Cell surface expression and turnover of the alpha -subunit of the epithelial sodium channel

Thomas R. Kleyman1, Jonathan B. Zuckerman2, Pamela Middleton3, Kathleen A. McNulty4, Baofeng Hu4, Xuefeng Su5, Bing An1, Douglas C. Eaton3, and Peter R. Smith5

1 Department of Medicine, University of Pittsburgh, Pittsburgh 15261; 2 Department of Medicine, Maine Medical Center, Portland, Maine 04102; 3 Department of Physiology, Emory University, Atlanta, Georgia 30322; 4 Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and 5 Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The renal epithelial cell line A6, derived from Xenopus laevis, expresses epithelial Na+ channels (ENaCs) and serves as a model system to study hormonal regulation and turnover of ENaCs. Our previous studies suggest that the alpha -subunit of Xenopus ENaC (alpha -xENaC) is detectable as 150- and 180-kDa polypeptides, putative immature and mature alpha -subunit heterodimers. The 150- and 180-kDa alpha -xENaC were present in distinct fractions after sedimentation of A6 cell lysate through a sucrose density gradient. Two anti-alpha -xENaC antibodies directed against distinct domains demonstrated that only 180-kDa alpha -xENaC was expressed at the apical cell surface. The half-life of cell surface-expressed alpha -xENaC was 24-30 h, suggesting that once ENaC matures and is expressed at the plasma membrane, its turnover is similar to that reported for mature cystic fibrosis transmembrane conductance regulator. No significant changes in apical surface expression of alpha -xENaC were observed after treatment of A6 cells with aldosterone for 24 h, despite a 5.3-fold increase in short-circuit current. This lack of change in surface expression is consistent with previous observations in A6 cells and suggests that aldosterone regulates ENaC gating and increases channel open probability.

epithelial sodium transport; epithelial sodium channels; ion channel; renal epithelium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RENAL TUBULAR EPITHELIAL CELLS transport enormous quantities of Na+ in a regulated manner, using a variety of Na+-selective transport proteins. The final site of renal Na+ reabsorption is the collecting tubule, and the rate of Na+ absorption across the collecting tubule is modified by hormones, such as aldosterone, that have key roles in the regulation of the extracellular fluid volume and blood pressure (1, 13). Epithelial Na+ channels (ENaCs) mediate the transport of Na+ across the apical plasma membrane of principal cells in renal collecting tubules (1, 13). These channels, originally cloned from rat distal colon, consist of at least three structurally related subunits, alpha , beta , and gamma .

The mineralocorticoid hormone aldosterone regulates transepithelial Na+ transport in the collecting tubule by activating ENaC (1, 13, 41). The mechanisms by which aldosterone regulates ENaC likely involve both translational as well as posttranslational events (1, 13, 41). In addition, the mechanism of ENaC activation appears to be dependent on the length of time that renal (or urinary) epithelia are exposed to aldosterone. Both early (~1.5 h) and late (>6 h) effects have been described and reflect changes in the biophysical properties of channels expressed at the cell surface or increases in ENaC protein expression at the apical cell surface (1, 12-13, 16). Patch-clamp analyses of single Na+ channels in the A6 amphibian renal epithelial cell line suggest that aldosterone activates channels by altering gating kinetics and increasing channel open probability (Po) (16). Noise analysis is an important and complementary method to examine ENaC Po and surface expression in A6 cells in response to aldosterone. Previous studies using this experimental approach suggested that aldosterone increases the density of channels expressed at the plasma membrane, rather than Po (15). Single-channel recordings from rat cortical collecting ducts also indicate that aldosterone increases the number of ENaC channels at the cell surface (27). Aldosterone increases the message or protein abundance of either the alpha -subunit alone (4, 23) or both the alpha - and beta -subunits in renal epithelia (9, 24). Subunit-specific ENaC antibodies have been used to localize Na+ channels in the kidneys of animals maintained on low- or high-NaCl diets (maneuvers that alter circulating aldosterone levels). An increase in ENaC alpha -subunit protein expression, as well as a redistribution of beta - and gamma -ENaC from the cytoplasm to the apical region of principal cells in the connecting tubule and cortical collecting duct of rat and mouse kidney (21, 23), in response to long-term dietary NaCl deprivation, has been reported by several groups. However, an earlier study suggested that long-term dietary NaCl deprivation did not alter the expression or localization of alpha - or gamma -ENaC in rat kidney (31), and a recent study demonstrated that activation of Na+ channels in rat cortical collecting duct in response to short-term (15 h) Na+ deprivation was not associated with a change in alpha -subunit protein expression (11). Taken together, these data suggest that aldosterone increases apical Na+ transport in the collecting duct, at least in part, by increasing ENaC cell surface expression in principal cells of the cortical collecting duct.

