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• W1994304837 endingPage "9779" @default.
• W1994304837 startingPage "9772" @default.
• W1994304837 abstract "The apically located epithelial Na+ channel (αβγ-ENaC) plays a key role in the regulation of salt and fluid transport in the kidney and other epithelia, yet its mode of trafficking to the plasma membrane and its cell surface stability in mammalian cells are poorly understood. Because the expression of ENaC in native tissues/cells is very low, we generated epithelial Madin-Darby canine kidney (MDCK) cells stably expressing αβγ-ENaC, where each subunit is tagged differentially at the intracellular C terminus and the β-subunit is also Myc-tagged at the ectodomain (αHAβMyc,T7γFLAG). ENaC expression in these cells was verified by immunoblotting with antibodies to the tags, and patch clamp analysis has confirmed that the tagged channel is functional. Moreover, using electron microscopy, we demonstrated apical, but not basal, membrane localization of ENaC in these cells. The glycosylation pattern of the intracellular pool of ENaC revealed peptide N-glycosidase F and endoglycosidase H sensitivity. Surprisingly, the cell surface pool of ENaC, analyzed by surface biotinylation, was also core glycosylated and lacked detectable endoglycosidase H-resistant channels. Extraction of the channel from cells in Triton X-100 demonstrated that both intracellular and cell surface pools of ENaC are largely soluble. Moreover, floatation assays to analyze the presence of ENaC in lipid rafts showed that both intracellular and cell surface pools of this channel are not associated with rafts. We have shown previously that the total cellular pool of ENaC is turned over rapidly (t 1/2 ∼ 1–2 h). Using cycloheximide treatment and surface biotinylation we now demonstrate that the cell surface pool of ENaC has a similarly short half-life (t 1/2 ∼1 h), unlike the long half-life reported recently for the Xenopus A6 cells. Collectively, these results help elucidate key aspects of ENaC trafficking and turnover rates in mammalian kidney epithelial cells. The apically located epithelial Na+ channel (αβγ-ENaC) plays a key role in the regulation of salt and fluid transport in the kidney and other epithelia, yet its mode of trafficking to the plasma membrane and its cell surface stability in mammalian cells are poorly understood. Because the expression of ENaC in native tissues/cells is very low, we generated epithelial Madin-Darby canine kidney (MDCK) cells stably expressing αβγ-ENaC, where each subunit is tagged differentially at the intracellular C terminus and the β-subunit is also Myc-tagged at the ectodomain (αHAβMyc,T7γFLAG). ENaC expression in these cells was verified by immunoblotting with antibodies to the tags, and patch clamp analysis has confirmed that the tagged channel is functional. Moreover, using electron microscopy, we demonstrated apical, but not basal, membrane localization of ENaC in these cells. The glycosylation pattern of the intracellular pool of ENaC revealed peptide N-glycosidase F and endoglycosidase H sensitivity. Surprisingly, the cell surface pool of ENaC, analyzed by surface biotinylation, was also core glycosylated and lacked detectable endoglycosidase H-resistant channels. Extraction of the channel from cells in Triton X-100 demonstrated that both intracellular and cell surface pools of ENaC are largely soluble. Moreover, floatation assays to analyze the presence of ENaC in lipid rafts showed that both intracellular and cell surface pools of this channel are not associated with rafts. We have shown previously that the total cellular pool of ENaC is turned over rapidly (t 1/2 ∼ 1–2 h). Using cycloheximide treatment and surface biotinylation we now demonstrate that the cell surface pool of ENaC has a similarly short half-life (t 1/2 ∼1 h), unlike the long half-life reported recently for the Xenopus A6 cells. Collectively, these results help elucidate key aspects of ENaC trafficking and turnover rates in mammalian kidney epithelial cells. epithelial Na+ channel endoglycosidase H peptideN-glycosidase F Madin-Darby canine kidney cells hemagglutinin N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine phosphate-buffered saline transferrin receptor The amiloride-sensitive epithelial Na+ channel (ENaC)1 is an apically located channel expressed primarily in salt-transporting epithelia of the kidney (distal nephron), distal colon, lung, ducts of exocrine glands, and other organs (for review, see Ref. 1Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1036) Google Scholar). Its critical role in regulating salt and fluid transport is underscored by the findings that inactivating mutations in ENaC cause the salt-wasting nephropathy pseudohypoaldosteronism type I, and gain-of-function mutations cause Liddle syndrome, a hereditary form of hypertension (for review, see Ref. 2Lifton R.P. Gharavi A.G. Geller D.S. Cell. 2001; 104: 545-556Abstract Full Text Full Text PDF PubMed Scopus (1368) Google Scholar). Liddle syndrome is caused by mutations in one of the PY motifs (PPPxY) of ENaC, leading to increased channel numbers and opening at the plasma membrane (3Firsov D. Schild L. Gautschi I. Merillat A.M. Schneeberger E. Rossier B.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15370-15375Crossref PubMed Scopus (396) Google Scholar); the increase in channel numbers is believed to be caused by impaired binding to and suppression by the ubiquitin ligase Nedd4 (4Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (740) Google Scholar, 5Schild L. Lu Y. Gautschi I. Schneeberger E. Lifton R.P. Rossier B.C. EMBO J. 1996; 15: 2381-2387Crossref PubMed Scopus (361) Google Scholar, 6Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 7Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Invest. 1999; 103: 667-673Crossref PubMed Scopus (327) Google Scholar, 8Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (250) Google Scholar, 9Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar) and by impaired endocytosis of the channel (10Shimkets R.A. Lifton R.P. Canessa C.M. J. Biol. Chem. 1997; 272: 25537-25541Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). ENaC consists of three subunits, αβγ (11Canessa C.M. Horisberger J.D. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (827) Google Scholar, 12Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1775) Google Scholar), arranged in a stoichiometry of 2α:1β:1γ (13, 14; for another view, see Ref.15Snyder P.M. J. Biol. Chem. 1998; 273: 681-684Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Each ENaC subunit is comprised of two transmembrane domains, a large ectodomain flanking them and containing numerousN-linked glycosylation sites, and short intracellular N and C termini (16Snyder P.M. McDonald F.J. Stokes J.B. Welsh M.J. J. Biol. Chem. 1994; 269: 24379-24383Abstract Full Text PDF PubMed Google Scholar, 17Renard S. Lingueglia E. Voilley N. Lazdunski M. Barbry P. J. Biol. Chem. 1994; 269: 12981-12986Abstract Full Text PDF PubMed Google Scholar, 18Canessa C.M. Merillat A.M. Rossier B.C. Am. J. Physiol. 1994; 267: C1682-C1690Crossref PubMed Google Scholar). The N termini of α- and γ-ENaC contain conserved Lys residues that serve as ubiquitin acceptor sites (19Staub O. Gautschi I. Ishikawa T. Breitschopf K. Ciechanover A. Schild L. Rotin D. EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (598) Google Scholar), and the C termini of all three subunits contain the above mentioned PY motifs (4Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (740) Google Scholar, 5Schild L. Lu Y. Gautschi I. Schneeberger E. Lifton R.P. Rossier B.C. EMBO J. 1996; 15: 2381-2387Crossref PubMed Scopus (361) Google Scholar, 20Snyder P.M. Price M.P. McDonald F.J. Adams C.M. Volk K.A. Zeiher B.G. Stokes J.B. Welsh M.J. Cell. 1995; 83: 969-978Abstract Full Text PDF PubMed Scopus (398) Google Scholar). Although all three ENaC chains are glycosylated in cells, the role of this glycosylation is not clear, and indeed mutation of all N-linked glycosylation sites in α-ENaC does not seem to affect channel function (16Snyder P.M. McDonald F.J. Stokes J.B. Welsh M.J. J. Biol. Chem. 1994; 269: 24379-24383Abstract Full Text PDF PubMed Google Scholar, 18Canessa C.M. Merillat A.M. Rossier B.C. Am. J. Physiol. 