The Carboxyl Terminus of the α-Subunit of the Amiloride-sensitive Epithelial Sodium Channel Binds to F-actin
2005; Elsevier BV; Volume: 281; Issue: 10 Linguagem: Inglês
10.1074/jbc.m509386200
ISSN1083-351X
AutoresChristopher Mazzochi, James K. Bubien, Peter R. Smith, Dale Benos,
Tópico(s)Electrolyte and hormonal disorders
ResumoThe activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between α-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of α-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of α-, β-, and γ-ENaC revealed co-localization of α-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with α-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of α-ENaC. A direct interaction between F-actin and the COOH terminus of α-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC. The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between α-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of α-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of α-, β-, and γ-ENaC revealed co-localization of α-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with α-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of α-ENaC. A direct interaction between F-actin and the COOH terminus of α-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC. The amiloride-sensitive epithelial sodium channel (ENaC) 2The abbreviations used are: ENaC, epithelial sodium channel; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; WGA, wheat germ agglutinin; PVDF, polyvinylidene difluoride; GST, glutathione S-transferase; mAb, monoclonal antibody.2The abbreviations used are: ENaC, epithelial sodium channel; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; WGA, wheat germ agglutinin; PVDF, polyvinylidene difluoride; GST, glutathione S-transferase; mAb, monoclonal antibody. is a member of the degenerin/epithelial sodium channel superfamily of ion channels. ENaC is expressed at the apical surface of polarized epithelia and is in part responsible for maintaining proper salt and water homeostasis in the body. A great deal of information is known about the biophysical properties of ENaC once it is inserted into the apical surface of an epithelial cell plasma membrane. However, less is known about the proteins that interact with ENaC. Data from the literature indicate an interaction between ENaC and components of the apical membrane cytoskeleton. A partially purified ENaC complex from bovine renal epithelia copurifies with ankyrin, spectrin, and actin (1Smith P.R. Saccomani G. Joe E.H. Angelides K.J. Benos D.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6971-6975Crossref PubMed Scopus (148) Google Scholar), suggesting that these cytoskeletal proteins may be associated with ENaC. In addition, α-rENaC has been shown to bind to α-spectrin, and this is mediated through direct interaction between the α-spectrin Src homology 3 domain and the second proline-rich region in the COOH terminus of α-rENaC (2Rotin D. Bar-Sagi D. O'Brodovich H. Merilainen J. Lehto V.P. Canessa C.M. Rossier B.C. Downey G.P. EMBO J. 1994; 13: 4440-4450Crossref PubMed Scopus (217) Google Scholar). Electrophysiological data provide further support for an interaction between ENaC and the actin-based cytoskeleton. In cell-attached patches of A6 renal epithelial cells treated with the actin filament disrupter cytochalasin D, an induction of ENaC activity was observed (3Cantiello H.F. Stow J.L. Prat A.G. Ausiello D.A. Am. J. Physiol. 1991; 261: C882-C888Crossref PubMed Google Scholar), thereby suggesting that changes in the actin cytoskeleton affect the activity of ENaC. ENaC activation was also observed when short F-actin filaments were added to excised patches, and this effect was increased with the addition of cytochalasin D and/or ATP. These effects were reversed by the addition of the G-actin binding protein, DNase I. In planar lipid bilayers, short F-actin filaments were demonstrated to increase the open probability of rENaC (4Berdiev B.K. Prat A.G. Cantiello H.F. Ausiello D.A. Fuller C.M. Jovov B. Benos D.J. Ismailov I. J. Biol. Chem. 1996; 271: 17704-17710Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), whereas