We previously raised an antibody directed against a 20-mer peptide corresponding to residues within the extracellular domain of Xenopus laevis ENaC (alpha - xENaC) that specifically recognizes in vitro translated alpha -xENaC. This antibody specifically recognized three polypeptides in A6 cell lysate with apparent molecular masses of ~70, ~150, and ~180 kDa (46). In the present study, we used this anti-alpha -xENaC antibody to examine the apical cell surface of alpha -xENaC in A6 epithelia. This antibody, as well as an anti-alpha -xENaC antibody raised against a 35-mer peptide corresponding to residues within the intracellular COOH-terminal domain of alpha -xENaC, specifically recognized only 180-kDa alpha -xENaC at the apical cell surface. A pulse (biotinylation)-chase protocol was used to examine the half-life of Na+ channel alpha -subunits expressed at the cell surface. The t1/2 was 24-30 h, suggesting that mature (i.e., the ~180-kDa polypeptide) alpha -xENaCs have a relatively slow turnover in A6 cells, in contrast to immature ENaC subunits that turn over within hours (24, 45). No significant change in surface expression of alpha -xENaC was observed after treatment of cells with aldosterone for 24 h to activate Na+ transport, despite a 5.3-fold increase in short-circuit current (Isc). These data suggest that increases in A6 cell Na+ transport in association with long-term (24 h) aldosterone treatment reflect changes in ENaC Po and are in agreement with the recent findings of Weisz et al. (45).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. A6 renal epithelial cells, derived from the distal nephron of X. laevis, were cultured as previously described (46). A6 cells were subcultured onto 4.7-cm2 Vitrogen-coated Costar polycarbonate filters (Corning, Acton, MA) for analysis of the effects of aldosterone on alpha -xENaC expression and for analysis of alpha -xENaC turnover. A6 cells were subcultured onto Millipore HAWP filter rings (Bedford, MA) for analysis by sucrose density gradient centrifugation. Monolayers were used 10-14 days after plating. To examine the effect of aldosterone on ENaC expression, monolayers were transferred to serum-free media for 3 days. The monolayers were then subsequently placed in either serum-free media supplemented with 1 µM aldosterone (experimental monolayers) or serum-free media alone (control monolayers) for 24 h before biochemical or electrophysiological studies. This concentration of aldosterone is sufficient to lead to saturating the binding of both the mineralocorticoid and glucocorticoid receptors. However, previous studies suggest that the response of A6 cells to aldosterone is mediated by glucocorticoid receptors, as these cells may not express mineralocorticoid receptors (33).

Sucrose density gradient centrifugation. A6 cell monolayers grown on HAWP filters were extracted in 0.5% Triton X-100 extraction buffer (26). The 0.5% Triton X-100-soluble fraction was concentrated to a volume of 1 ml using Centricon 10 concentrators (Amicon, Beverly, MA). Linear sucrose gradients (5-20%) were overlain with 200 µl of the concentrated 0.5% Triton X-100-soluble fraction and centrifuged in a SW 50.1 rotor (Beckman Instruments, Fullerton, CA) at 40,000 rpm for 22 h at 4°C, as previously described (46). Gradients were fractionated from bottom to top into 20 fractions (250 µl/fraction). Gradient fractions (1-3, 4-8, 12-13) were pooled before analysis. The distribution of alpha -xENaC within the pooled fractions was determined by immunoblot analysis.

Cell surface biotinylation. Cell surface biotinylation was used to examine both the turnover of alpha -xENaC and aldosterone-mediated changes in alpha -xENaC cell surface expression. Briefly, A6 cell monolayers grown on Costar filters were washed extensively with amphibian Ringer solution. Sulfonated N-hydroxysuccinimide-biotin (sulfo-NHS-biotin; Pierce, Rockford, IL), 0.5 mg/ml, in a borate-buffered solution (85 mM NaCl, 4 mM KCl, 15 mM Na2B4O7, pH 9.0) was added to the apical compartment and incubated with gentle shaking at 4°C (7, 8). The basolateral compartment received an equivalent volume of tissue culture medium with 5% FCS to bind the sulfo-NHS-biotin that leaked across the epithelium. Monolayers were then extensively washed with amphibian Ringer solution containing protease inhibitors (1 µM antipain, 1 µM leupeptin, 1 µM pepstatin A, and 100 µM phenylmethylsulfonyl fluoride). Filters were excised from their supports, and cells were solubilized in a lysis buffer containing 0.4% sodium deoxycholate, 1% Nonidet P-40, 50 mM EGTA, and 10 mM Tris · HCl (pH 7.4) supplemented with protease inhibitors. Lysates (500 µl/well) were precleared for 30 min by inverting on a rotator at 4°C with 50 µl of a 50% suspension of protein A-agarose beads. Lysates were subsequently centrifuged for 2 min, transferred to new tubes, and incubated with a 1:100 dilution of the anti-alpha -xENaC antibody for 12-16 h at 4°C. Protein A-agarose beads (50 µl) were then added for an additional 30 min. Immunoprecipitates were washed extensively with high-stringency wash buffer and were followed by low-stringency wash buffer (26). The beads were resuspended in 50 µl of 2× sample buffer containing 100 mM dithiothreitol, heated at 95°C for 2 min, and centrifuged for 2 min to pellet the beads. Supernatants were analyzed by 7.5% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Alternatively, after cell surface biotinylation, streptavidin-agarose beads were used to precipitate biotinylated proteins, as previously described. The precipitated proteins were subjected to 7.5% SDS-PAGE and transferred to PVDF membranes.

