Department of Physiology and Biophysics and Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294
Submitted 15 April 2004 ; accepted in final form 20 May 2004
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ABSTRACT |
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epithelial sodium channel surface density; chloride channels; cystic fibrosis transmembrane conductance regulator
Recently, the inhibition by cystic fibrosis transmembrane conductance regulator (CFTR) of transepithelial Na+ absorption via ENaC has been studied intensely because it is hypothesized to explain the pathophysiology of cystic fibrosis in airway epithelia (11, 38). The inhibitory effects of CFTR on Na+ transport are also observed in other organs affected by this disease and in a variety of epithelia that express both transporters (21, 27, 33, 45). Several potential mechanisms by which CFTR inhibits ENaC have been proposed. For example, CFTR may interfere with protein kinase A-dependent regulation of ENaC (55), or CFTR may control ENaC through additional regulatory proteins such as PDZ-binding domain proteins (5) or by direct physical binding (28). Alternatively, the inhibitory effect of CFTR in some preparations may be due to the rise it produces in intracellular Cl concentration ([Cl]i) rather than a direct molecular interaction of CFTR with ENaC (32). Kunzelmann and colleagues (6, 30, 32) showed that the inhibition of ENaC in Xenopus laevis oocytes by CFTR coexpression required a high extracellular [Cl]. Furthermore, this inhibition was not specific to CFTR but could also be produced by coexpression of ClC-0 or ClC-2, and it occurred even when amphotericin was used to permeabilize the cell membrane nonspecifically (30), leading to the conclusion that ENaC can be inhibited by any agent that increases [Cl]i (30, 32). The importance of [Cl]i has also been demonstrated in whole cell patch-clamp studies of M-1 cells (34), in excised patches of salivary duct in which a variety of anions including NO3 on the cytosolic side inhibited ENaC (14), and in sweat gland ducts in which the basolateral membrane had been permeabilized by -toxin (49).
As pointed out by Kunzelmann (32), the effect of activating CFTR or other Cl channels in a particular epithelium will depend on the direction of the resulting change in [Cl]i and on whether that epithelium is primarily involved in NaCl absorption or secretion. In epithelial cells, [Cl]i is determined by the steady-state relationship between Cl transporters in apical and basolateral membranes (25). In most epithelia that actively secrete Cl, Cl is actively accumulated across the basolateral membrane by cotransporters such as NKCC and NCC that maintain [Cl]i above its electrochemical equilibrium value. Stimulation of Cl secretion in such epithelia (e.g., by cAMP, forskolin, or cholera toxin) could be produced by increased apical membrane Cl channel activity, which would result in a fall in [Cl]i, or by an increase in the activity of the basolateral cotransporter in the presence of a finite Cl conductance in the apical membrane, in which case [Cl]i would rise. For example, stimulation of Cl secretion by cAMP results in a fall in [Cl]i in Necturus gallbladder (47), spiny dogfish rectal gland tubules (22), canine trachea (53), and human colonic epithelium (7, 39), but [Cl]i increases with stimulation in human colonic epithelial cells (37).
We previously developed a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) expressing "flagged" rat ENaC subunits, i.e., subunits labeled in the extracellular loop-domain with FLAG epitope as described by Firsov et al. (17). The FLAG epitope allows the number of ENaC subunits residing in the apical membrane of the transfected MDCK cells to be quantified by the surface binding of 125I-labeled anti-FLAG antibodies. Our initial studies with intact FL-MDCK monolayers in DMEM medium showed they had a high short-circuit current (Isc) and that cAMP treatment produced a rapid transient peak within 5 min followed by a broad peak that decayed over 20 min (43), as also described in A6 and M-1 cultures (8, 29, 34, 44). The biphasic response to cAMP in all of these epithelia has been attributed to rapid Cl secretion by CFTR activation followed by a slower cAMP-dependent activation of ENaC that is blunted by the inhibitory effect of CFTR. However, in a Cl-free medium, cAMP produced a monotonic and sustained increase in Isc in the FL-MDCK monolayers (43). For that reason, Morris and Schafer (43) used a Cl-free medium to examine the effect of cAMP on the surface density of ENaC subunits and established that it increased in proportion to the increase in amiloride-sensitive (AS)-Isc, demonstrating that cAMP increased Na+ transport by an increase in the number rather than a change in the intrinsic activity of the ENaC in the apical membrane (43).