application of DNase I prevented the activation of rENaC. The application of gelsolin, a Ca2+-activated protein that severs actin filaments and caps the plus end of the actin filament, preventing the repolymerization of actin and keeping it in a gel-like state, was found to cause a sustained activation of rENaC. In addition, actin was required for the transient activation of rENaC by protein kinase A and ATP when ENaC was reconstituted into planar lipid bilayers. These data indicate that a direct interaction between actin and ENaC may underlie the regulation of ENaC by short actin filaments. Identification of regions involved in a direct protein interaction between actin and α-ENaC has only been deduced from biophysical methods. Current evidence suggests that actin interacts with the C-terminal domain of α-ENaC. A C-terminal truncation mutant (R613X) of α-rENaC was not responsive to the addition of actin. The single channel recordings of the αR613Xβγ-rENaC treated with actin were the same as wild type αβγ-rENaC that was not treated with actin (5Jovov B. Tousson A. Ji H.L. Keeton D. Shlyonsky V. Ripoll P.J. Fuller C.M. Benos D.J. J. Biol. Chem. 1999; 274: 37845-37854Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The deletion of 14 amino acids, Glu631-Phe644,in a chimeric rat-bovine α-ENaC (α-rbENaC), consisting of α-rENaC residues 1-615 and α-bENaC residues 570-650, nullified the effect of actin normally seen with the chimeric α-bENaC (6Copeland S.J. Berdiev B.K. Ji H.L. Lockhart J. Parker S. Fuller C.M. Benos D.J. Am. J. Physiol. 2001; 281: C231-C240Crossref PubMed Google Scholar). The deleted 14-amino acid sequence of α-bENaC has an 11-amino acid sequence identity to the same region of amino acids in the COOH terminus of α-rENaC. This high degree of sequence identity suggests that this amino acid sequence in α-rENaC may also participate in the regulation of ENaC by actin. However, to date, there is no definitive biochemical evidence for a direct interaction between F-actin and α-ENaC. In order to investigate the hypothesis that the actin cytoskeleton interacts directly with the carboxyl terminus of α-ENaC, we have used a combination of gel overlay and F-actin co-sedimentation assays to demonstrate binding of actin to the COOH terminus of α-ENaC. Actin was found to co-immunoprecipitate with α-ENaC from MDCK cell lysates, thereby providing in vivo data supporting an association between actin and ENaC. Moreover, co-localization of actin and α-ENaC in the apical membrane of MDCK cells stably expressing functional ENaC was demonstrated using laser-scanning confocal microscopy. These three independent lines of evidence support an interaction between actin and α-ENaC, which is mediated by the direct binding of actin to the carboxyl terminus of α-ENaC. Cell Culture—Stably transfected MDCK cells expressing the rat isoforms of αβγ-ENaC and MDCK parental cells were obtained as a kind gift from Drs. R. G. Morris and J. A. Schafer. The α-rENaC subunit was tagged with a single FLAG tag in the extracellular loop as described by Firsov et al. (7Firsov D. Schild L. Gautschi I. Merillat A.M. Schneeberger E. Rossier B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15370-15375Crossref PubMed Scopus (395) Google Scholar); both β- and γ-subunits were wild type. Cells were grown at 37 °C in a 5% CO2 humidified incubator. Cells were initially grown in T75 flasks in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone) and antibiotics (penicillin and streptomycin (1%), G418 (800 μg/ml), hygromycin (300 μg/ml), and puromycin (5 μg/ml)). Once the cells were at least 80% confluent, cells were seeded on poly-l-lysine-coated semipermeable supports (Transwell; 24-mm diameter, 0.4-μm pore size; Costar). Cells were used ∼5-14 days later when polarized monolayers were formed. MDCK cells used for Western blots and co-immunoprecipitations were plated in 100-mm plastic Petri dishes. In some experiments, in order to increase ENaC expression, the media were supplemented with 2 μm dexamethasone (Sigma) and 2 mm sodium butyrate (Fluka) 24 h before the cells