Cell surface biotinylation of A6 cells grown on Millipore HAWP filters was performed, as described by Prince et al. (28), using biotin-LC-hydrazide. Briefly, monolayers were cooled to 4°C, washed with amphibian PBS containing 0.1 mM CaCl2 and 1 mM MgCl2, and then incubated for 30 min with 10 mM NaIO4 in the dark. Monolayers were washed with PBS, and the apical surface was labeled at 4°C with 2 mM biotin-LC-hydrazide (Pierce) in 100 mM sodium acetate (pH 5.5) for 30 min with continuous gentle shaking. The basolateral compartment received an equivalent volume of PBS. After being labeled, the monolayers were extensively washed with PBS-containing protease inhibitors and the filters were excised from their supports. The cells were lysed in Triton X-100 extraction buffer (26), and the cell lysates were subjected to sucrose density centrifugation, as described above.

Aliquots of pooled gradient fractions 4-8 containing 200 µg of total protein were applied to a 250-µl column of immobilized monomeric Avidin (Pierce) (28) that had been equilibrated with PBS containing 0.5% Triton X-100. After application of the pooled fractions to the column, the column was washed with 2 ml of PBS containing 0.5% Triton X-100 to remove nonbiotinylated proteins. To elute bound biotinylated proteins, 1.25 ml of 2 mM biotin in PBS-0.5% Triton X-100 were added to the column. The eluant fractions were collected, pooled (2 ml total volume), and concentrated to a volume of 100 µl before analysis by SDS-PAGE.

Antibodies. The rabbit polyclonal antibody directed against the alpha -subunit of xENaC was generated by Lofstrand Laboratories (Bethesda, MD) by using a 20-mer synthetic peptide corresponding to amino acids 107-125 (CQNDLQELDKETQRTLYEL) in the extracellular loop of alpha -xENaC, as previously described (46). The immunizing peptide was coupled to Sulfolink agarose (Pierce), and anti-alpha -xENaC antibodies were affinity purified according to the manufacturer's instructions. A chicken polyclonal antibody raised against the COOH-terminal 33 residues of alpha -xENaC and purified using a gamma yolk purification kit (Amersham Pharmacia Biotech) was provided by Dr. J. Johnson (Univ. of Pittsburgh). The characteristics of these antibodies have been previously described (32).

Immunoblotting. Sucrose gradient fractions (30 µg/lane) or biotinylated proteins were separated by 7.5% SDS-PAGE and transferred to Immobilon PVDF paper (Millipore) as described (38). Blots of sucrose gradient factions were probed with rabbit anti-alpha -xENaC (15 µg/ml) antibody, followed by the appropriate secondary antibody coupled to biotin. Bound antibodies were detected by using a Western-Lite Plus chemiluminescent detection system (Tropix, Bedford, MA). Controls consisted of preincubation of anti-alpha -xENaC antibodies with excess-free peptide (100 µg/ml). Blots of biotinylated proteins were either probed with the rabbit anti-alpha -xENaC (15 µg/ml) antibody followed by the appropriate horseradish peroxidase-conjugated secondary antibody or with streptavidin-alkaline phosphatase conjugate followed by chemiluminescent detection (Tropix). A chicken anti-alpha -xENaC antibody directed against the COOH terminus was used to probe blots as previously described (32). The blots were scanned and analyzed using the IPLab Gel densitometry program (Scanalytics, Vienna, VA). The half-life of ENaC recovered from the cell surface was calculated using the equation T0.5 = -0.693 t/ln (A/A0), where t = time, A = recovery of ENaC at time t, and A0 = recovery of ENaC at time 0. Recovery of alpha -xENaC at 24 and 48 h was normalized to alpha -xENaC recovered from the apical membrane at time 0, which is defined as 100%. The linear range of the densitometer was determined by scanning film exposed to slot blot of increasing amounts of biotinylated A6 cell extract probed with streptavidin-alkaline phosphatase. For experiments using anti-alpha -xENaC antibodies to probe blots of streptavidin-precipitated surface proteins, the scanned densities of the bands were within the linear range of the densitometer.

Measurement of Isc and transepithelial potential difference. A6 monolayers grown on Costar filter inserts were placed in a modified Ussing chamber, and the transepithelial potential and Isc were measured using a DVC-1000 voltage clamp (World Precision Instruments, Sarasota, FL), as previously described (18).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously raised and characterized an antibody is directed against residues 107-125 within the extracellular domain of alpha -xENaC. This antibody specifically recognized in vitro translated alpha -xENaC, and it did not recognize in vitro translated beta - or gamma -xENaC (46). Three distinct polypeptides (relative molecular mass of ~70, ~150, and ~180 kDa) were specifically recognized when an immunoblot of A6 cell Triton X-100 extract was probed with the anti-alpha -xENaC antibody (46). The 70-kDa polypeptide is consistent with the predicted size of nonglycoslyated alpha -xENaC (30). We have previously demonstrated that the 180-kDa polypeptide was recognized by antibodies directed against two distinct domains within alpha -xENaC: the antibody directed against the extracellular domain of alpha -xENaC and an antibody directed against the COOH-terminal intracellular domain of alpha -xENaC (46). In addition, beta -xENaC was shown to coimmunoprecipitate with the 180-kDa polypeptide (46).