The present studies were designed to examine whether increased [Cl]i inhibits ENaC-mediated Na+ absorption in epithelial monolayers of FL-MDCK cells as it does in oocytes and patch-clamped membranes from other epithelia. We also examined whether such a mechanism could account for the later decay in Na+ absorption after cAMP stimulation observed in previous studies with this and similar epithelia (8, 29, 34, 43, 44) and the effect of increases in [Cl]i on the density of ENaC subunits in the apical membrane of these FL-MDCK cells.
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METHODS |
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Electrophysiological studies. The membranes supporting the high-resistance FL-MDCK monolayers were carefully cut from the plastic insets and mounted in Ussing-type chambers in a 37°C incubator. In experiments with intact monolayers, both sides were bathed with 10 ml of Krebs-Ringer bicarbonate (KRB) solution containing (in mM) 113 NaCl, 1.2 Na2HPO4, 25 NaHCO3, 1.1 CaCl2, 1.2 MgCl2, 4.5 KCl, and 10 glucose. All solutions were gassed with 95% O2-5% CO2 at 37°C, and the pH was 7.4.
Transepithelial Isc (µA/cm2) and conductances (Gte; mS/cm2) were measured as described previously (43). The AS-Isc was defined as the change in Isc produced by the addition of 10 µM amiloride to the apical solution, and the remaining Isc was defined as the non-amiloride-sensitive short-circuit current (NS-Isc). In those experiments involving cAMP treatment, 100 µM 8-(4-chlorophenylthio)-cAMP (Sigma) plus 100 µM IBMX were added to both the apical and basolateral solutions. In some experiments, the monolayers were kept under open-circuit conditions during the course of the experiment except for intermittent voltage clamping to 0 mV and ±2 mV for a total of 4 s every 30 s to measure Isc and Gte.
Permeabilization of the basolateral membrane with -toxin or nystatin.
To short-circuit the apical membrane directly and to manipulate the ionic composition of the cytoplasm, we permeabilized the basolateral membrane with the pore-forming agents
-toxin or nystatin. FL-MDCK monolayers were incubated with 200 U/ml of
-toxin in the basolateral solution at 37°C for 30 min. The agent was then removed before the FL-MDCK monolayers were studied in Ussing chambers. As demonstrated in other epithelia (46), short-term
-toxin treatment of the basolateral membrane did not permeabilize the apical membrane or the junctional complexes because cAMP was able to activate transepithelial transport when added to the basolateral but not to the apical side. In other experiments, nystatin (Ca2+ 50 mg/ml stock solution in DMSO; Calbiochem, La Jolla, CA) was added to the basolateral solution at a final concentration of 350 µg/ml after the monolayer was mounted in the Ussing chamber (see METHODS in Ref. 36). On nystatin addition, the open-circuit voltage and Isc fell to zero in monolayers bathed in symmetrical apical and basolateral solutions, indicating that active transport was abolished by the permeabilization of the basolateral membrane. The transepithelial conductance remained low and stable even 60 min after nystatin addition to the basolateral solution.
After permeabilization of the basolateral membrane with either agent, Isc was allowed to stabilize for 1520 min before the beginning of each experiment. In the experiments with -toxin, the Ringer-like apical solution contained (in mM) 120 Na+ gluconate, 1.2 CaCl2, 1.2 MgCl2, 2.4 K2HPO4, 0.6 KH2PO4, 10 glucose, and 10 HEPES, whereas the basolateral solution contained 120 KCl, 1.2 CaCl2, 1.2 MgCl2, 2.4 K2HPO4, 0.6 KH2PO4, 10 glucose, and 10 HEPES. Thus there were large transepithelial chemical gradients favoring Na+ absorption and Cl secretion. In the experiments with nystatin permeabilization, the monolayers were bathed in variants of Ringer solution that produced a Na+ concentration gradient in the absorptive direction. The apical solution contained (in mM) 120 NaCl, 1.2 CaCl2, 1.2 MgCl2, 2.4 K2HPO4, 0.6 KH2PO4, 10 glucose, and 10 HEPES, whereas the basolateral solution contained (in mM) 120 KCl, 1.2 CaCl2, 1.2 MgCl2, 2.4 K2HPO4, 0.6 KH2PO4, 10 glucose, and 10 HEPES. The chloride concentration in these two solutions was varied by replacement with gluconate.