were harvested. Electrophysiology—Whole cell clamp bath solution was RPMI culture medium. The pipette solution was 100 mm potassium gluconate, 30 mm KCl, 10 mm NaCl, 20 mm HEPES, 0.5 mm EGTA, free Ca2+ less than 10 nm, 4 mm ATP, at a pH of 7.2. The bath contained serum-free RPMI 1640 cell culture medium (133 mm Na+, 5.3 mm K+, 108.3 mm Cl-). These solutions approximate normal ionic gradients in situ. After formation of a gigaohm seal, the membrane within the seal was ruptured by an additional suction pulse. The whole-cell configuration was confirmed by an increase in the capacitance with no change in resistance. After the additional capacitance was balanced, the cells were held at -60 mV and clamped to membrane potentials ranging from -160 to +40 mV sequentially for 1 s, returning to the holding potential (-60 mV) for 1 s between each test voltage. Currents were recorded digitally using pClamp hardware and software (Axon Instruments, Sunnyvale, CA). During bath perfusion to change to amiloride-supplemented medium, the cells were held at -60 mV and pulsed sequentially to -120 and +60 mV for 0.5 s, returning to the holding potential between each test pulse (see Fig. 1C). This provided a continuous record and shows in real time the inhibition of inward current by amiloride. Single channel currents were recorded using the patch clamp technique. For cell-attached patches the pipette solution was RPMI culture medium, and for outside-out patches the pipette solution was 150 mm KCl. In all cases, the bath solution was RPMI culture medium. Data were recorded and analyzed using fetchex, fetchan, and pstat software (Axon Instruments, Sunnyvale, CA). Immunocytochemistry—MDCK cells were grown on poly-l-lysine-coated semipermeable supports as described above. Culture medium was aspirated from the monolayer, cells were rinsed twice with 1× phosphate-buffered saline (PBS) (137 mm NaCl, 27.7 mm KCl, 1.5 mm KH2PO4, 8 mm Na2HPO4), and monolayers were then fixed with 3% paraformaldehyde (prepared from 20% EM Grade solution; Electron Microscopy Services) for 15 min at 37 °C. Cells were rinsed three times with 1× PBS and then permeabilized using 1× PBS with 0.1% Triton X-100 (PBST) for 5 min at room temperature. The blocking step was done with 1× PBS plus 10% normal serum (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. The wheat germ agglutinin/Alexa594 conjugate (WGA594) (Molecular Probes, Inc., Eugene, OR) was used at a concentration of 5 μg/ml diluted in 1× PBS and incubated with the samples for 10 min at room temperature. The anti-FLAG M2 antibody (Stratagene) was diluted 1:100 with 1× PBST from a stock concentration of 2 mg/ml with 1× PBST and incubated with the samples for 1 h at room temperature. Specificity of the anti-FLAG M2 antibody was demonstrated using the FLAG peptide (Sigma). The antibody was preabsorbed with the FLAG peptide, 10 μg of peptide, 1 μg of anti-FLAG M2 antibody for a minimum of 15 h prior to use. All phalloidin conjugates (Molecular Probes) were diluted 1:100 with 1× PBST from a stock concentration of ∼6.6 μm and incubated with the samples for 1 h at room temperature. Monolayers were then washed with 1× PBS, three times for 10 min at room temperature. Secondary antibody conjugated to Alexa488 or Alexa594 (Molecular Probes) was diluted 1:100 with 1× PBST from a stock of 2 mg/ml and incubated with sample for 1 h at room temperature. After application of primary/probe and secondary antibody, samples were washed in 1× PBS three times for 10 min each at room temperature. Counterstaining was performed using Hoechst 33258 (20 μg/mlin1× PBS) for 3-5 min at room temperature. Cells were rinsed once with 1× PBS. Monolayers were mounted with 1% para-phenylenediamine in 1:9 (v/v) 1× PBS/glycerol and coverslipped. Images were viewed using laser-scanning confocal microscopy (Leica DM IRBE microscope, mounted with a Leica TCS SP scanhead). The Leica DM IRBE microscope was equipped with oil, PlanApochromat 40×, 63×, and 100× objectives. The 100× objective with a numerical aperture of 1.4 was used to acquire the final images. Visualization of blue fluorophores (Hoechst 33258) was achieved by using a dedicated UV laser (Coherent) for excitation at 350 nm. Green fluorophore (Alexa488) excitation at 488 nm was achieved by using an argon laser (Leica). Red fluorophore (Alexa 594) excitation at 568 nm was achieved by using a krypton laser (Leica). Energy emission in the form of light by blue fluorophores (380-494 nm), green fluorophores (500-575 nm), and red fluorophores (596-722 nm) was detected using three independent photomultiplier tubes. Color channels for the final double label images were captured sequentially and then merged using the Leica TCS NT software. Adobe Photoshop version 7.0 was used for image processing. All confocal microscopy was done at the University of Alabama High Resolution Imaging Facility and the Veterans Affairs Hospital (Birmingham, AL). SDS-PAGE, Immunoprecipitations/Co-immunoprecipitations, and Immunoblotting—Whole cell lysates prepared from MDCK cells stably expressing ENaC and MDCK parental cells were used. Cells were grown in three 100-mm plastic Petri dishes until confluent. Cells were washed twice with 2 ml of cold 1× PBS. Petri dishes were placed on ice for 10 min with 1 ml each of 1× lysis buffer (50 mm Tris, pH 7.4, 150 mm NaCl, and 1% Triton X-100) supplemented with Complete protease inhibitor mixture (Roche Molecular Biochemicals). Cells were scraped from the Petri dishes and placed into microcentrifuge tubes on ice. Lysates were sheared a minimum of three times with a 22-gauge needle, and incubated on ice for 1 h. Sheared lysates were then spun at 15,800 × g at 4 °C for 5 min. The supernatant was removed, and a BCA protein assay (Pierce) was performed to quantify the amount of total protein in the samples. A maximum of 200 μg of whole cell lysate diluted in 1× PBS was used per immunoprecipitation reaction and incubated overnight at 4 °C on a rotator with 3 μg of anti-FLAG mAb (Stratagene), 40 μl of a 50% slurry of protein G-agarose beads (Roche Applied Science) and Complete protease inhibitor mixture (final volume of 500 μl). After overnight incubation, beads were centrifuged for 2 min at 15,800 × g. The supernatant was aspirated, and beads were washed and pelleted three times (2 min at 15,800 × g) in 1× lysis buffer supplemented with Complete protease inhibitor mixture. Samples were diluted 1:1 (v/v) with Laemmli sample buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromphenol blue, 5% β-mercaptoethanol), heated at 95 °C for 5 min, and separated by SDS-PAGE with constant voltage at room temperature. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The Western transfer was done in running buffer (25 mm Tris base, pH 8.0, 192 mm glycine, 1% (w/v) SDS, 20% (v/v) methanol) at 4 °C for 1 h at constant voltage. Blots were either blocked in 1× TBST (10 mm Tris base, pH 8.0, 150 mm NaCl, 0.1% Tween 20) with 5% nonfat dry milk overnight at 4 °C or for 1 h at room temperature. Following immunoprecipitation with the mAb anti-FLAG antibody, Western blot detection of α-rENaC was performed using anti-FLAG mAb (1:500 dilution) or a rabbit polyclonal anti-α-ENaC antibody (2 μg/ml final concentration) (Affinity BioReagents). Specificity of the anti-α-ENaC antibody was demonstrated using its immunizing peptide (Affinity BioReagents). The antibody was preabsorbed with its immunizing peptide 2 μg peptide/1 μg of α-ENaC antibody for a minimum of 15 h prior to use. Primary antibodies were diluted with 1× TBST, 1% nonfat dry milk and incubated with blots for 1 h at room temperature. Blots were washed a minimum of 3 times with 1× TBST, 10 min each at room temperature. Secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories) were used at dilutions of 1:10,000 or 1:20,000 in 1× TBST and incubated for 1 h at room temperature. Blots were washed a minimum of three times with 1× TBST, 10 min each at room temperature. Immunoreactivity was visualized using enhanced chemiluminescence (Super Signal West Pico; Pierce) and imaged onto Eastman Kodak Co. X-OMAT AR film (Fisher). Controls for immunoprecipitations consisted of ChromPure normal IgG serum (Jackson ImmunoResearch Laboratories) added in a