We previously reported that the distributions of the 70-, 150-, and 180-kDa polypeptides recognized by the anti-alpha -xENaC antibody peaked in distinct fractions after centrifugation of Triton X-100 extracts of A6 cell monolayers on a 5-20% linear sucrose density gradient (46). In the present study, we determined which of these polypeptides is expressed at the cell surface of A6 cells. To first confirm that 180- and 150-kDa alpha -xENaC are present within distinct fractions after sedimentation through the sucrose gradient, peak fractions containing 180- (fractions 4-8) and 150-kDa alpha -xENaC (fractions 12 and 13), as well as fractions 1-3 (control), were subjected to SDS-PAGE and transferred to PVDF paper. The blots were probed with the anti-alpha -xENaC antibody directed against the ectodomain of alpha -xENaC in the presence or absence of a peptide immunogen. As shown in Fig. 1, 180- and 150-kDa alpha -xENaC were in distinct fractions and were specifically recognized by the anti-alpha -xENaC antibody. Polypeptides with apparent molecular masses of 120 and 66 kDa were recognized in a nonspecific manner. A polypeptide with an apparent molecular mass of 50 kDa, present in fractions 1-3, was specifically recognized by the anti-alpha -xENaC antibody and may represent a degradation product. Several groups have suggested that ENaC has a subunit stoichiometry of alpha 2, beta 1, gamma 1, (10, 19), although conflicting results have been reported (35). We previously speculated that 180-kDa alpha -xENaC represents a dimer of mature alpha -subunits, whereas 150-kDa alpha -xENaC represents a dimer of immature alpha -subunit (46).


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Fig. 1.   Sucrose density gradient analysis of Xenopus epithelial Na+ channel (alpha -xENaC) extracted from A6 cell monolayers grown on permeable supports. Sucrose density gradient fractions 1-3, 4-8, and 12 and 13 were pooled and alpha -xENaC was immunoprecipitated under high-stringency conditions using the anti-alpha -xENaC antibody. Immunoprecipitated proteins were separated on 7.5% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) paper. Immunoblots were probed with the anti-alpha -xENaC antibody (left); or probed with the anti-alpha -xENaC antibody in the presence of excess peptide (right). The anti-alpha -xENaC antibody specifically recognized a polypeptide with an apparent molecular mass of 180 kDa in fractions 4-8 and a 150-kDa polypeptide in fractions 12 and 13. A 50-kDa polypeptide specifically recognized by the anti-alpha -xENaC antibody was present in fractions 1-3 and may represent a degradation product. Data presented are representative of 3 independent experiments.

To determine whether 180-kDa alpha -xENaC is expressed at the cell surface, A6 cell monolayers grown on Millipore HAWP filters were labeled on their apical surfaces with biotin-LC-hydrazide and extracted with Triton X-100. The extracts were subjected to 5-20% linear sucrose density centrifugation. Gradient fractions 4-8 were pooled, and biotinylated proteins were captured by passing the pooled fractions over an avidin column. Biotinylated proteins were subsequently eluted, subjected to SDS-PAGE, and transferred to PVDF paper. The resulting blots were probed with the anti-alpha -xENaC antibody in the presence or absence of excess free peptide immunogen. As shown in Fig. 2A, 180-kDa alpha -xENaC was specifically recognized by the anti-alpha -xENaC antibody. To further confirm that 180-kDa alpha -xENaC is expressed at the cell surface, A6 cell monolayers were grown on Costar supports, and apical plasma membrane proteins were labeled with sulfo-NHS-biotin. Cells were then solubilized and subjected to immunoprecipitation with the anti-alpha -xENaC antibody in the absence or presence of free peptide immunogen. The immunoprecipitated proteins were separated by SDS-PAGE, transferred to PVDF paper and then probed with alkaline phosphatase-conjugated streptavidin. As shown in Fig. 2B, biotinylated 180-kDa alpha -xENaC was specifically immunoprecipitated by the anti-alpha -xENaC antibody, but 150-kDa alpha -xENaC was not detected.