Surface labeling of flagged ENaC subunits. ENaC subunits surface density was measured as described by Morris and Schafer (43). Briefly, anti-FLAG antibody (Sigma; referred to below as M2 Ab) was radiolabeled with 125I in the UAB Radiolabeling Core Facility in the Comprehensive Cancer Center at the University of Alabama at Birmingham. Labeled M2 Ab was dialyzed for at least 24 h in PBS, and the Ab concentration was determined by a microprotein assay. Specific binding assays were performed on FL-MDCK cells grown on Transwell inserts. After incubation with low- or high-[Cl] bathing solutions containing 210 µg/ml nystatin in the basolateral solution for 20 min, 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). Binding was started on addition of 4 nM M2 Ab in a volume of 500 µl blocking solution per insert. To determine the nonspecific binding, M2 Ab was added to paired inserts together with a 100-fold excess (by weight) of FLAG peptide (Sigma). After 1-h incubation on ice, the inserts were washed four times with 1.5 ml of blocking solution. The M2 antibody remaining bound to the apical surface of the monolayers was then removed with 750 µl of ice-cold acid washing solution (0.5 M NaCl, 0.2 M Na+ acetate, pH 2.4). Two acid strips were performed, and the counts were combined for data analysis.
Immunoprecipitation and Western blotting. For immunoprecipitation of CFTR, FL-MDCK monolayers were washed twice with ice-cold PBS, scraped from the Transwell inserts, and lysed in PBS containing 1% Triton X-100 and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml aprotinin) for 10 min at 4°C. The lysate was centrifuged for 10 min at 14,000 g, and the supernatant containing the solubilized proteins was incubated overnight at 4°C with a polyclonal rabbit antibody against the highly conserved nucleotide binding domain (NBD-1) of human CFTR (gift of Dr. J. Collawn, Dept. of Cell Biology, University of Alabama at Birmingham). This IgG antibody was cross-linked to protein A/G-agarose beads with 10 mM dimethylpimelimidate (Pierce Chemical) for 30 min at room temperature, and 500 ng were used per lysate sample. After immunoprecipitation, samples were incubated in Laemmli buffer for 15 min at 37°C and run on 412% polyacrylamide gels. The separated proteins were transferred to a nitrocellulose membrane (Nitrobind, Osmonics) and processed for immunoblotting. The membrane blots were blocked with Blotto for 1 h at room temperature and incubated overnight at 4°C with 2 µg/ml of a monoclonal mouse anti-human CFTR NBD-1 antibody (Chemicon, Temecula, CA). They were then incubated with secondary antibody horseradish peroxidase conjugate at 1:4,000 dilution followed by ECL detection (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
For immunodetection of ENaC subunits, FL-MDCK cell lysates (40 µg protein) were separated by electrophoresis on a 412% polyacrylamide gel, transferred to nitrocellulose, and probed with antibodies specific to the three rat ENaC subunits (a kind gift from Dr. M. Knepper, National Institutes of Health) at 1:2,000 dilutions.
Intracellular Cl measurements. Measurements of [Cl]i in monolayers of FL-MDCK cells were made using 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ; Molecular Probes) as a fluorescent Cl probe. FL-MDCK monolayers grown on Transwell inserts were incubated overnight at 37°C in a humidified CO2 incubator with DMEM medium containing 5 mM SPQ. After being loaded, the monolayers were rapidly washed several times and the membranes were cut from the inserts and mounted on custom-made rectangular supports, which were then placed in the cuvette of a Delta-Scan fluorometer (Photon Technologies, Princeton, NJ) at a 45° angle to the excitation beam. The supports had cutouts so that the basolateral as well as the apical surfaces of the monolayers were bathed by the KRB that was perfused continuously through the cuvette at 2 ml/min. Experiments were performed at 37°C, and SPQ fluorescence was measured every second at an emission wavelength of 440 nm in response to the excitation wavelength of 344 nm using Photon Technologies software.
The [Cl]i was determined from the fluorescence values using the methods described by Verkman and his collaborators (9, 31). At the end of every experiment, the fluorescence intensity in the absence of Cl (F0) was determined by perfusion with a Cl-free solution containing nigericin (5 µM) and tributylin chloride (10 µM). To determine the fluorescence background, potassium thiocyanate (150 mM) and valinomycin (5 µM) were added to quench intracellular SPQ fluorescence. The fluorescence that was not quenched was subtracted from the measured fluorescence throughout each experiment.
