeled M2, defined in the manner just described, was demonstrated to be directly proportional to the amiloride-sensitive Isc in the
FßF
F MDCK cells (Fig. 6).
The effects of cAMP on the binding of M2 antibody to the apical membrane of FßF
F MDCK cells were examined in two separate series of experiments in chloride-free medium. First, the results shown in Fig. 7 demonstrated that the specific binding of 125I-labeled M2 antibody was greater in cAMP-stimulated cells than in controls over a range of antibody concentrations with no change in the binding affinity. Furthermore, Bmax, obtained by nonlinear fitting to the Michaelis-Menten equation, was 40% higher than in controls (P < 0.001).
Second, in the experiments summarized by Table II, Isc measurements and binding assays were conducted in trays of cultured cells on inserts plated at the same time and treated in the same manner. The binding assays in this series of experiments were performed using a single concentration (1.7 nM) of 125I-labeled M2 antibody. The specific surface binding of radioligand was significantly higher by 86 ± 19% in FßF
F MDCK cells treated with cAMP compared with untreated controls (Table II). Extrapolation of these binding values obtained with 1.7 nM M2 antibody gives Bmax values comparable to the Bmax values obtained from the previous saturation binding experiments (Table II). In the parallel electrophysiological experiments, cAMP stimulation also significantly increased the amiloride-sensitive Isc by 61 ± 10% (Table II). The difference between the fractional increases in cAMP-stimulated Isc and M2 antibody binding (-25 ± 21%) was not statistically significant. Put another way, the increases in the surface density of ENaC subunits in the apical membrane and the amiloride-sensitive Isc were proportional.
One might argue, however, that the increase in M2 Ab binding produced by cAMP treatment was due to some allosteric rearrangement of ENaC subunits, which were already present in the apical membrane, that made more FLAG binding sites accessible to the antibody. If this putative molecular rearrangement were accompanied by an increase in channel Po, our results would not necessarily be inconsistent with other findings that PKA, the intermediate mediator of cAMP effects, can increase Po of Na+ channels in planar lipid bilayers (Berdiev et al., 1996; Jovov et al., 1999
) or detached membrane patches (Prat et al., 1993
). However, there are no data in support of any molecular rearrangement. Furthermore, it seems unlikely that the number of sites newly exposed by cAMP action would be in direct proportion to the increase in AS-Isc, or that there would be no change in the apparent affinity of binding, as we have found in our studies. For these reasons, we feel that our data are more easily explained by an increase in the surface density of ENaC channels.
Quantitative Comparison of the ENaC Subunit Density with Single Channel Characteristics
Because there is one FLAG epitope per ENaC subunit, the M2 binding data permit an estimate of the molar density of ENaC subunits in the apical membrane assuming one bound antibody per subunit as shown by Firsov et al. (1996). Thus, the Bmax estimates from Fig. 7 and Table II give a range of 2.42.5 fmoles of ENaC subunits/cm2 of apical membrane in the control cells, and 3.44.5 fmol/cm2 in the cAMP-treated cells. Based on a cell density of
2.5·106 cells/cm2 in the confluent cultures, the subunit density would be on the order of 500600 per cell under control conditions, and 8001,100 per cell in the presence of cAMP.
The analysis presented in Table II shows that 0.22 fmoles of ENaC subunits are present per microampere of AS-Isc both in the presence and absence of cAMP. Thus,
130 subunits are present in the membrane for every picoampere of AS-Isc, This result can be compared with the number of subunits predicted from the single channel kinetic data obtained from patch clamp and fluctuation analyses. Ishikawa et al. (1998)
obtained a single channel conductance of 4.7 pS when unmodified rat ENaC subunits were transfected into the same MDCK cell line used in our studies. This conductance agrees well with the range of 3.74.9 pS observed in a variety of epithelia possessing the highly selective Na+ channel (Garty and Palmer, 1997
). Assuming an apical membrane voltage of -80 mV under short-circuit conditions, a 4.7 pS conductance would predict a single channel current of 0.38 pA. Using the average Po of 0.5 typically reported for the highly selective Na+ channel (Garty and Palmer, 1997
), our estimate of 130 subunits/pA indicates that
25 subunits are present in the membrane for each channel.
This estimate of the subunit number per channel exceeds that expected based on estimates of the channel composition. The majority of studies indicate that the channel is a heterotetrameric assembly of ENaC subunits, but stoichiometries involving up to nine subunits per channel have been proposed (Firsov et al., 1998; Kosari et al., 1998
; Snyder et al., 1998
). Although our calculated number of subunits per channel may be compromised by the accuracy of the Bmax values and the assumptions made, Firsov et al. (1996)
also found that the membrane density of ENaC subunits transfected into Xenopus oocytes exceeded that expected from the amiloride-sensitive current. There are two possible explanations for the apparent excess number of subunits. A fraction of the subunits in the membrane may be unassembled, i.e., not associated with a functional channel. Firsov et al. (1996)
discounted this possibility as well as the presence of a pool of assembled but electrically silent channels in their oocyte expression studies based on the fact that the relationship between current and binding was linear and had a zero intercept. Although the Y-intercept of our correlation plot (Fig. 5) is not significantly different from zero, the error of this estimate cannot exclude the possibility that 2030% of the specific binding was not associated with transporting channels.