concentration equivalent to the primary antibody. Co-immunoprecipitation of α-rENaC with actin was performed as above with the following exceptions: 1) the lysis buffer used contained 50 mm Tris base, 15 mm Na2HPO4, 150 mm NaCl, 0.5% Triton X-100, 0.5% deoxycholate, pH 7.41; 2) following the BCA assay, the 200 μg of supernatant was precleared with 50 μl of protein A-agarose beads and 50 μl of protein G-agarose beads for 2 h at 4°C; 3) after preclearing, the samples were spun, and the anti-FLAG antibody alone was added for overnight incubation at 4 °C; 4) the α-rENaC/FLAG complex was captured by the addition of 40 μl of protein G-agarose and incubated for 2 h at 4°C;5) the beads were washed three times with the lysis buffer plus 500 mm KCl, pH 9.1; 6) the co-immunoprecipitation control consisted of an anti-His antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) added in a concentration equivalent to that of the primary anti-FLAG antibody. The monoclonal anti-actin antibody (Chemicon) was used at a 1:1,500 dilution for 1 h at room temperature. Immunoblotting and primary antibody detection for the co-immunoprecipitations was done exactly as described above. Construction of GST-ENaC Fusion Constructs—PCR amplification of the γ-rENaC COOH terminus (amino acids Ala563-Ile650, bp 1,785-2,051), the α-rENaC NH2 terminus (amino acids Met1-Asn90, bp 82-351), and the α-rENaC COOH terminus (amino acids Arg613-Leu698, bp 1,918-2,178) consisted of using full-length α- and γ-rENaC cDNAs (a kind gift of Dr. B. Rossier, Lausanne, Switzerland). The PCR (50-μl total volume) consisted of 5 μlof10× buffer, 0.8 μl of Mg2+ (1.5 mm), 1 μl of dNTP (25 mm/nucleotide), 1 μl of primer (15 pmol/primer), 100 ng of DNA template, and 0.5 μl of native Pfu polymerase (Stratagene). PCR products were analyzed on 2% agarose gels (NuSeive 3:1 agarose) for gel purification (Qiagen). After purification, the insert was subcloned into the pCR4.0 TOPO-TA vector (Invitrogen) and transformed into chemically competent TOP10 Escherichia coli (Invitrogen). Transformed bacteria were grown overnight at 37 °C on LB/ampicillin plates. Positive clones were screened by restriction enzyme digestion and grown up overnight again, and the plasmid DNA was isolated (Promega). Following confirmation by sequencing, the amplified cDNA was excised from the pCR4.0 vector and ligated into linearized pGEX 5X-1 GST expression vector (AP Biotech). The ligation products were then transformed into the JM109 maintenance strain (Promega), plated overnight, and screened for positive clones by restriction enzyme digestion. DNA from positive clones was isolated (Qiagen), sequenced, and then transformed into E. coli BL21-CodonPlus-RP (Stratagene) cells for the production of GST fusion proteins. Generation and Purification of GST Fusion Proteins—GST-α-hENaC C-terminal fusion protein (a generous gift of Dr. F. J. McDonald, Dunedin, New Zealand), GST-γ-rENaC C-terminal, GST-α-rENaC NH2-terminal, and GST were produced in E. coli BL21-CodonPlus-RP (Stratagene). Cells were induced with 0.1 mm isopropyl-β-d-thiogalactopyranoside for 3 h at 37 °C. Bacterial cultures were spun at 10,000 × g for 10 min at 4 °C. Bacterial pellets were snap frozen with liquid N2 and stored overnight at -80 °C. Pellets were lysed with BugBuster (Novagen) in the presence of benzonase (Novagen) and Complete protease inhibitor mixture (Roche Molecular Biochemicals) for 20 min at room temperature or at 4 °C for 45 min and then spun at 16,000 × g for 20 min at 4 °C. Supernatant was poured over a glutathione-Sepharose 4B (AP Biotech) gravity flow column five times. Bound GST fusion protein or GST was eluted with 10 mm reduced glutathione (Sigma) in three 2-ml fractions. The purified fractions were assayed by SDS-PAGE, and the gel was stained with Gel Code (Pierce). The fractions were then pooled, concentrated, and dialyzed overnight at 4 °C in 1× PBS with three buffer changes. Labeling and Polymerization of Nonmuscle Actin—Nonmuscle actin (Cytoskeleton) was labeled with Alexa Fluor 488 carboxylic acid succinimidyl ester (Molecular Probes) according to the manufacturer. The polymerization of the nonmuscle actin was done as previously described by Hitt et al. (8Hitt A.L. Laing S.D. Olson S. Anal. Biochem. 