1994; 267: C1682-C1690Crossref PubMed Google Scholar). In native tissues, ENaC is a rare protein with very low expression levels (21Loffing J. Pietri L. Aregger F. Bloch-Faure M. Ziegler U. Meneton P. Rossier B.C. Kaissling B. Am. J. Physiol. 2000; 279: F252-F258Crossref PubMed Google Scholar, 22Masilamani S. Kim G.H. Mitchell C. Wade J.B. Knepper M.A. J. Clin. Invest. 1999; 104: R19-R23Crossref PubMed Scopus (630) Google Scholar), permitting electrophysiological and limited immunofluoresence analyses to be performed but precluding biochemical studies on such low abundance proteins. Thus, several groups have investigated aspects of ENaC trafficking and protein stability in cell lines and heterologous expression systems, primarily inXenopus oocytes, Xenopus A6 cells, and mammalian fibroblasts transiently expressing the ENaC subunits (23–26; for review, see Ref. 27Rotin D. Kanelis V. Schild L. Am. J. Physiol. 2001; 281: F391-F399Crossref PubMed Google Scholar). ENaC trafficking in mammalian kidney (polarized) epithelial cells stably expressing αβγ-ENaC has not been reported, hence this was the focus of our studies. The ENaC chains appear to assemble together early on in the endoplasmic reticulum (28Adams C.M. Snyder P.M. Welsh M.J. J. Biol. Chem. 1997; 272: 27295-27300Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), but details of the route of trafficking to the cell surface are lacking. A major obstacle in following ENaC maturation biochemically has been the lack of an apparent endoglycosidase H (Endo H)-resistant pool, which typically marks mature transmembrane proteins that have acquired complex glycosylation at the medial Golgi compartment. This is unlike the ENaC relative Phe-Met-Arg-Phe-amide-activated Na channel (FaNaC), in which an Endo H-resistant pool is easily detectable during its maturation process (29Coscoy S. Lingueglia E. Lazdunski M. Barbry P. J. Biol. Chem. 1998; 273: 8317-8322Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Thus, an important issue to be resolved is whether ENaC does not acquire complex glycosylation, or whether it does so but the Endo H-resistant pool is below our detection limits. In addition, it has been suggested that in COS cells, the mature ENaC is stripped of its core glycosylation and becomes insoluble in non-ionic detergents upon arrival at the plasma membrane (23Prince L.S. Welsh M.J. Biochem. J. 1998; 336: 705-710Crossref PubMed Scopus (47) Google Scholar, 24Prince L.S. Welsh M.J. Am. J. Physiol. 1999; 276: C1346-C1351Crossref PubMed Google Scholar). Our previous work (19Staub O. Gautschi I. Ishikawa T. Breitschopf K. Ciechanover A. Schild L. Rotin D. EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (598) Google Scholar) has demonstrated that the total cellular pool of ENaC expressed heterologously in mammalian MDCK or NIH-3T3 cells has a short half-life (t 1/2 ∼1–2 h), as also shown for A6 cells, which express ENaC endogenously (25Weisz O.A. Wang J.M. Edinger R.S. Johnson J.P. J. Biol. Chem. 2000; 275: 39886-39893Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 30May A. Puoti A. Gaeggeler H.P. Horisberger J.D. Rossier B.C. J. Am. Soc. Nephrol. 1997; 8: 1813-1822Crossref PubMed Google Scholar). InXenopus oocytes expressing ectopic ENaC, which are grown at a much lower temperature, this half-life is ∼10 h (31Valentijn J.A. Fyfe G.K. Canessa C.M. J. Biol. Chem. 1998; 273: 30344-30351Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Despite the relatively short half-life of the intracellular pool of ENaC in cultured cells, it has been recently suggested that the half-life of the surface pool of ENaC in A6 cells is quite long (>24 h for α or γ, and ∼6 h for the β subunit) (25Weisz O.A. Wang J.M. Edinger R.S. Johnson J.P. J. Biol. Chem. 2000; 275: 39886-39893Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 26Kleyman T.R. Zuckerman J.B. Middleton P. McNulty K.A. Hu B. Su X. An B. Eaton D.C. Smith P.R. Am. J. Physiol. 