Epithelial Sodium Channel Exit from the Endoplasmic Reticulum Is Regulated by a Signal within the Carboxyl Cytoplasmic Domain of the α Subunit
2007; Elsevier BV; Volume: 282; Issue: 46 Linguagem: Inglês
10.1074/jbc.m707339200
ISSN1083-351X
AutoresGunhild M. Mueller, Ossama B. Kashlan, James B. Bruns, Ahmad B. Maarouf, Meir Aridor, Thomas R. Kleyman, Rebecca P. Hughey,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoEpithelial sodium channels (ENaCs) are assembled in the endoplasmic reticulum (ER) from α, β, and γ subunits, each with two transmembrane domains, a large extracellular loop, and cytoplasmic amino and carboxyl termini. ENaC maturation involves transit through the Golgi complex where Asn-linked glycans are processed to complex type and the channel is activated by furin-dependent cleavage of the α and γ subunits. To identify signals in ENaC for ER retention/retrieval or ER exit/release, chimera were prepared with the interleukin α subunit (Tac) and each of the three cytoplasmic carboxyl termini of mouse ENaC (Tac-Ct) or with γ-glutamyltranspeptidase and each of the three cytoplasmic amino termini (Nt-GGT). By monitoring acquisition of endoglycosidase H resistance after metabolic labeling, we found no evidence of ER retention of any chimera when compared with control Tac or GGT, but we did observe enhanced exit of Tac-αCt when compared with Tac. ER exit of ENaC was assayed after metabolic labeling by following the appearance of cleaved α as cleaved α subunit, but not non-cleaved α, is endoglycosidase H-resistant. Interestingly ER exit of epitope-tagged and truncated α (αΔ624–699-V5) with full-length βγ was similar to wild type α (+βγ), whereas ER exit of ENaC lacking the entire cytoplasmic carboxyl tail of α (αΔ613–699-V5 +βγ) was significantly reduced. Subsequent analysis of ER exit for ENaCs with mutations within the intervening sequence 613HRFRSRYWSPG623 within the context of the full-length α revealed that mutation αRSRYW620 to AAAAA significantly reduced ER exit. These data indicate that ER exit of ENaC is regulated by a signal within the α subunit carboxyl cytoplasmic tail. Epithelial sodium channels (ENaCs) are assembled in the endoplasmic reticulum (ER) from α, β, and γ subunits, each with two transmembrane domains, a large extracellular loop, and cytoplasmic amino and carboxyl termini. ENaC maturation involves transit through the Golgi complex where Asn-linked glycans are processed to complex type and the channel is activated by furin-dependent cleavage of the α and γ subunits. To identify signals in ENaC for ER retention/retrieval or ER exit/release, chimera were prepared with the interleukin α subunit (Tac) and each of the three cytoplasmic carboxyl termini of mouse ENaC (Tac-Ct) or with γ-glutamyltranspeptidase and each of the three cytoplasmic amino termini (Nt-GGT). By monitoring acquisition of endoglycosidase H resistance after metabolic labeling, we found no evidence of ER retention of any chimera when compared with control Tac or GGT, but we did observe enhanced exit of Tac-αCt when compared with Tac. ER exit of ENaC was assayed after metabolic labeling by following the appearance of cleaved α as cleaved α subunit, but not non-cleaved α, is endoglycosidase H-resistant. Interestingly ER exit of epitope-tagged and truncated α (αΔ624–699-V5) with full-length βγ was similar to wild type α (+βγ), whereas ER exit of ENaC lacking the entire cytoplasmic carboxyl tail of α (αΔ613–699-V5 +βγ) was significantly reduced. Subsequent analysis of ER exit for ENaCs with mutations within the intervening sequence 613HRFRSRYWSPG623 within the context of the full-length α revealed that mutation αRSRYW620 to AAAAA significantly reduced ER exit. These data indicate that ER exit of ENaC is regulated by a signal within the α subunit carboxyl cytoplasmic tail. Epithelial Na+ channels (ENaCs) 2The abbreviations used are: ENaC, epithelial Na+ channel; Ct, carboxyl-terminal tail; Endo H, endoglycosidase H; ER, endoplasmic reticulum; GGT, γ-glutamyltranspeptidase; MDCK, Madin-Darby canine kidney; Nt, amino-terminal tail; Tac, interleukin receptor α subunit; TGN, trans-Golgi network; WT, wild type; DMEM, Dulbecco'ns modified Eagle'ns medium; MTSET, 2-(trimethylammonium)ethyl methanethiosulfonate bromide; COP, coat protein complex; NMDA, N-methyl-d-aspartate; ERGIC, endoplasmic reticulum-Golgi intermediate compartment. are found in apical membranes of Na+-transporting epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels consist of three structurally related subunits, termed α, β, and γ, with a presumed α1β1γ1 subunit stoichiometry (1Kosari F. Sheng S. Li J. Mak D.