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Fig. 2.   Expression of the 180-kDa alpha -xENaC polypeptide at the apical surface of A6 cell monolayers. A: recovery of biotinylated 180-kDa alpha -xENaC from pooled sucrose density gradient fractions 4-8. A6 cell monolayers were biotinylated apically and extracted in 0.5% Triton X-100. Triton X-100 extracts were subjected to 5-20% sucrose density gradient centrifugation, and peak 180-kDa alpha -xENaC containing fractions 4-8 were pooled and passed over an Avidin column. Biotinylated proteins were eluted, separated by SDS-PAGE, and transferred to PVDF paper. Blots were probed with anti-alpha -xENaC antibody (lane 1) or anti-alpha -xENaC antibody plus excess free peptide (lane 2). Only the 180-kDa polypeptide was recognized by the anti-alpha -xENaC antibody. B: immunoprecipitation of surface-expressed alpha -xENaC from apically-biotinylated A6 cells with the anti-alpha -xENaC antibody (lane 1) or anti-alpha -xENaC antibody preincubated with excess-free peptide (lane 2). Immunoprecipitated proteins were separated by 7.5% SDS-PAGE and transferred to PVDF paper. The protein blots were subsequently probed with streptavidin. A single polypeptide with an Mr of 180 kDa, corresponding to mature alpha -xENaC, was detected by the streptavidin in the immunoprecipitate. Immunoprecipitation of alpha -xENaC was inhibited by preincubation of the anti-alpha -xENaC antibody with excess free peptide immunogen (lane 2). Data presented are representative of 3 independent experiments. C: A6 cell monolayers were biotinylated apically. After precipitation of biotinylated proteins with streptavidin-agarose, proteins were separated by 7.5% SDS-PAGE and transferred to PVDF paper. The protein blots were probed with anti-alpha -xENaC antibodies directed against an extracellular epitope (lane 1) or a COOH-terminal epitope (lane 2). Both antibodies recognized a 180-kDa polypeptide. D: alternatively, after apical surface biotinylation, streptavidin-agarose precipitation, SDS-PAGE, and transfer to PVDF, blots were probed with the anti-alpha -xENaC antibody directed against a COOH-terminal epitope in the presence (lane 2) or absence (lane 1) of the peptide immunogen. Both antibodies recognized a 180-kDa polypeptide. This polypeptide was not recognized when the antibodies were preincubated with their respective immunogens.

To provide additional evidence that 180-kDa alpha -xENaC is expressed at the cell surface, we used an antibody raised against the COOH-terminal 33 residues of alpha -xENaC (32). A6 apical cell surface proteins were biotin labeled and precipitated with streptavidin-agarose. The precipitated proteins were subjected to SDS-PAGE and transferred to PVDF paper. The blot was probed with either the antibody directed against the COOH terminus or the antibody against an extracellular domain of alpha -xENaC. Both antibodies recognized 180-kDa alpha -xENaC (Fig. 2C). Furthermore, a blot probed with the anti-alpha -xENaC antibody directed against its COOH-terminal domain in the presence or absence of peptide immunogen confirmed that this antibody specifically recognized 180-kDa alpha -xENaC (Fig. 2D). Taken together, these data provide convincing evidence that 180-kDa alpha -xENaC is expressed at the apical plasma membrane of A6 epithelial cells.

Previous studies have suggested that ENaC is rapidly degraded after its biosynthesis, and that its half-life may be on the order of hours (24, 40, 45). However, these studies that examined ENaC biosynthesis and maturation using pulse-chase metabolic labeling protocols in conjunction with immunoprecipitation identified immature subunits, as it was apparently difficult to detect mature subunits (24, 40). We used cell surface biotinylation as an alternative pulse-chase protocol to examine the turnover of cell surface-expressed alpha -xENaC. A6 epithelia were grown on permeable supports, and apical membrane proteins were labeled with NHS-biotin. Cells were then solubilized or, alternatively, they were placed back in an incubator for 24 or 48 h before solubilization. alpha -xENaC present at the apical surface at the time of biotinylation was detected by immunoprecipitation with the anti-alpha ENaC antibody directed against the extracellular domain followed by probing protein blots with alkaline phosphatase-conjugated streptavidin (Fig. 3A). The recovery of biotinylated alpha -ENaC decreased as cells remained in culture for 24-48 h after surface labeling (Fig. 4A). When compared with the recovery of cell surface (i.e., biotinylated) alpha -xENaC immediately after surface biotinylation, recovery of biotinylated alpha -xENaC after 24 h was 61 ± 15, and was 28 ± 8% after 48 h (Fig. 3B). The half-life of biotinylated alpha -xENaC (i.e., expressed at the apical surface at t = 0) was 30 h.


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Fig. 3.   Cell surface expression of alpha -xENaC. A: representative blot illustrating the amount of biotinylated 180-kDa alpha -xENaC that was recovered from A6 cells grown on permeable supports at 0, 24, and 48 h after apical membrane biotinylation. Cells were subsequently lysed, and alpha -xENaC was immunoprecipitated under high stringency conditions. Immunoprecipitated proteins were separated on a 7.5% SDS-PAGE gel, transferred to PVDF paper, and the blot was probed with streptavidin. A single polypeptide with an Mr of 180 kDa was detected (arrow). B: quantitation of the recovery of biotinylated 180-kDa alpha -xENaC from the apical surface of A6 cells grown on permeable supports. As biotinylation efficiency may vary between experiments, the amount of apical membrane 180-kDa alpha -xENaC recovered at 24 and 48 h was normalized to apical membrane alpha -xENaC at time 0 (defined as 100%; n = 4, performed in triplicate). C: recovery of biotinylated 180 kDa alpha -xENaC from A6 cells grown on permeable supports at 0 (lane 1) or 24 h (lane 2) after apical membrane biotinylation. Streptavidin-agarose was used to precipitate biotinylated proteins. After 7.5% SDS-PAGE and transferred to PVDF paper, the blot was probed with an anti-alpha -xENaC antibody directed against an extracellular epitope (n = 5).