2001; 281: F213-F221Google Scholar). This contrasts previous work which suggested, based on functional studies in Xenopusoocytes after brefeldin A treatment, that the active cell surface pool of ENaC is short lived (10Shimkets R.A. Lifton R.P. Canessa C.M. J. Biol. Chem. 1997; 272: 25537-25541Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 19Staub O. Gautschi I. Ishikawa T. Breitschopf K. Ciechanover A. Schild L. Rotin D. EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (598) Google Scholar), although this was not tested biochemically. The stability of ENaC at the cell surface of mammalian epithelial cells, most relevant for ENaC function, has not been investigated. In this study, we describe the generation of kidney epithelial MDCK cells stably expressing epitope-tagged αβγ-ENaC under an inducible promoter. Each subunit was tagged with a different tag at the intracellular C terminus, and an additional tag was added to the extracellular domain of β-ENaC. These cells express functional ENaC at the apical membrane. Using this cell line, we show here that the cell surface, mature ENaC is Endo H-sensitive (similar to intracellular ENaC), suggesting that the channel does not acquire complex glycosylation during trafficking to the cell surface. We also show that ENaC is not associated with lipid rafts, and its intracellular and cell surface pools are primarily detergent soluble. Moreover, we demonstrate that unlike A6 cells, the cell surface ENaC has a very short half-life, about 1 h. These studies are important for our understanding of the regulation of channel numbers at the plasma membrane, which play a key role in regulating ENaC function under physiological and pathophysiological conditions, such as Liddle syndrome. MDCK clones expressing rat ENaC chains bearing different epitope tags (see Fig. 1 A) were generated from high resistance MDCK cells as follows. For α-ENaC, a triple HA tag (YPYDVPDY) was introduced intracellularly just upstream of the stop codon, and the cDNA was subcloned into pLKneo, which possesses a dexamethasone-inducible promoter and neomycin resistance gene (32Hirt R.P. Poulain-Godefroy O. Billotte J. Kraehenbuhl J.P. Fasel N. Gene (Amst.). 1992; 111: 199-206Crossref PubMed Scopus (53) Google Scholar). After selection in G418, the stably expressing α-ENaC-MDCK cells were used as a template for the introduction of FLAG-tagged γ-ENaC. FLAG-γ-ENaC was generated by introducing an intracellular FLAG tag (DYKDDDDK) upstream of the stop codon and subcloning into pCEP4 (Invitrogen), which possesses a hygromycin resistance gene. After selection in hygromycin (in the presence of G418), the αHAγFLAG-ENaC-MDCK cells were used to introduce a double-tagged β-ENaC into them. For β-ENaC, a Myc tag (AEEQKLISEEDL) was inserted in the ectodomain (between amino acids 138 and 139, in a position previously described to have little effect on channel activity (3Firsov D. Schild L. Gautschi I. Merillat A.M. Schneeberger E. Rossier B.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15370-15375Crossref PubMed Scopus (396) Google Scholar)), and an intracellular T7 (MASMTGGQQMG) tag was added just upstream of the stop codon. The tagged β-ENaC was subcloned into pBabe-puro, which expresses the puromycin resistance gene, and the cDNA was introduced into the above cells. Puromycin selection (in the presence of G418 and hygromycin) yielded the αHAβMyc,T7γFLAG-ENaC-MDCK cells (see Fig. 1 A). The αβγFLAG-ENaC-MDCK cells have been described previously (19Staub O. Gautschi I. Ishikawa T. Breitschopf K. Ciechanover A. Schild L. Rotin D. EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (598) Google Scholar). MDCK cells were maintained in Dulbecco's modified Eagle's medium containing 10%, fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2-containing humidified air. The αHAβMyc,T7γFLAG-ENaC-MDCK cells were maintained in the above medium supplemented with 300 μg/ml G418, 5 μg/ml puromycin, 100 μg/ml hygromycin, and 10 μm amiloride. To verify protein expression, confluent monolayers grown on 10-cm plates were induced overnight (with 2 mm sodium butyrate, 1 μm dexamethasone, and 10 μm amiloride), washed, and lysed in lysis buffer (150 mm NaCl, 50 mm HEPES, 1% Triton X-100 (w/v), 10% glycerol (w/v), 1.5 mm MgCl2, 1.0 mm EGTA plus protease inhibitors (10 μg/ml leupeptin and aprotinin, 1 μg/ml pepstatin A, 1 mm phenylmethylsulfonyl fluoride)). Lysates were cleared by centrifugation at 14,000 × g for 10 min at 4 °C; a 250-μg aliquot (for α- or β-ENaC) was boiled in sample buffer, and proteins separated on