Calibration solutions of varying Cl concentrations were prepared as mixtures of a solution containing 150 mM KCl plus 10 mM D-glucose and a Cl-free solution containing 150 mM KNO3 plus 10 mM D-glucose. These solutions also contained nigericin and tributylin to ensure rapid equilibration of the cytoplasmic Cl concentration with that of the extracellular solution. The value of (F0/F) 1, where F is the fluorescence at a given Cl concentration and F0 is the fluorescence in the Cl-free solution, was plotted as a function of the Cl concentration, and the Stern-Volmer constant Ksv (M1) was calculated as the slope of the linear regression fit of the data (see Ref. 9).
Data analysis. StatView for Macintosh (SAS Institute) was used for standard statistical analyses: ANOVA with Bonferroni/Dunn post hoc testing for multiple comparisons, and paired or nonpaired t-tests as appropriate for single comparisons; n represents the number of experiments. Kaleidagraph for Macintosh (Synergy Software) was used for linear fitting and regression analysis, and the correlation coefficient (r) and associated P value of the slope were calculated. In all cases, significance was assumed for P < 0.05.
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RESULTS |
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Because of these limitations with the -toxin permeabilization method, we used nystatin permeabilization for those experiments in which AS-Isc was measured. Nystatin is known to create aqueous pores of
4
radius in thin lipid membranes, which exclude Ca2+ but not smaller monovalent ions such as Na+, K+, and Cl (16, 26). In the experiments shown in Fig. 5, FL-MDCK cell monolayers were mounted in Ussing chambers with KRB on both sides, and Isc was measured as usual. Nystatin (350 µg/ml) was then added to the basolateral solution, and both Isc and the transepithelial voltage (measured under open-circuit conditions) fell to zero within 15 min. A short-circuit current was then produced by replacing Na+ with K+ in the basolateral solution and thus imposing an external gradient for a Na+ flux in the absorptive direction.
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Effect of cAMP on [Cl]i. Given the marked inhibition of AS-Isc by [Cl]i in the preceding experiments (Fig. 5), we tested whether a change of [Cl]i might explain the late inhibition of ENaC when anion secretion is stimulated by cAMP. We measured [Cl]i in FL-MDCK cells grown on Transwell inserts with the fluorescent dye SPQ using the double ionophore technique (9, 31) as described in METHODS. The fluorescence was measured using standard solutions with varying extracellular Cl concentrations, and a Stern-Volmer plot for the relationship between (F0/F 1) vs. [Cl]i gave a Ksv of 9.3 M1. Based on the SPQ data for five experiments such as the one shown in Fig. 6A, [Cl]i was estimated to be 76 ± 14 mM in the absence of cAMP treatment (control) and rapidly decreased to 35.7 ± 8.7 mM (P = 0.03) after cAMP treatment (Fig. 6B). The response was sustained, and [Cl]i returned to basal levels after cAMP was washed out. The decrease in [Cl]i that was produced by cAMP could also be reversed to the basal level by 200 µM glibenclamide (Fig. 6C), indicating again that the Cl secretion activated by cAMP was mediated through CFTR.
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As shown in Fig. 7A, increasing the Cl concentration in the basolateral solution (and thus presumably [Cl]i) from 15 to 145 mM decreased AS-Isc from 24.5 ± 1.0 to 10.2 ± 1.6 µA/cm2 (n = 6, P < 0.001). Because one FLAG epitope was inserted into the extracellular domain of every ENaC subunit, the apical membrane surface density of ENaC subunits could be measured by the specific binding of 125I-labeled anti-FLAG (M2) antibody. As shown in Fig. 7B, the specific binding of 4 nM M2 antibody was decreased from 0.65 ± 0.05 to 0.43 ± 0.05 fmol/cm2 (n = 7, P < 0.01), when Cl concentration was increased from 15 to 145 mM. To estimate the surface density of ENaC subunits from saturation binding (Bmax), we compared the concentration dependence of M2 Ab binding to that we had observed previously in DMEM (43) and verified the constant for half-maximal binding k0.5 of 7.9 nM (data not shown). Using the Michaelis-Menten equation (43), Bmax was estimated, respectively, to be 1.91 ± 0.16 or 1.32 ± 0.17 fmol/cm2 (P < 0.05) with 15 or 145 mM Cl in the basolateral solution (Fig. 8). In summary, the 58% reduction in AS-Isc that was produced by this increase in [Cl]i was explained by a 31% decrease in the density of ENaC in the apical membrane and a 38% reduction in the intrinsic channel activity (AS-Isc/n).