-O.D. Foskett J.K. Kleyman T.R. J. Biol. 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The subunits share common structure features with two membrane-spanning and adjacent extracellular hydrophobic domains (M1H1 and H2M2) separated by a large extracellular loop and cytoplasmic amino (Nt) and carboxyl (Ct) termini (2Firsov D. Gautschi I. Meriallat A.M. Rossier V.C. Schild L. EMBO J. 1998; 17: 344-352Crossref PubMed Scopus (369) Google Scholar, 4Canessa C.M. Meriallat A.M. Rossier B.C. Am. J. Physiol. 1994; 267: C1682-C1690Crossref PubMed Google Scholar, 5Snyder P.M. McDonald F.J. Stokes J.B. Welsh M.J. J. Biol. Chem. 1994; 269: 24379-24383Abstract Full Text PDF PubMed Google Scholar). ENaCs have important roles in the regulation of extracellular fluid volume, blood pressure, and airway surface liquid volume. ENaC subunits are thought to assemble in the endoplasmic reticulum (ER) where they undergo Asn-linked glycosylation and form disulfide bridges (5Snyder P.M. McDonald F.J. Stokes J.B. Welsh M.J. J. Biol. 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Quality control of ENaC subunit folding and assembly involves interactions with both Hsc70 and the small heat shock protein αA-crystallin (11Goldfarb S.B. Kashlan O.B. Watkins J.N. Suaud L. Yan W. Kleyman T.R. Rubenstein R.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5817-5822Crossref PubMed Scopus (130) Google Scholar, 12Kashlan O.B. Mueller G.M. Qamar M.Z. Poland P.A. Ahner A. Rubenstein R.C. Hughey R.P. Brodsky J.L. Kleyman T.R. J. Biol. Chem. 2007; 282: 28149-28156Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). There is also clear evidence that ENaC transits the Golgi complex during its maturation. Expression of active channels at the plasma membrane is blocked by treatment of cells with the fungal metabolite brefeldin A that disrupts assembly of cytosolic coats that are required for intra-Golgi transport (13Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1542) Google Scholar, 14Shimkets R.A. 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Frindt G. J. Gen. Physiol. 1996; 107: 35-45Crossref PubMed Scopus (100) Google Scholar, 19Sheng S. Carattino M.D. Bruns J.B. Hughey R.P. Kleyman T.R. Am. J. Physiol. 2006; 290: F1488-F1496Crossref PubMed Scopus (133) Google Scholar). Furin cleavage of the α subunit releases an inhibitory 26-mer peptide (20Carattino M.D. Sheng S. Bruns J.B. Pilewski J.M. Hughey R.P. Kleyman T.R. J. Biol. Chem. 2006; 281: 18901-18907Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Processing of the γ subunit by furin and prostasin releases an additional 43-mer inhibitory peptide (21Bruns J.B. Carattino M.D. Sheng S. Maarouf A.B. Weisz O.A. Pilewski J.M. Hughey R.P. Kleyman T.R. J. Biol. Chem. 2007; 282: 6153-6160Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Furin is a well characterized proprotein convertase that is localized primarily in the trans-Golgi network (TGN), although it shuttles between the TGN and plasma membrane, and prostasin is a glycosylphosphatidylinositol-anchored serine protease likely found on the cell surface (22Jean F. Thomas L. Molloy S.S. Liu G. Jarvis M.A. Nelson J.A. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2864-2869Crossref PubMed Scopus (67) Google Scholar, 23Adachi M. Kitamura K. Miyoshi T. Narikiyo T. Iwashita K. Shiraishi M. Nonoguchi H. Tomita K. J. Am. Soc. Nephrol. 2001; 12: 1114-1121Crossref PubMed Google Scholar). ENaC assembly clearly begins in the ER as immature non-processed forms of all three subunits are found in co-immunoprecipitates (10Hughey R.P. Mueller G.M. Bruns J.B. Kinlough C.L. Poland P.A. Harkleroad K.L. Carattino M.D. Kleyman T.R. J. Biol. Chem. 2003; 278: 37073-37082Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Co-expression of αβγENaC with chimera designed to properly orient either the α extracellular loop, the two membrane-spanning and adjacent extracellular hydrophobic domains (αM1H1 or αH2M2), or α cytoplasmic domains blocks both surface expression of ENaC and amiloride-sensitive sodium currents in Xenopus oocytes, indicating that subunit folding, channel assembly, and channel exit from the ER are interdependent (24Bruns J.B. Baofeng H. Ahn Y.J. Sheng S. Hughey R.P. Kleyman T.R. Am. J. Physiol. 