an 8% SDS-PAGE. Because the expression of the γFLAG was very low, a larger amount of protein (6 mg) was used, and proteins were concentrated by methanol/chloroform precipitation (33Wessel D. Flugge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3170) Google Scholar) prior to separation on a 7% Tricine SDS-PAGE. For comparison, 600 μg of protein from the αβγFLAG-ENaC-expressing cells was also run on the same gel. After SDS-PAGE separation, proteins were immunoblotted with antibodies to the tags (from Sigma (anti-FLAG), BAbCO (anti-HA), Novagen (anti-T7), PharMingen (anti-Myc)) and detected by chemiluminescence (ECL from Amersham Biosciences, Inc. or SuperSignal from Pierce). In all experiments, MDCK cells were grown either on permeable filters (2.4-cm diameter, 0.4-μm pore size, Costar) or in 3.25-cm wells or 10-cm plates (plastic, Falcon) to confluence, and assays were performed 4 days later to ensure complete polarization and formation of domes (on plates) in the monolayers of cells. Several assays were performed using both systems, with similar results (see “Results”). Whole cell current recordings were made from αHAβMyc,T7γFLAG-ENaC-expressing MDCK cells grown on cover glass using the standard whole cell configuration of the patch clamp technique as reported previously (34Ishikawa T. Marunaka Y. Rotin D. J. Gen. Physiol. 1998; 111: 825-846Crossref PubMed Scopus (118) Google Scholar). An Axopatch 1D patch clamp amplifier (Axon Instruments, Foster City, CA) was used to measure whole cell currents. The amplifier was driven by PCLAMP 6 software to allow the delivery of voltage step protocols with concomitant digitization of the whole cell current. The patch clamp pipettes, which were pulled from glass capillaries (LG 16, Dagan, Minneapolis) using a vertical puller (model PP-830, Narishige, Tokyo), had resistances of ∼2–3 megohms when filled with a standard cesium-glutamate-rich solution described below. The reference was an Ag/AgCl electrode, which was connected to the bath via an agar bridge filled with a standard NaCl-rich bathing solution. Current-voltage (I-V) relations were studied using voltage ramps. The command voltage was varied from −124 mV to +16 mV over a duration of 800 ms every 30 s. 10 μm amiloride-sensitive currents were estimated by subtraction of currents measured under identical conditions except for the addition of 10 μmamiloride. The pipette solution contained (in mm) 100 or 120 cesium-glutamate, 10 CsCl, 1 MgCl2, 10 HEPES, 0 or 10 Na2-ATP, and 10 EGTA. The pH of the solution was adjusted with CsOH to 7.4. The cells were initially immersed in a bath solution (pH 7.4) containing (in mm) 140 NaCl, 4.3 KCl, 1MgCl2, and 10 HEPES. Before the establishment of whole cell configuration, the bath solution was changed to the one containing (in mm) 145 lithium-glutamate, 1 MgCl2, and 19 HEPES. The pH of the solution was adjusted with LiOH. All experiments were performed at room temperature (∼20 °C). Bath solution changes were accomplished by gravity feed from reservoirs. The results are reported as the means ± S.E. of several experiments (n), and n refers to the number of cells patched in the different plate. Confluent monolayers grown on either a permeable filter or a solid support were induced overnight and then kept on ice throughout the experiment. Cells were washed three times with ice-cold PBS-CM (PBS with 1 mm MgCl2and 0.1 mm CaCl2) and incubated 15 min with 1.0 mg/ml EZ-linkTM Sulfo-NHS-S-S-biotin (Pierce) in cold biotinylation buffer (10 mm triethanolamine, 2 mm CaCl2, 150 mm NaCl, pH 9.0) with gentle agitation. Cells were washed once with quenching buffer (192 mm glycine, 25 mm Tris in PBS-CM) and incubated for 10 min with quenching buffer with light agitation. Cells were then rinsed twice with PBS-CM, scraped in cold PBS, and pelleted at 2,000 rpm at 4 °C. They were lysed in lysis buffer and incubated on ice 30 min before centrifugation (10 min at 14,000 × g, 4 °C). Supernatants were transferred to new tubes and after the addition of 50 μl of 50% slurry of streptavidin-agarose beads (Sigma), were rotated for 2 h at 4 °C. Beads were pelleted by brief centrifugation and aliquots of the supernatant were taken to represent