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DISCUSSION |
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Anion channels associated with NS-Isc. As expected, the FL-MDCK clone used in these studies expressed all three of the transfected ENaC subunits (Fig. 1, left), and the presence of the FLAG epitope in the extracellular domain of each subunit was verified by RT-PCR. Epithelial monolayers of these cells uniformly exhibited a high basal Isc, most of which was eliminated by 10 µM apical amiloride (Fig. 2) as previously observed by Morris and Schafer (43). We hypothesized that anion secretion mediated the NS-Isc because in the presence of apical amiloride, Cl and HCO3 were the only ions present in sufficient concentration to account for the Isc observed, and previous studies showed that the apical membrane of MDCK cells has no measurable K+ permeability (1). The biphasic time course of the Isc response to cAMP (Fig. 2) was consistent with previous studies in MDCK cells (43, 54) and in A6 cells (4, 8, 29, 44, 58) that showed a rapid increase in anion secretion followed by a more gradual increase in Na+ absorption.
The secretory anion current, represented by NS-Isc, was partially inhibited by 200 µM glibenclamide (Figs. 2 and 3), which is a well-established inhibitor of CFTR at this concentration (33, 45). The expression of CFTR protein in FL-MDCK cells was confirmed by RT-PCR and immunoprecipitation experiments (Fig. 1, right). This finding is in good agreement with Mohamed et al. (42), who previously reported the presence of CFTR in MDCK type 1 cells (the progenitor for the FL-MDCK cells used in these studies) but not MDCK type 2 cells. In experiments such as that shown in Fig. 3, in which -toxin was used to permeabilize the basolateral membrane and isolate the apical transporters, NS-Isc was increased by cAMP and inhibited by glibenclamide added to the apical solution as would be expected if CFTR was present in the apical membrane. Also, in the
-toxin-permeabilized monolayers, the selectivity sequence of the cAMP-activated anion conductance was NO3 > Br > Cl > I (Fig. 4), which is a unique characteristic of CFTR (2, 3).
In preliminary experiments that are not shown here, we also found evidence for a Ca2+-activated Cl channel (CaCC) and the ClC-2 channel in these cells. Thapsigargin, which moderately increases [Ca2+]i in MDCK cells (35), produced a sustained increase in anion secretion that could be completely blocked by apical DIDS as expected for CaCC (19). Thus, given the fact that cAMP can increase intracellular [Ca2+]i in the MDCK cells (10), CaCC may contribute to the fraction of NS-Isc that is not sensitive to glibenclamide (Figs. 2 and 3). A Ca2+-activated Cl conductance has been described in mouse M-1 cortical collecting duct cells (41) and in primary cultures of rabbit proximal and distal tubule cells (52). We were unable to detect an RT-PCR product using various primer pairs based on the CaCC sequence from human or mouse; however, it is quite possible that the dog sequence is significantly different. Immunoblot and RT-PCR studies (data not shown) showed that these FL-MDCK cells also expressed the ClC-2 isoform of the Cl channel that is widely distributed in many similar epithelia (27, 45) and that can be activated by cAMP acting via PKA as well as by cell swelling and a low extracellular pH (12, 27). However, we have no information about the localization of ClC-2 to the apical vs. basolateral membrane or about its possible contribution to NS-Isc. Based on these results, we conclude that CFTR is the major but not the only Cl channel in the apical membrane of FL-MDCK cells and at least one other Cl channel may contribute to anion secretion and its augmentation by cAMP.
Effect of intracellular Cl on ENaC.
Kunzelmann and collaborators (6, 30, 32) showed that the inhibition of ENaC in X. laevis oocytes by coexpression of CFTR is not specific to this Cl channel but can be produced by an increase in intracellular [Cl]i due to coexpression of other Cl channels or even nonspecific membrane permeabilization by amphotericin. The inhibitory effect of [Cl]i on ENaC channels has also been demonstrated in patch-clamp studies of other mammalian epithelia (e.g., 14, 34) and in sweat gland ducts in which the basolateral membrane was permeabilized by -toxin (49).
