The Activity of the Epithelial Sodium Channels Is Regulated by Caveolin-1 via a Nedd4-2-dependent Mechanism
2009; Elsevier BV; Volume: 284; Issue: 19 Linguagem: Inglês
10.1074/jbc.m809737200
ISSN1083-351X
AutoresIl‐Ha Lee, Craig Campbell, Sung‐Hee Song, Margot L. Day, Sharad Kumar, David I. Cook, Anuwat Dinudom,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoIt has recently been shown that the epithelial Na+ channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the β- and γ-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel. It has recently been shown that the epithelial Na+ channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the β- and γ-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel. Amiloride-sensitive epithelial Na+ channels (ENaC) 3The abbreviations used are: ENaC, epithelial Na+ channel; Cav-1, caveolin-1; MβCD, methyl-β-cyclodextrin; E3, ubiquitin-protein isopeptide ligase; FRT, Fischer rat thyroid; siRNA, small interfering RNA. are membrane proteins that are expressed in salt-absorptive epithelia, including the distal collecting tubules of the kidney, the mucosa of the distal colon, the respiratory epithelium, and the excretory ducts of sweat and salivary glands (1Duc C. Farman N. Canessa C.M. Bonvalet J.P. Rossier B.C. J. 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The physiological role of lipid rafts in the regulation of ENaC has been the subject of many recent investigations. Most of these studies used a pharmacological agent, methyl-β-cyclodextrin (MβCD), to promote redistribution of proteins away from the cholesterol-enriched membrane domains. The results were, however, inconclusive. In some studies, MβCD treatment was found to inhibit open probability (42Balut C. Steels P. Radu M. Ameloot M. Driessche W.V. Jans D. Am. J. Physiol. 2006; 290: C87-C94Crossref PubMed Scopus (37) Google Scholar) or cell surface expression of ENaC (35Hill W.G. Butterworth M.B. Wang H. Edinger R.S. Lebowitz J. Peters K.W. Frizzell R.A. Johnson J.P. J. Biol. Chem. 2007; 282: 37402-37411Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), whereas others found no direct effect of MβCD on the channel (33Shlyonsky V.G. Mies F. Sariban-Sohraby S. Am. J. Physiol. 2003; 284: F182-F188Crossref PubMed Scopus (36) Google Scholar, 43West A. Blazer-Yost B. Cell. Physiol. Biochem. 2005; 16: 263-270Crossref PubMed Scopus (21) Google Scholar). Despite a number of studies into the role of lipid rafts on the regulation of ENaC, little is known about the physiological relevance of caveolins to the function of this ion channel. In the present study, we use gene interference and gene expression techniques to determine the role of Cav-1 in the regulation of ENaC activity. We provide evidence of the association of Cav-1 with ENaC and evidence that Cav-1 negatively regulates both activity and abundance of ENaC at the surface of epithelial cells. Importantly, we demonstrate, for the first time, that the mechanism by which Cav-1 regulates activity of ENaC involves the E3 ubiquitin protein ligase, Nedd4-2. DNA Constructs—Mouse α-, β-, and γ-ENaC (in pBluescript) were a gift from Thomas R. Kleyman (University of Pittsburgh, Pittsburgh, PA). ENaC subclones with C-terminal FLAG tags were provided by Angeles Sanchez-Perez (University of Sydney, Sydney, Australia). Cav-1 fused with monomeric red protein (mRed) in pcDNA3.1 was obtained from Richard E. Pagano (Mayo Clinic and Foundation, Rochester, MN), Cav-1 S80E was obtained from Michael B. Robinson (University of Pennsylvania, Philadelphia, PA). Wild-type Nedd4-2 in pcDNA3.1 was generated as described previously (11Fotia A.B. Dinudom A. Shearwin K.E. Koch J.P. Korbmacher C. Cook D.I. Kumar S. FASEB J. 2003; 17: 70-72Crossref PubMed Scopus (88) Google Scholar). Cell Culture and Transfection and Reagents—Fischer rat thyroid (FRT) cells were a gift from Lucio Nitsch (University of Naples, Naples, Italy), and M1 mouse collecting duct cells, originally generated by Stoos et al. (44Stoos B.A. Naray-Fejes-Toth A. Carretero O.A. Ito S. Fejes-Toth G. Kidney