the unbound, intracellular pool. Beads were then washed three times with HNTG (20 mm HEPES, pH 7.5, 150 mmNaCl, 10% glycerol, 0.1% Trtion X-100). Biotinylated proteins were eluted by boiling in sample buffer supplemented with 5% β-mercaptoethanol, and proteins were separated on 8% SDS-PAGE and immunoblotted as above. In some experiments, cells were surface biotinylated by treating with prechilled 10 mm sodium periodate on ice for 30 min in the dark. Cells were then incubated with 1.0 mg/ml EZ-linkTM biotin-LC-hydrazide (Pierce) in prechilled 100 mm sodium acetate, pH 5.5, on ice with slight agitation for 30 min. For all experiments analyzing cell surface expression using the Sulfo-NHS-S-S-biotin for surface biotinylation, to ensure the absence of leakage of biotin into the cell (which could label the large intracellular pool of ENaC thus skewing the results), an internal control was used for each plate/well of cells by measuring surface biotinylation of an intracellular protein (e.g. actin, annexin II, enolase). Only plates/wells showing no labeling of intracellular proteins (i.e. no leakage of biotin) were included in our data analysis. Cells were grown on plates and induced overnight prior to experiments. Plates were surface biotinylated and lysed, and biotinylated proteins were precipitated and eluted as described above. Aliquots of supernatant from the streptavidin-agarose bead incubations, representing the intracellular pool of proteins, were boiled in sample buffer. Samples were then either left untreated or were treated with 100 mU Endo H or 15 mU peptide N-glycosidase F (PNGaseF), according to the manufacturer's instructions (New England Biolabs). Proteins were separated by SDS-PAGE and transferred to nitrocellulose for immunoblotting. Cells were grown on plates or filters and induced overnight. The following day, the indicated plates/filters were incubated with fresh induction medium supplemented with 20 μg/ml cycloheximide (ICN) for 1–6 h at 37 °C. We used one 10-cm plate or six filters (pooled) for each time point. At each time point, the appropriate plates/filters were placed on ice, surface biotinylated, and lysed in lysis buffer as described above. Lysates for each time point were quantitated using the Bio-Rad assay, and total protein was normalized before addition of streptavidin-agarose beads. Lysates were then rotated for 2 h at 4 °C. After a brief centrifugation to pellet the beads, aliquots were taken to represent the unbound, intracellular pool. Beads were washed three times with HNTG, and proteins were eluted by boiling in sample buffer containing 5% β-mercaptoethanol. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to the tags followed by chemiluminescence detection. Band quantitation was then performed using Fluorchem™ equipment and software (Alpha Innotech Corp., San Leandro, CA). Bands were quantified using spot densitometry, where only density values within the linear range were used. Polarized αHAβMyc,T7γFLAG-ENaC-expressing MDCK cells grown on filters or plates were induced overnight and placed on ice. Cells were rinsed three times with PBS-CM, surface biotinylated, and quenched as described above, then scraped in PBS. Cells were pelleted and lysed in lysis buffer for 30 min on ice. Samples were centrifuged for 10 min at 14,000 rpm, and pellets (detergent-insoluble fraction) were resuspended in a volume of lysis buffer equal to the supernatants (detergent-soluble fractions). Re- suspended pellets were sonicated three times for 15 s each on ice to solubilize the insoluble fraction (35Adair-Kirk T.L. Cox K.H. Cox J.V. J. Cell Biol. 1999; 147: 1237-1248Crossref PubMed Scopus (23) Google Scholar). Samples were then centrifuged for 10 min at 14,000 rpm, and the supernatants (representing the insoluble fraction) were collected. For ENaC at the cell surface, biotinylated proteins were precipitated with 50 μl of 50% streptavidin-agarose bead slurry by rotating for 2 h at 4 °C. After brief centrifugation, aliquots of supernatants were taken to represent the intracellular pool and were boiled in sample buffer. Beads were washed, and biotinylated proteins were eluted from the detergent-soluble and -insoluble fractions