To determine whether [Cl]i had an inhibitory effect on ENaC in FL-MDCK cells, we permeabilized the basolateral membranes with nystatin, which allowed us to study the apical membrane transporters without the complication of the basolateral membrane and to vary the cytosolic Cl concentration. Nystatin produces aqueous pores of 4-
radius that allow rapid equilibration of small monovalent ions but not Ca2+ (16, 26). Thus, in the presence of a transepithelial Na+ gradient, there was a large AS-Isc in the nystatin-permeabilized cells (Fig. 5) but not in the
-toxin-permeabilized cells (Fig. 3), in which the elevation of [Ca2+]i inhibits ENaC. In similar experiments with sweat ducts, Reddy et al. (48) avoided this inhibition of ENaC during
-toxin permeabilization by decreasing the basolateral Ca2+ concentration. However, in our experiments, we could not reduce the Ca2+ concentration in the basolateral solution without complete disruption of the epithelium. In our experiments, we found that the addition of nystatin to the basolateral solution in the Ussing chamber rapidly reduced both Isc and the open-circuit voltage to zero in the absence of any transepithelial ion concentration gradients as expected, and the transepithelial conductance remained low, indicating that the basolateral membrane was permeabilized but the paracellular junction remained intact.
In the experiments shown in Fig. 5, we manipulated the direction and magnitude of Cl current by varying the Cl concentrations in the external solutions. An Na+ concentration gradient from the apical (120 mM Na+, 0 K+) to basolateral (0 Na+, 120 mM K+) was used to produce a net driving force for a Na+-absorptive flux, because the apical membrane potential difference is clamped to zero when these permeabilized cells are short-circuited, AS-Isc is a direct measure of Na+ absorption via ENaC and is not complicated by the possible effects of changes in the anion conductance on the electrochemical potential driving force for Na+ across the apical membrane (see Ref. 24). Under these conditions, we found that increases in the basolateral Cl concentration, and hence in [Cl]i, inhibited ENaC, but that changes in the Cl concentration in the apical solution had no effect. Furthermore, the inhibitory effect of [Cl]i on ENaC was solely dependent on the intracellular Cl concentration rather than the magnitude or direction of the Cl current.
In the experiments with nystatin-permeabilized monolayers, the Cl concentration was raised by replacing gluconate in the apical or basolateral solution. Expecting that Cl is much more permeant than gluconate, one might argue that cell swelling was, in some way, responsible for the inhibitory effect of elevated [Cl]i on ENaC [e.g., ClC-2 is activated by cell swelling (27)]; however, when cell swelling was prevented or cell shrinkage was produced by adding mannitol with Cl, we obtained the same inhibitory effect of [Cl]i (data not shown).
Effect of cAMP on [Cl]i. Although increased [Cl]i inhibited ENaC in the permeabilized FL-MDCK cells in this study as it did in the oocyte and patch-clamp studies, the inhibitory effect of increased anion secretion on ENaC that we observed in intact FL-MDCK monolayers (43) could not be attributed to an increase in [Cl]i if the activation of Cl channels in the apical membrane decreased [Cl]i as reported in other epithelia (7, 53).
To determine the changes in the intracellular Cl concentration with cAMP stimulation, we developed a method to measure [Cl]i in intact monolayers of our FL-MDCK cells on permeable supports using the Cl-sensitive fluorescent dye SPQ. Because the two sides of these monolayers were not electrically isolated in the cuvette of the fluorometer, they were effectively short-circuited as they were in the electrophysiological studies. Under control conditions, i.e., in KRB medium and in the absence of cAMP treatment (Fig. 7), [Cl]i was 76 ± 14 mM, and cAMP treatment decreased it to 36 ± 9 (P = 0.03). Although the control [Cl]i is somewhat higher than measured in some epithelia that secrete Cl, e.g., 47 mM in canine tracheal epithelium (53), it is quite close to the measurement in others, e.g., 61 mM in salivary acinar cells (18). However, the important point is that [Cl]i falls on treatment with cAMP and should activate rather than inhibit ENaC. Thus the late fall in ENaC activity after cAMP treatment, which was observed previously in this (43) and similar epithelia (8, 29, 34, 44), cannot be attributed to the effect of stimulated anion secretion on [Cl]i.
Mechanism of the inhibitory effect of [Cl]i on ENaC.