Int. 1991; 39: 1168-1175Abstract Full Text PDF PubMed Scopus (164) Google Scholar), were a gift from Christoph Korbmacher (Universität Erlangen, Nürnberg, Germany). Both cell types were cultured in Dulbecco's modified Eagle's medium/F-12 medium with 100 units/ml penicillin and 100 μg/ml streptomycin at 37 °C. The medium for FRT cells contained 5% fetal bovine serum, whereas the medium for M1 cells contained 10% fetal bovine serum and 100 nm dexamethasone. The cells were seeded onto permeable filter supports (Millicell PCF, 0.4-μm pore size; Millipore). One day after seeding, FRT cells were cotransfected with cDNA of α-, β-, and γ-mENaC-FLAG (0.7 μg/ml each). When appropriate, FRT and M1 cells were transfected with cDNA of mRED-tagged Cav-1 (3 μg/ml), siRNA against Nedd4-2 (0.5 μg/ml) or siRNA against Cav-1 (0.5 μg/ml). In short, cDNA or siRNA were mixed with Lipofectamine 2000 (Invitrogen) in Opti-MEM reduced serum medium (Invitrogen) and incubated for 20 min at room temperature before being transferred to the apical side of the monolayer and further incubated for 4 h at 37 °C. The transfection medium was then replaced with Dulbecco's modified Eagle's medium/F-12 medium containing fetal bovine serum and antibiotics. In addition, the medium also contained dexamethasone (100 nm) for M1 cells or amiloride (10 μm) for FRT cells. All of the siRNAs were obtained from Qiagen, and all of the reagents were obtained from Sigma-Aldrich unless otherwise specified. Water-soluble cholesterol for the cholesterol replenishment study was from Sigma (catalogue number C4951). Immunoblotting—Two days after transfection, the cells were washed twice with phosphate-buffered saline before treatment with a lysis buffer containing 50 mm Tris-HCl, 150 mm NaCl, 10 mm EDTA with 10% glycerol and 1% Triton X-100 plus Complete protease inhibitor mixture (Roche Applied Science). After the protein concentration of each lysate was determined, an equal amount of protein lysate was loaded onto a 12% SDS-polyacrylamide gel. Following electrophoresis, the protein was transferred to a nitrocellulose membrane and incubated overnight with anti-caveolin-1 or anti-β-actin monoclonal antibody (Santa Cruz Biotechnology) or anti-FLAG M2 monoclonal antibody. The blots were washed to remove unbound antibodies before incubating with a horseradish peroxidase-conjugated secondary antibody. The blots were then washed with a Tris-buffered saline buffer containing 0.1% Tween 20. The proteins of interest were then visualized using an ECL™ Western blotting kit (GE Healthcare) and quantitated by densitometric analysis with ImageJ software (National Institutes of Health). The data are representative of at least three experiments. Immunoprecipitation—The lysates obtained from 1–2 × 107 cells were mixed with anti-FLAG antibodies (2 μg/ml) for 4 h or overnight and then incubated with 40 μl of protein A/G-agarose (Santa Cruz Biotechnology) for 4 h at 4 °C. Immune complexes were washed five times with lysis buffer containing 50 mm Tris-HCl, 150 mm NaCl, 10 mm EDTA, 10% glycerol, and 1% Triton X-100 plus Complete protease inhibitor mixture (Roche Applied Science). After boiling in SDS-PAGE sample buffer, the samples were subjected to SDS-PAGE and electro-transferred to nitrocellulose membranes. The membranes were immunoblotted with the indicated primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. The bands were visualized by chemiluminescence. Quantitation of Amiloride-sensitive Na+ Current—After the monolayer became confluent, normally within 2–3 days following transfection, the Millicell PCF insert was transferred to a modified Ussing chamber. The apical and basolateral surfaces of the monolayer were simultaneously perfused with a solution containing 130 mm NaCl, 1 mm CaCl2, 1 mm KCl, 1 mm MgCl2, 5 mm glucose, 10 mm HEPES, pH 7.4, maintained at 37 °C. The experiments were carried out under open circuit conditions (45Kunzelmann K. Beesley A.H. King N.J. Karupiah G. Young J.A. Cook D.I. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10282-10287Crossref PubMed Scopus (112) Google Scholar). Transepithelial resistance was measured by applying short (1 s) repetitive 10-μA current pulses across the epithelium. The transepithelial potential differences (Vte) were measured with reference to the luminal side of the epithelium, and the equivalent short circuit current was calculated according to Ohm's law. Amiloride-sensitive equivalent short circuit current (Iami) was determined as the change in current following the addition of amiloride (10 μm) to the apical bathing solution. The data were normalized by dividing the amiloride-sensitive short circuit current by that observed in control cells transfected with ENaC and studied on the same day. This ratio was reported as relative amiloride-sensitive equivalent short circuit current (Iami (relative)). The data for each experiment were obtained from at least three different batches of cells and are reported as the means ± S.E. with the number of experiments in parentheses. Statistical significance was assessed using Student's t test. Quantitation of Na+/K+ ATPase Activity—M1 cell monolayers were mounted in a modified Ussing chamber bathed symmetrically with the physiological solution. The chamber was connected to a VCC MC8 multichannel voltage/current clamp amplifier (Physiologic Instruments, San Diego, CA) together with the Acquire & Analyze data acquisition system (V2.3.177; Physiologic Instruments) and used to current clamp the monolayers at zero current (open circuit), monitor the transepithelial voltage with reference to the basolateral side of the epithelium, and inject intermittent current pulses to determine the transepithelial resistance. The equivalent short circuit current was calculated using Ohm's law and plotted by the software. Activity of the Na+/K+-ATPase was determined as previously described (46Ramminger S.J. Baines D.L. Olver R.E. Wilson S.M. J. Physiol. 2000; 524: 539-547Crossref PubMed Scopus (38) Google Scholar). In short, amiloride (10 μm) was added to the apical bathing solution to inhibit activity of ENaC. Nystatin (360 μg/ml) was then added to the apical solution to permeablize this membrane and to eradicate any rate limitation associated with the apical Na+ entry. Isc was allowed to stabilize for 30 min before the addition of 1 mm ouabain, an inhibitor of the Na+/K+-ATPase, to the basolateral solution. The change in short circuit current (Isc) following the addition of ouabain was used to estimate the activity of the Na+/K+-ATPase. Surface Expression of ENaC—FRT cells were transfected with FLAG-tagged α-, β-, and γ-mENaC. Two days after transfection, the cells were washed three times with ice-cold phosphate-buffered saline and then incubated for 30 min in 5 ml of cell-impermeant Sulfo-NHS-SS-Biotin solution (0.5 mg/ml; Pierce) at 4 °C. The reaction was stopped by quenching with Tris-buffered saline. The cells were solubilized in lysis buffer, and the lysate was centrifuged at 14,000 rpm for 5 min at 4 °C. The supernatant was collected and mixed with 250 μl of NeutrAvidin™ gel slurry (Pierce) before incubating with gentle rocking for 60 min at room temperature. After incubation, the sample was centrifuged at 1,000 rpm for 2 min. The precipitant, containing the biotinylated proteins, was washed five times with lysis buffer. Finally, the biotinylated proteins were eluted by the addition of SDS sample buffer and analyzed by Western blot using anti-FLAG M2 monoclonal antibody. Caveolin-1 Down-regulates Activity of ENaC—To determine the physiological role of Cav-1 in the regulation of ENaC activity and membrane surface expression, we first investigated whether interfering with Cav-1 expression could alter the activity of endogeneous ENaC in M1 cells. Immunoblot analysis revealed that transfecting the M1 cells with an siRNA directed against Cav-1 substantially suppressed endogenous Cav-1 protein level (Fig. 1A). The effect of Cav-1 knockdown on the activity of ENaC was then determined in Ussing chamber experiments (Fig. 1, B and C). We found that the normalized amiloride-sensitive