2002; 310: 67-71Crossref PubMed Scopus (6) Google Scholar). Gel Overlays—Samples were heated at 95 °C for 5 min and separated using 12% Tris/glycine gels with constant voltage at room temperature. Proteins were transferred onto PVDF membrane in running buffer (25 mm Tris base, pH 8.0, 192 mm glycine, 20% (v/v) methanol) at 4 °C for 1 h at constant voltage. Gel overlays were blocked in 1× TBST, 5% nonfat dry milk overnight at 4 °C. The blots were probed with 25 μg/ml F-actin/Alexa488 in 1× TBST, 5% nonfat dry milk for 2 h at room temperature, washed briefly in 1× TBST, allowed to air-dry, and scanned using a FujiFilm FLA-5100 Scanner (FujiFilm). Images were processed using Adobe Photoshop 7.0. Nonmuscle F-Actin Co-sedimentation—Nonmuscle F-actin co-sedimentation experiments were done according to the manufacturer's instructions (Cytoskeleton). All samples were incubated at room temperature for 30 min. Briefly, 40 μl of a 23 μm nonmuscle F-actin stock was incubated with 10 μl of GST-α-hENaC C-terminal fusion protein (0.4 μg of full-length fusion protein). Negative controls consisted of 40 μl of F-actin buffer incubated with 10 μl of GST-α-hENaC C-terminal fusion protein (0.4 μg of full-length fusion protein) and an equal amount of GST protein that was used for the GST-α-hENaC C-terminal fusion protein sample. Positive controls consisted of 1 μl of α-actinin (2.5 μg), 40 μl of nonmuscle F-actin, 8 μl of F-actin buffer, and 1 μl of Tris-HCl. Following the 30-min incubation, samples were subjected to ultracentrifugation for 1.5 h, 150,000 × g at 24 °C. Supernatant was removed, and pellets were resuspended in 50 μl of Laemmli sample buffer and heated at 95 °C for 5 min. Equal volumes of supernatant and pellet were loaded into the wells, separated using 12% Tris/glycine gels with constant voltage at room temperature. Gels were stained using GelCode (Pierce). Determination of Functional ENaC—Wild type MDCK cells do not endogenously express amiloride-sensitive epithelial sodium channel (9Ishikawa T. Marunaka Y. Rotin D. J. Gen. Physiol. 1998; 111: 825-846Crossref PubMed Scopus (118) Google Scholar, 10Morris R.G. Schafer J.A. J. Gen. Physiol. 2002; 120: 71-85Crossref PubMed Scopus (89) Google Scholar). MDCK cells stably transfected with rat αβγ-ENaC were examined by whole-cell patch clamp. Three stably transfected MDCK cells (induced to increase expression of ENaC) were whole-cell-clamped. Each cell had inward sodium current that was inhibited by 2 μm amiloride, thereby confirming the presence of ENaC in the plasma membrane. Fig. 1 (A and B) shows a representative example of whole-cell currents before and after treatment with amiloride. Fig. 1C shows the amiloride block in a real time pulse record. Fig. 1D is the summary of the amiloride-blockable current-voltage data. The amiloride-sensitive current reversed at a positive membrane potential, indicating sodium selectivity. Fig. 1B shows the slight reduction in outward current after amiloride treatment. This may be due to inhibition of outward potassium flux through ENaC. The result is a shift to the left in the I-V relation. In the cell-attached configuration, small single channel currents with relatively long open times were observed (Fig. 1E). Moreover, cAMP characteristically increased channel activity (NPo 0.5 versus 1.5). The single channel conductance was 8 picosiemens, which is typical of human ENaC channels (11Bubien J.K. Watson B. Khan M.A. Langloh A.L. Fuller C.M. Berdiev B. Tousson A. Benos D.J. J. Biol. Chem. 2001; 276: 8557-8566Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). To confirm independently the identity of the single channels, outside-out patches were formed, and amiloride (100 nm) was added to the bath solution (Fig. 1F). 