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Fig. 4.   Representative immunoprecipitation of surface-expressed alpha -xENaC (180-kDa polypeptide) from apically biotinylated A6 cell monolayers maintained in media with (lane 1) or without (lane 2) aldosterone (1 µM) for 24 h. A: after lysis of the cells, alpha -xENaC was immunoprecipitated under high-stringency conditions. Immunoprecipitated proteins were separated on a 7.5% SDS-PAGE gel, transferred to PVDF paper, and the blot was probed with streptavidin. B: after lysis of the cells, biotinylated proteins were precipitated, separated on a 7.5% SDS-PAGE gel, transferred to PVDF paper, and the blot was probed with an anti-alpha -xENaC antibody. No significant change in the cell surface recovery of the 180-kDa alpha -ENaC polypeptide (arrow) was observed in response to aldosterone.

To confirm these observations, A6 cells were detergent solubilized 0 or 24 h after apical surface biotinylation. A6 apical cell surface proteins were precipitated with streptavidin-agarose, subjected to SDS-PAGE and transferred to PVDF paper. The blots were probed with the anti-alpha -xENaC antibody directed against the extracellular domain. The recovery of biotinylated alpha -xENaC after a 24-h chase period was 52 ± 15% of the amount of biotinylated alpha -xENaC recovered immediately after surface biotinylation (Fig. 3C), consistent with a half-life of biotinylated alpha -xENaC of 24 to 30 h.

Several recent studies suggest that expression of alpha -ENaC mRNA or protein is increased in response to aldosterone (4, 9, 23-24). An increase in immunoreactive alpha -ENaC localized to the region of the apical plasma membrane was observed in rat or mouse connecting tubules or cortical collecting tubules in response to dietary NaCl restriction (21, 23). However, conflicting data regarding changes in ENaC surface expression in A6 cells in response to aldosterone have been reported (15-16, 45). We examined whether apical plasma membrane expression of alpha -xENaC was altered by long-term (24 h) exposure of A6 cells to aldosterone. Cells were grown on permeable supports in serum-free media for >72 h to deplete cells of steroid hormones. Cells were then placed in serum-free media supplemented with 1 µM aldosterone, or maintained in serum-free medium in the absence of aldosterone. A6 monolayers exposed to aldosterone for 24 h clearly showed activation of transepithelial Na+ transport, compared with monolayers that were not exposed to aldosterone. Aldosterone treatment resulted in a 5.3-fold increase in Isc (P < 0.0001 aldosterone vs. control) and a 6.6-fold increase in transepithelial potential difference (P < 0.0001 aldosterone vs. control). To examine whether aldosterone treatment led to changes in apical plasma membrane expression of the alpha -subunit, A6 cell monolayers were apically labeled with NHS-biotin, lysed, and alpha -xENaC was immunoprecipitated from cell lysates using the antibody directed against the extracellular domain of alpha -xENaC. The surface pool of alpha -xENaC was detected by probing blots of the immunoprecipitates with alkaline phosphatase-conjugated streptavidin. Cell surface expression of alpha -xENaC was modestly (1.4-fold) but not significantly increased when cells were exposed to aldosterone, compared with nonaldosterone-treated controls (n = 22, P > 0.05, aldosterone vs. control; Table 1, Fig. 4A). To confirm these observations, A6 apical cell surface proteins were precipitated with streptavidin-agarose after surface biotinylation. The precipitated proteins were subjected to SDS-PAGE and transferred to PVDF paper, and the blots were probed with the anti-alpha -xENaC antibody directed against the extracellular domain. Cell surface expression of alpha -xENaC was unchanged in response to 24-h aldosterone exposure (n = 5, P > 0.05, aldosterone vs. control; Table 1, Fig. 4B).