2003; 285: F600-F609Crossref PubMed Scopus (22) Google Scholar). Although ER exit signals for transmembrane cargo that interact with the prebudding coat protein complex II (COPII) (Sar1p, Sec23p, and Sec24p) are diacidic motifs, dibasic motifs, short hydrophobic motifs, or combinations of these, the signals for ER exit of multitransmembrane proteins and/or multisubunit proteins are more complex using a hierarchy of arginine-based ER retention signals and diverse ER exit (release) signals (for reviews, see Refs. 25Bonifacino J.S. Glick B.S. Cell. 2004; 116: 153-166Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar, 26Michelsen K. Yuan H. Schwappach B. EMBO Rep. 2005; 6: 717-722Crossref PubMed Scopus (195) Google Scholar, 27Tang B.L. Wang Y. Ong Y.S. Hong W. Biochim. Biophys. Acta. 2005; 1744: 293-303Crossref PubMed Scopus (76) Google Scholar). ENaCs released from the ER reach the cell surface by both transit through the Golgi complex and by a non-conventional route that bypasses glycan processing and furin-dependent activation in the Golgi complex and TGN (28Hughey R.P. Bruns J.B. Kinlough C.L. Harkleroad K.L. Tong Q. Carattino M.D. Johnson J.P. Stockand J.D. Kleyman T.R. J. Biol. Chem. 2004; 279: 18111-18114Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 29Hughey R.P. Bruns J.B. Kinlough C.L. Kleyman T.R. J. Biol. Chem. 2004; 279: 48491-48494Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Non-processed ENaC can be activated by proteolytic cleavage at the cell surface by serine proteases such as exogenous trypsin or endogenous elastase or prostasin (21Bruns J.B. Carattino M.D. Sheng S. Maarouf A.B. Weisz O.A. Pilewski J.M. Hughey R.P. Kleyman T.R. J. Biol. Chem. 2007; 282: 6153-6160Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 30Vallet V. Chraïbi A. Gaeggeler H.-P. Horisberger J.-D. Rossier B.C. Nature. 1997; 389: 607-610Crossref PubMed Scopus (453) Google Scholar, 31Knight K.K. Olson D.R. Zhou R. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2805-2808Crossref PubMed Scopus (126) Google Scholar, 32Caldwell R.A. Boucher R.C. Stutts M.J. Am. J. Physiol. 2005; 288: L813-L819Crossref PubMed Scopus (212) Google Scholar, 33Harris M. Firsov D. Vuagniaux G. Stutts M.J. Rossier B.C. J. Biol. Chem. 2007; 282: 58-64Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Thus, factors that regulate the assembly, retention, and release of ENaC from the ER could represent key regulatory steps in the ability of cells to modulate Na+ transport. As a first step toward understanding how ER exit of ENaC is regulated, we followed the acquisition of Endo H resistance for chimera made with each of the six cytosolic domains of α, β, and γ mouse ENaC subunits. Although we found no evidence for a retention signal in any of the cytoplasmic domains, an ER exit signal was clearly present within the carboxyl terminus of the α subunit. We then analyzed ER exit of ENaC containing wild type or mutant α subunits and found that an ER exit signal is present within a short peptide motif of the α cytoplasmic carboxyl-terminal tail. Vector Preparation—Chimeras of mouse ENaC cytoplasmic domains were constructed in pcDNA5/FRT (Invitrogen) using a two-step PCR method (34Sheng S. Li J. McNulty K.A. Avery D. Kleyman T.R. J. Biol. Chem. 2000; 275: 8572-8581Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The Nt cytoplasmic domains of the α (residues 1–109), β (residues 1–49), or γ (residues 1–53) ENaC subunits were linked to the transmembrane and ectodomain of the rat γ-glutamyltranspeptidase (GGT) mutant T380N that lacks autocatalytic cleavage (residues 5–568) and named αNt-GGT, βNt-GGT, and γNt-GGT, respectively (35Kinlough C.L. Poland P.A. Bruns J.B. Hughey R.P. Methods Enzymol. 