100 nm is slightly above the IC50 (75 nm) for inhibition of ENaC. This concentration was chosen, because it would produce a partial inhibition of the channels, changing the long open times into rapidly transitioning channels due to the binding and unbinding of the inhibitor. Based on these observations (conductance and appropriate inhibition by amiloride), we conclude that the stably transfected MDCK cells expressed functional ENaC in their apical plasma membrane. Co-localization of α-ENaC with the Cortical F-actin Cytoskeleton—Initially, stably transfected MDCK cells expressing the α-rENaC subunit with a FLAG epitope tag on the extracellular loop and wild type β- and γ-rENaC subunits, were labeled with a monoclonal anti-FLAG epitope antibody to determine the membrane localization of α-ENaC. Fig. 2, A-E, show representative images of the pattern of expression for α-rENaC in this cell line. As these images illustrate, the level of α-rENaC expression varied between cells. Fig. 2E is a XZ reconstruction, illustrating that ENaC immunoreactivity was primarily situated in the apical domain of this cell line. In contrast to the anti-FLAG antibody, the peptide competition controls (Fig. 2, F and G) exhibited little or no detectable immunoreactivity at the surface of the monolayer (Fig. 2F) or within the cytoplasm (Fig. 2G). In order to determine where the apical anti-FLAG immunoreactivity was located relative to the apical plasma membrane, double labeling experiments were performed. Fig. 2H is an XZ reconstruction of a monolayer labeled with anti-FLAG antibody to detect α-rENaC. The monolayer was labeled with wheat germ agglutinin (WGA) conjugated to Alexa594 (WGA594) prior to fixation to delineate the apical plasma membrane (Fig. 2I). Fig. 2J is an overlay of H and I, which demonstrates co-localization of α-rENaC with WGA within the apical plasma membrane. To localize the cortical F-actin cytoskeleton with respect to the apical membrane in MDCK cells stably expressing ENaC, monolayers were labeled with WGA594 and the specific F-actin probe, phalloidin, conjugated to Alexa488. Fig. 3, A-D, are optical sections from a Z-stack of MDCK ENaC cells. As the optical sections were taken deeper into the cells, the fluorescence from the WGA594 largely disappeared, leaving only the basolateral labeling of the phalloidin/Alexa488 in the majority of the field of view. An XZ reconstruction (Fig. 3, E-G) revealed co-localization of actin and WGA in some of the cells, demonstrating that a population of F-actin is associated with the apical membrane. We then performed double labeling experiments to determine whether α-rENaC and F-actin are co-localized in this model system. The representative XZ reconstructions in Fig. 4, C and F, shows the overlay of the FLAG (Fig. 4, A and D) and phalloidin (Fig. 4, B and E) signals. When the two signals are merged, partial co-localization can be observed, indicating that both F-actin and α-rENaC segregate to the same region of the apical domain. Western Blotting of α-ENaC—In order to examine whether α-rENaC and actin are found in the same protein complex, we attempted to co-immunoprecipitate FLAG-tagged α-ENaC and actin from the MDCK cell line. To first demonstrate that we were able to immunoprecipitate specifically FLAG-tagged α-ENaC, we used the monoclonal anti-FLAG antibody; the α-subunit was immunoprecipitated using the anti-FLAG antibody, and the resulting immunoblot was then probed with the same primary antibody (Fig. 5A). As shown in lane 3, FLAG tagged α-ENaC was only detected by the anti-FLAG antibody in lysates immunoprecipitated with the anti-FLAG antibody. It was not detected in the beads only control (lane 1) or the nonimmune mouse IgG control (lane 2) or when blots were probed with the secondary antibody alone (lanes 4-6). To further demonstrate that the 100-kDa polypeptide immunoprecipitated by the anti-FLAG antibody was indeed α-rENaC, samples were immunoprecipitated with the anti-FLAG antibody and the blot was probed using a commercially available anti-α-rENaC antibody (Fig. 5B). In addition to the stably transfected MDCK ENaC cell lysate (designated E), lysates prepared from the original MDCK parental strain (designated P) were subjected to immunoprecipitation with the anti-FLAG antibody. The parental strain has pre
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