                              
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Table 1.   Effect of 1 µM aldosterone on alpha -xENaC surface expression, Isc, and PD in A6 cell monolayers grown on permeable supports


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous work examining ENaC biosynthesis in A6 cells using a metabolic labeling, pulse-chase protocol suggested that alpha -xENaC has an apparent molecular mass of 70 kDa and has a relatively short half-life (~1 to 2 h) that is not altered by aldosterone (24). Although the 70-kDa polypeptide was shown to coimmunoprecipitate with beta - and gamma -xENaC, it was unclear whether this polypeptide represented mature alpha -xENaC (24). Weisz and co-workers (45) have recently confirmed the rapid turnover of whole cell alpha -, beta -, and gamma -xENaC in A6 cells. Valentijn and co-workers (40) also examined ENaC biosynthesis in Xenopus oocytes. They demonstrated that ENaC was synthesized in the endoplasmic reticulum and that the vast majority of newly synthesized ENaC remained in pre-Golgi compartments and were degraded via proteasomes. Only a minor fraction of newly synthesized ENaC had complex oligosaccharides and were expressed at the cell surface. Kosari and co-workers (19) and Firsov and co-workers (10) have suggested that ENaC has a subunit stoichiometry of alpha 2, beta 1, and gamma 1. We speculate that 180-kDa alpha -xENaC specifically recognized by the two distinct anti-alpha -xENaC antibodies used in the present study represent a dimer of alpha -xENaC subunits that is expressed at the apical surface of A6 cells. Using our anti-alpha -xENaC antibody directed against an extracellular epitope, Weisz and co-workers (45) also observed the 180-kDa alpha -xENaC expressed at the apical plasma membrane. The 180-kDa alpha -xENaC may represent a terminally glycosylated dimer of alpha -subunits, and the 150-kDa alpha -xENaC may represent a dimer of core or nonglycosylated alpha -subunits. However, Weisz et al. (45) observed that the 180-kDa polypeptide was resistant to treatment with either N-glycanase or endoglycosidase H, and Prince and Welsh (29) suggested that ENaC expressed at the cell surface of Chinese hamster ovary cells is deglycosylated. Our previous observations (46) suggest that 180-kDa alpha -xENaC is not an alpha  and/or beta  heterodimer, and Weisz et al. (45) suggest that it is not an alpha - and/or a gamma -heterodimer. We have not been successful in dissociating these putative alpha -subunit dimers, despite treatment with dithiothreitol (100 mM), urea (8 M), and SDS (3%). It was surprising that the putative alpha -xENaC dimers resisted dissociation into monomers, particularly in light of published observations (4, 23) indicating that mouse, rat, and human alpha -ENaC migrate with an apparent Mr of ~90 to 100 kDa. However, there are examples in the literature of oligomeric integral membrane proteins, including ion channels, that are resistant to disruption by conventional methods used to dissociate protein oligomers. For example, the K+ channel of Streptomyces lividans is a stable tetramer in the presence of SDS and beta -mercaptoethanol (14). Glycophorin A, the major integral membrane protein of the erythrocyte, is a dimer that is resistant to disruption by heat and SDS (20). Bacterial outer membrane porins are stable trimers in the presence of SDS and 8 M urea but dissociate into monomers when heated (39). The stability of these multimers may be conferred through secondary structural interactions (i.e., alpha -helices or beta -sheets). Secondary structural interactions between alpha -xENaC monomers might have a role in conferring stability to the 180-kDa alpha -xENaC.

Given the difficulties in identifying the pool of cell surface-expressed ENaC in epithelia, limited information exists regarding the turnover of mature ENaC. A half-life of 3.6 h has been reported for functional Na+ channels expressed at the plasma membrane of Xenopus oocytes (40). The functional half-life of ENaC increases to 30 h when channels express a Liddle's mutation (40). However, these studies did not address the turnover of the biochemical pool of channels, which may differ from the measured half-life of functional channels if the channels recycle between endosomal and plasma membrane compartments. Data from a recent study examining Fischer rat thyroid cells transiently transfected with alpha -, beta -, and gamma -ENaC have suggested that ENaC is inserted and retrieved from the plasma membrane in a regulated manner (34) and support the notion that ENaC recycles between endosomal and plasma membrane compartments. Our data suggest that the half-life of mature alpha -xENaC is ~24 to 30 h. Weisz and co-workers (45) examined the turnover of cell surface-expressed alpha -xENaC in A6 cells. Their data indicated that the recovery of biotinylated (i.e., cell surface-expressed) alpha -xENaC was unchanged over a 24-h period, suggesting a half-life of considerably longer than 24 h. Taken together, these data are consistent with the notion that once ENaC is expressed at the plasma membrane, it has a relatively slow turnover rate. As mentioned above, this clearly differs from the reported half-life of ENaC subunits in A6 epithelia determined by metabolic labeling and pulse-chase analyses, which was on the order of 1-2 h (24, 45). It also differs from the biochemical half-life of 9.5 to 11 h observed in Xenopus oocytes by Valentjin and co-workers (40). We agree with their findings, which suggested that studies using metabolic labeling and pulse-chase protocols to examine ENaC turnover in Xenopus oocytes are limited to analyses of immature subunits and that the assembly and processing of ENaC to a mature form are inefficient. However, once ENaC matures and reaches the plasma membrane, our data suggest that the turnover is slow and is similar to that reported for "mature" cystic fibrosis transmembrane conductance regulator (CFTR) (22, 44).