Returning to the effect of elevated [Cl]i on ENaC that we observed in the permeabilized monolayers, two mechanisms might account for the inhibition of ENaC by [Cl]i: a decrease in the number of ENaC subunits (n) in the apical membrane, or a decrease in the intrinsic single-channel activity, i.e., a decrease in the single-channel conductance () or the open probability (Po). Because one FLAG epitope was inserted into the extracellular domain of every ENaC subunit, the binding of 125I-labeled monoclonal antibody (M2) to the FLAG epitope permitted an estimate of the molar density of ENaC subunits (17, 43). As shown in Fig. 8, the Bmax estimates for the experiments in Fig. 7 were 1.91 ± 0.16 and 1.32 ± 0.17 fmol/cm2 in the presence of, respectively, low and high [Cl]i (P < 0.05). Based on a calculated cell density of
2.6·106 cells/cm2 in the confluent monolayers, the subunit density would be
460 subunits per cell at low [Cl]i and 320 subunits per cell at high [Cl]i.
As shown in Fig. 8, top, the ratio of AS-Isc to Ab bound, i.e., the average current per subunit, depends on the single-channel properties of ENaC and is proportional to the product ·Po. The analysis presented in Fig. 8 shows that the average current per subunit is significantly decreased from 13.3 ± 1.2 to 8.2 ± 1.4 µA/fmol by increased [Cl]i, indicating that either
or Po, or both, is inhibited by [Cl]i. Using the
value of 4.7-pS conductance observed in rENaC-transfected MDCK cells from the same MDCK cell line (26), estimates of the range of Po for ENaC can be calculated. In this analysis, it is recognized that the electrochemical potential gradient for Na+ across the short-circuited apical membrane is equal to the chemical potential difference (
Na) imposed by the transepithelial Na+ concentration difference. With a nominal cytosolic Na+ concentration of 6 mM,
Na is 80 mV. If one assumes, for example, that there are four subunits per active ENaC, Po would be 0.22 and 0.14 in the presence of, respectively, low and high [Cl]i.1 It should be noted that these estimates of Po are considerably higher than those in our previous study (43) or that of Firsov et al. (17). In other words, it appears that an elevation of [Cl]i decreases both the number of ENaC subunits (n) and the intrinsic ENaC channel activity (
·Po). Our conclusion that [Cl]i reduces ENaC-mediated Na+ transport due to a decrease in n as well as
·Po is consistent with that of Marunaka et al. (40), who reported that an increase in [Cl]i decreased the Po of the amiloride-sensitive nonselective cation channel by increasing the closing rate without any significant change in the opening rate in fetal rat alveolar epithelium, but different from that of Dinudom et al. (13) who showed that trafficking is not involved in the feedback control of Na+ channels by intracellular anions in mouse mandibular gland ducts.
In addition to its inhibitory effect on ENaC shown in the present study, intracellular Cl has been reported to regulate the activity of several other ion transporters, including NKCC1, the Na+/H+ exchanger, and a nonselective cation channel (50). Several laboratories identified Cl-sensitive proteins that may mediate the effect of changes in [Cl]i on ion transporters, such as Cl-sensitive kinases (15, 56) and GTP-binding proteins (23) that may regulate amiloride-sensitive Na+ channels. Alternatively, a direct and nonspecific interaction of intracellular Cl with amiloride-sensitive, nonselective cation channel activity has been proposed in fetal rat alveolar epithelium (40). Our results do not speak to these possibilities, but they do show that an elevation of [Cl]i inhibits ENaC-mediated Na+ absorption by approximately proportional decreases in the density of ENaC in the apical membrane and in the intrinsic activity of these channels.
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GRANTS |
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
1 This calculation takes the number of channels in the apical membrane to be the number of subunits (n) divided by the number of subunits per channel (), and, using the equation in Fig. 8, Po can be estimated as [(AS-Isc·
)/(n·
·
Na)]. The tacit assumption in this calculation is that changes in total subunit density are proportional to changes in the density of ENaC channels in the apical membrane. This would not be the case if, for example, only one subunit was trafficked into the membrane and that subunit caused the assembly active ENaC channels from pools of the other two subunits already present. Then the change in the surface density of subunits would considerably underestimate the change in active Na+ channels. However, based on presently available evidence (51), it is most likely that only assembled channels are trafficked to the membrane.
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REFERENCES |
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