Na+ current in Cav-1 siRNA transfected cells (1.92 ± 0.26, n = 6) is significantly higher than that of the nontransfected cells (1.00 ± 0.08, n = 7; p < 0.01). The absence of any detectable effect of the scrambled siRNA on either the level of endogenous expression of Cav-1 protein or on the amiloride-sensitive Na+ current (1.11 ± 0.09, n = 5; p > 0.05) indicates that increased activity of ENaC in Cav-1 knockdown cells is not attributable to a nonspecific effect of the siRNA used and, hence, that the basal activity of Cav-1 may be physiologically a negative regulator of ENaC. To confirm this finding, we inhibited Cav-1 activity in M1 cells by overexpressing Cav-1 S80E (Fig. 1D), a known dominant-negative variant that mimics the phosphorylated form of Cav-1 (47Schlegel A. Arvan P. Lisanti M.P. J. Biol. Chem. 2001; 276: 4398-4408Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) and that has a profound inhibitory effect on the function of Cav-1 (48Trouet D. Hermans D. Droogmans G. Nilius B. Eggermont J. Biochem. Biophys. Res. Commun. 2001; 284: 461-465Crossref PubMed Scopus (55) Google Scholar, 49Gonzalez M.I. Krizman-Genda E. Robinson M.B. J. Biol. Chem. 2007; 282: 29855-29865Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Expression of Cav-1 S80E increases activity of ENaC (Fig. 1D) by 66 ± 0.62% (n = 6, p < 0.001). On the other hand, overexpression of mRed-tagged wild-type Cav-1 significantly inhibited activity of ENaC (Fig. 1, B and C). It has been previously reported that interfering with basolateral cholesterol content alters Na+ transport in A6 amphibian kidney cells (43West A. Blazer-Yost B. Cell. Physiol. Biochem. 2005; 16: 263-270Crossref PubMed Scopus (21) Google Scholar), suggesting that lipid rafts may indirectly regulate Na+ transport via ENaC by modulating the activity of the basolateral Na+/K+-ATPase. In addition, Cav-1 has been shown to play an important role in regulating the internalization of the Na+/K+-ATPase in renal epithelia (50Liu J. Liang M. Liu L. Malhotra D. Xie Z. Shapiro J.I. Kidney Int. 2005; 67: 1844-1854Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 51Liu J. Shapiro J.I. Pathophysiology. 2007; 14: 171-181Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To eliminate the possibility that modifying expression of Cav-1 might alter activity of the basolateral Na+/K+-ATPase, which would, in turn, affect the electrochemical driving force for apical influx of Na+ via ENaC, we examined the effect of Cav-1 overexpression on the activity of the Na+/K+-ATPase in M1 cells (Fig. 2A) using the method described under "Experimental Procedures." As shown in Fig. 2B, the normalized ouabain-sensitive current, which represents activity of the Na+/K+-ATPase, in cells transfected with Cav-1 (0.95 ± 0.02, n = 8) is not significantly different from that of the control cells (1.0 ± 0.05, n = 9; p > 0.05), suggesting that the activity of Cav-1 does not significantly influence the activity of the basolateral Na+/K+-ATPase. Cav-1 Regulates Activity of ENaC in FRT Cells—Cav-1 has been shown to interact with and regulate the function and trafficking of an array of membrane proteins (52Hommelgaard A.M. Roepstorff K. Vilhardt F. Torgersen M.L. 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Cav-1 and ENaC Are Associated—To determine the relationship between Cav-1 and ENaC, FRT cells were cotransfected with Cav-1-mRed and FLAG-tagged ENaC. Cav-1 in lysates from the cotransfected cells was then immunoprecipitated, and the product was probed with an anti-FLAG antibody (Fig. 4A, upper panel). In a complementary study, ENaC in the cell lysates was immunoprecipitated with anti-FLAG antibody, and the precipitant was probed with an antibody specific to Cav-1 (Fig. 4A, lower panel). The presence of FLAG-tagged protein in the precipitant pulled down by antibody against Cav-1 (Fig. 4A, upper panel) and Cav-1 in the precipitant pulled down by anti-FLAG antibody (Fig. 4A, lower panel) suggests that Cav-1 and ENaC are physically associ
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