Several groups have examined mechanisms of regulation of ENaC by aldosterone. Aldosterone dramatically increases Na+ transport in the mammalian collecting duct and distal colon (13, 41) and increases transepithelial Na+ transport across epithelial cell lines derived from amphibian and mammalian distal nephron (4, 9, 16, 18). Previous work in cultured renal cell lines and in rat kidney and colon suggests that aldosterone regulates Na+ channels by two distinct mechanisms: 1) increases in Na+ channel Po (16) and 2) increases in ENaC protein expression at the plasma membrane (4, 9, 23, 31). Aldosterone also enhances transcription and translation of Na+-K+-ATPase, and increases in basolateral Na+-K+-ATPase expression also have an important role in the activation of transepithelial Na+ transport by aldosterone (42-43). A serum and growth factor-regulated kinase (sgk) was recently identified as an aldosterone-regulated gene product that activates ENaC by increasing the number of channels expressed at the cell surface (2, 6, 25). Consistent with this notion, rats maintained on a low-NaCl diet to induce extracellular volume depletion and stimulate aldosterone secretion were found to have an increase in the expression of Na+ channels in the apical region of the collecting duct principal cells (27). Mechanisms by which aldosterone regulates Na+ channel Po are poorly understood, although several regulatory pathways that likely participate in this process have been identified, including methylation, activation of the ras signaling pathway, and activation of phosphoinositide 3-kinase, (3, 5, 36-37, 41).

Patch-clamp analyses of Na+ channels in A6 epithelia suggested that both the early and late phases of ENaC activation by aldosterone are associated with an increase in channel Po (16). Consistent with the notion that aldosterone activates ENaC in A6 epithelia by increasing Po, we previously observed that long-term (24 h) treatment of A6 cells with aldosterone did not result in large changes in the cell surface pool of putative Na+ channels, when compared with nonaldosterone-treated cells or with cells that were treated with the aldosterone antagonist spironolactone (18). In this previous study, we used an anti-idiotypic antibody that recognized a putative amiloride-binding domain within the alpha -subunit of ENaC to examine whether aldosterone treatment altered plasma membrane expression of ENaC (17). In the present study, no significant changes in the plasma membrane expression of alpha -xENaC were observed in response to 24-h aldosterone exposure (Table 1 and Fig. 4), suggesting that the 5.3-fold increase in Isc observed with aldosterone primarily reflects an increase in ENaC Po (16). Also consistent with this notion, levels of expression of alpha -, beta -, and gamma -xENaC are, at best, only modestly altered by aldosterone in A6 epithelia (Middleton P, Al-Khalili O, Yue G, Zuckerman J, Kleyman TR, and Eaton DC, unpublished observations). A6 epithelia express the kinase sgk in a dexamethasone-regulated manner; however, a time course of sgk protein expression in response to dexamethasone suggests that sgk expression increases early (0.5 to 6 h) but falls after 24 h (6). Given these observations, we were not surprised that the plasma membrane pool of alpha -xENaC was essentially unchanged following a 24-h treatment with aldosterone. Weisz and co-workers (45) also observed that cell surface expression of alpha -xENaC was unchanged after a 3- or 18-h exposure to aldosterone. Cell surface expression of gamma -xENaC was unchanged as well in response to aldosterone; however, surface expression of beta -xENaC increased by ~60% after an 18-h exposure to aldosterone, raising the possibility that aldosterone might alter ENaC subunit stoichiometry. ENaCs are heterotetrameric channels (10, 19). Assuming that alpha -, beta -, and gamma -channels as well as alpha beta - or alpha gamma -channels have an alpha -subunit stoichiometry of two, our results and the observations reported by Weisz et al. (45) suggest that exposure of A6 cells to aldosterone for up to 24 h does not alter the number of channels expressed at the plasma membrane.

In summary, mature alpha -xENaC exists at the apical surface of A6 cells as a 180-kDa polypeptide. The half-life of mature alpha -xENaC was found to be 24 to 30 h, suggesting that once alpha -xENaC is expressed at the plasma membrane, its turnover rate is similar to that reported for mature CFTR. No significant change in apical surface expression of alpha -xENaC was observed after a 24-h exposure to aldosterone, despite a 5.3-fold increase in Isc. These data support the notion that the increase in Isc in A6 epithelial cells in response to aldosterone is mediated via regulatory pathways that alter alpha -xENaC Po (3, 37). The increases in apical membrane expression of ENaC in rat and mouse cortical collecting ducts observed in response to long-term dietary Na+ deprivation, and the increases in surface expression of ENaC in response to the kinase sgk, suggest that aldosterone-mediated regulation of ENaC involves regulation of both the surface pool of channels as well as channel Po. The predominant effect likely depends on the cell type as well as the duration of exposure to aldosterone.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-51391 (to T. R. Kleyman), DK-50268 (to D. C. Eaton), and DK-56596 (to P. R. Smith). J. B. Zuckerman and B. Hu were supported by postdoctoral fellowships from the Cystic Fibrosis Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: T. R. Kleyman, Renal-Electrolyte Div., A919 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (E-mail: kleyman{at}pitt.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 22 January 2001; accepted in final form 23 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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