An alternative explanation for the apparent excess of subunits is that the Po of the channels is <0.5. Firsov et al. (1996) proposed that the ENaC channels expressed in oocytes had a Po in the range of 0.0040.014. In the present experiments, assuming a heterotetrameric stoichiometry, Po would have to be <0.02 for the subunit density to be consistent with the macroscopic currents measured. As argued by Firsov et al. (1996)
, the long open and closed times that characterize ENaC channels make the determination of the number of channels in a patch difficult and bias the Po determination to higher values. In their review of such studies, Garty and Palmer (1997)
noted that the average Po of 0.5 is misleading because individual measurements exhibit a bimodal distribution with the majority of the measurements either >0.7 or <0.3.
An interesting sidelight to this issue is raised by our binding results in DMEM, in which the AS-Isc is calculated to be 10.3 µA/cm2 (Table I), whereas Bmax is 7.4 fmol/cm2. The ratio of binding to AS-Isc is thus 0.7 fmol/µA or >400 subunits/pA, which is threefold higher than calculated above for experiments in chloride-free medium. In other words, it appears that in DMEM the number of electrically silent subunits is greater or the Po lower than in the absence of chloride. However, these calculations should be regarded with caution for two reasons. First, the electrophysiological parameters were not measured in the same experiments as the binding for the experiments in DMEM. Second, we did not measure Isc but rather VT and RT in the electrophysiological experiments, and thus we could compute only an "equivalent Isc" (VT/RT) under open circuit conditions (Table I). These values would be expected to underestimate the true Isc, but certainly not by an amount sufficient to explain a threefold higher estimate of the subunits per µA. Thus, the possibility that the channel density per µA is greater in the presence of chloride is deserving of examination in further studies.
Conclusions
The results summarized above lead us to conclude that increases Na+ transport produced by cAMP are due to a proportional increase in the number of ENaC channels in the apical membrane, which is in agreement with the previous studies using less quantitative methods of determining the ENaC surface density as described in the introduction (Marunaka and Eaton, 1991; Kleyman et al., 1994
; Snyder, 2000
). Thus, our findings support the view that membrane trafficking is responsible for the regulation of ENaC activity that is mediated by cAMP.
Vesicular trafficking as a mechanism of ENaC regulation has received recent support from observations that all ENaC is present in cytoplasmic vesicles in subapical membrane region and that trafficking events are stimulated by cAMP. Using confocal and electron microscopy in immunohistochemical studies of the rat kidney, Hager et al. (2001) have shown that all three ENaC subunits are present in the apical membrane and cytoplasmic membrane vesicles of principal cells in the cortical collecting duct, and that cytoplasmic
ENaC was found exclusively in the apical membrane region, where it colocalized with the aquaporin-2 water channel. Butterworth et al. (2001)
used a membrane-specific fluorescent probe and confocal microscopy in A6 cells to show that elevation of intracellular cAMP increases the rate of endo- and exocytosis, with the increase exocytosis dominating.
Other studies have shown that the half-life of ENaC channels in the apical membrane is on the order of one to several hours (Weisz et al., 2000; Rotin et al., 2001
), implying that ultimately ENaC activity must be maintained by trafficking. The pulse-chase experiments of Weisz et al. (2000)
have also shown that aldosterone and ADH increase the membrane surface expression of ßENaC in A6 cells, but not that of
- or
ENaC, which contrasts with the apical localization of
ENaC in the experiments of Hager et al. (2001)
. Our results, however, do not support the view that trafficking of a single subunit is responsible for cAMP regulation of ENaC because they show an increase in total (
, ß, and
) ENaC in the apical membrane. As proposed by Firsov et al. (1996)
, it seems more likely that a preassembled complex of all three ENaC subunits is trafficked to the apical membrane.
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FOOTNOTES |
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Portions of this work were previously published in abstract form (Morris, R.G., and J.A. Schafer. 2000. J. Am. Soc. Nephrol. 11:34A; and Morris, R.G., and J.A. Schafer. 2001. J. Am. Soc. Nephrol. 12:37A).
* Abbreviations used in this paper: ADH, antidiuretic hormone; AS-Isc, amiloride-sensitive short-circuit current; Bmax, maximal specific binding; CCD, cortical collecting duct; CPT, 8-p-chlorophenylthio; DMEM, Dulbecco's modified Eagle's medium; ENaC, epithelial (amiloride-sensitive) Na+ channel; FLAG, octapeptide epitope DYKDDDDY; IBMX, isobutylmethylxanthine; Isc, short-circuit current; MDCK, Madin-Darby canine kidney; PKA, protein kinase A; RT, transepithelial resistance; VT, transepithelial voltage.
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ACKNOWLEDGMENTS |
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This study was supported by National Institutes of Health grant DK-25519-21. This work was part of a dissertation submitted in partial fulfillment of the requirements for the Ph.D. degree awarded to R.G. Morris by the School of Graduate Studies at the University of Alabama at Birmingham.
Submitted: 27 December 2001
Revised: 19 April 2002
Accepted: 6 May 2002
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REFERENCES |
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