as de- scribed above. Proteins were separated by SDS-PAGE and transferred to nitrocellulose for immunoblotting and quantitation of chemiluminescence, as described above. Confluent monolayers grown on permeable filters or solid support (six multiwell plates) were washed three times with PBS, scraped in PBS, and pelleted at 2,000 rpm, 4 °C. Cells were lysed in 200 μl of TN (25 mm Tris-HCl, pH 7.5, 150 mmNaCl, 1 mm dithiothreitol, 10 μg/ml leupeptin and aprotinin, 1 μg/ml pepstatin A, 1 mm phenylmethylsulfonyl fluoride, 10% sucrose, and 1% Triton X-100) on ice and incubated for 30 min on ice. For some experiments, cells were surface biotinylated as described above prior to lysis in TN. Samples were mixed with 0.4 ml of cold OptiprepTM, transferred into SW60 centrifuge tubes, and overlaid with 0.6 ml of each 35, 30, 25, 20, and 0% Optiprep in TN, as described by Oliferenko et al. (36Oliferenko S. Paiha K. Harder T. Gerke V. Schwarzler C. Schwarz H. Beug H. Gunthert U. Huber L.A. J. Cell Biol. 1999; 146: 843-854Crossref PubMed Scopus (355) Google Scholar). The gradients were centrifuged at 35,000 rpm in an SW60 rotor for 12 h at 4 °C. Fractions were collected from the top to the bottom of the centrifuge tubes, and proteins were methanol/chloroform precipitated. After resuspension of the pellets in equal volumes of 5% SDS, sample buffer was added, and the fractions were boiled. Proteins were separated by SDS-PAGE and analyzed by immunoblotting. Antibodies to annexin II and caveolin were from Santa Cruz and for the tranferrin receptor (TfnR) from Zymed Laboratories Inc. Proteins in the top two fractions (20 and 25%) are considered to be raft-associated (36Oliferenko S. Paiha K. Harder T. Gerke V. Schwarzler C. Schwarz H. Beug H. Gunthert U. Huber L.A. J. Cell Biol. 1999; 146: 843-854Crossref PubMed Scopus (355) Google Scholar). For experiments in which cells were first surface biotinylated, 50 μl of 50% streptavidin-agarose bead slurry was added to each sample after resuspension of the pellets and rotated for 2 h at room temperature. After brief centrifugation, supernatants were used as described above, and beads were washed three times with HNTG. Biotinylated proteins were eluted by boiling in sample buffer supplemented with 5% β-mercaptoethanol. Confluent, filter-grown αHAβMyc,T7γFLAG-ENaC-expressing MDCK cells were induced overnight and rinsed three times with cold PBS-CM. Immunogold surface labeling was performed by incubating monolayers on ice for 3 h with monoclonal anti-Myc antibodies in PBS-CM (1:10, PharMingen) in both the apical and basal compartments (4 °C). Cells were washed three times (5 min each) with cold PBS-CM and incubated with goat anti-mouse 15 nm gold in PBS-CM (1:12, British Biolabs) at 4 °C for 1 h on ice. Cells were rinsed three times (5 min each) with ice-cold PBS and incubated in Karnovsky's fixative (2.5% glutaraldehyde, 3.2% paraformaldehyde in 0.1 m phosphate buffer, pH 7.2) for 2 h at room temperature. Samples were rinsed three times with PBS and incubated with 1% OsO4 for 15 min in the dark. Following PBS washes, filters were incubated with 2% uranyl acetate for 15 min in the dark. Samples were then washed x3 with distilled water and dehydrated using ethanol incubations in 50, 70, 95, and 100% ethanol (2 times 5 min each). Filters were infiltrated with Epon resin:ethanol (1:1 then 3:1, 30 min each) followed by 100% Epon overnight, 4 °C. Fresh Epon was applied to filters and incubated at room temperature for 5 h. Epon was again replaced and polymerized for 48 h at 65 °C. Blocks containing filter were sectioned (90-nm thick- ness) and placed on slotted grids. Sections were viewed using a Hitachi H600 transmission electron microscope. The expression of the ENaC protein in native kidney epithelia is very low, thus precluding biochemical analyses of the channel in these tissues. To analyze ENaC function in cultured kidney epithelial cells, we utilized high resistance MDCK cells, which form polarized monolayers and which are well characterized with respect to cellular trafficking (37Bacallao R. Antony C. Dotti C. Karsenti E. Stelzer E.H. Simons K. J. Cell B" @default.
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