2005; 401: 426-449Crossref PubMed Scopus (38) Google Scholar). The Ct cytoplasmic domains of the α (residues 613–699), β (residues 555–638), or γ (residues 573–655) ENaC subunits were linked to the transmembrane and ectodomain of the human interleukin receptor α subunit (referred to as Tac, residues 1–266) and named Tac-αCt, Tac-βCt, and Tac-γCt, respectively (36Marks M.S. Roche P.A. van Donselaar E. Woodruff L. Peters P.J. Bonifacino J.S. J. Cell Biol. 1995; 131: 351-369Crossref PubMed Scopus (178) Google Scholar). Wild type α subunit with both amino-terminal HA and carboxyl-terminal V5 epitope tags (named αWT), wild type β subunit with a carboxyl-terminal FLAG epitope tag (β-FLAG), wild type γ subunit with a carboxyl-terminal myc epitope tag (γ-myc), and mutant γG542C were generated and characterized previously (1Kosari F. Sheng S. Li J. Mak D.-O.D. Foskett J.K. Kleyman T.R. J. Biol. Chem. 1998; 273: 13469-13474Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 10Hughey R.P. Mueller G.M. Bruns J.B. Kinlough C.L. Poland P.A. Harkleroad K.L. Carattino M.D. Kleyman T.R. J. Biol. Chem. 2003; 278: 37073-37082Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). αWT was transferred into pcDNA6, and truncation mutants α624 (HA-αΔ624–699-V5) and α613 (HA-αΔ613–699-V5) as well as site-specific mutations of HA-α-V5 named αHRF (αHRF615 to AAA), αRSRYW (αRSRYW620 to AAAA), or αSPG (αSPG623 to AAA) were constructed using a PCR-based approach (34Sheng S. Li J. McNulty K.A. Avery D. Kleyman T.R. J. Biol. Chem. 2000; 275: 8572-8581Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Cell Culture and Transfections—Clonal cultures of flip-in Chinese hamster ovary cells expressing each Nt-GGT or Tac-Ct chimera were prepared by co-transfection of cells with plasmid POG44 and recombinant plasmids constructed in pcDNA5/FRT (Invitrogen) followed by clonal selection and maintenance in medium containing hygromycin (0.5 mg/ml) (37Poland P.A. Kinlough C.L. Rokaw M.D. Magarian-Blander J. Finn O.J. Hughey R.P. Glycoconj. J. 1997; 14: 89-96Crossref PubMed Scopus (17) Google Scholar). All Chinese hamster ovary cell lines were maintained in Dulbecco'ns modified Eagle'ns medium (DMEM) and Ham'ns F-12 (1:1) (DMEM/Ham'ns F-12 medium) with 3% fetal bovine serum at 37 °C in 5% CO2. All tissue culture reagents were purchased from Invitrogen. MDCK type 1 cells were obtained from both Barry M. Gumbiner (Memorial Sloan-Kettering Cancer Center, New York, NY) and Daniela Rotin (University of Toronto, Toronto, Canada) (38Morris R.G. Schafer J.A. J. Gen. Physiol. 2002; 120: 71-85Crossref PubMed Scopus (89) Google Scholar). Experiments were carried out in MDCK cells from both sources, but no differences were observed. MDCK cells were maintained in DMEM with 10% fetal bovine serum as described previously (10Hughey R.P. Mueller G.M. Bruns J.B. Kinlough C.L. Poland P.A. Harkleroad K.L. Carattino M.D. Kleyman T.R. J. Biol. Chem. 2003; 278: 37073-37082Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar) and transiently transfected with either αWT, mutant α624, or mutant α613 in combination with β-FLAG and γ-myc using Lipofectamine 2000 (Invitrogen). Cells were cultured in the presence of 20 μm amiloride 4 h after transfection and used the following day for experiments described in Figs. 3 and 5 (10Hughey R.P. Mueller G.M. Bruns J.B. Kinlough C.L. Poland P.A. Harkleroad K.L. Carattino M.D. Kleyman T.R. J. Biol. Chem. 2003; 278: 37073-37082Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Inclusion of amiloride in the medium after transfection improved the signal obtained in experiments using radiolabeling.FIGURE 5ENaC exit from the endoplasmic reticulum is reduced by mutation of a five-residue motif in the α subunit carboxyl-terminal cytoplasmic tail.A, MDCK cells transiently expressing wild type βγ with wild type (αWT) or mutant α subunits, αHRF (αHRF615 to AAA), αRSRYW (αRSRYW620 to AAAA), or αSPG (αSPG623 to AAA), were assayed for ER exit as described in Fig. 3. Representative SDS gel profiles are presented. Levels of non-cleaved (▸) and cleaved (◃) α subunit bands were used to calculate the percentage of cleaved subunit at the 2-h chase period relative to the full-length α subunit at t = 0. The asterisk (*) denotes background bands present near the cleaved α subunit band. B, data obtained for ENaC with mutant α subunits were normalized to ENaC with wild type α in each experiment, and the mean and S.E. from multiple experiments (n = 7–18) is presented. Processing of ENaC containing mutant αRSRYW was significantly reduced when compared with ENaC containing wild type α (*, p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Radiolabeling, Immunoprecipitation, and Immunoblotting—Confluent cultures of flip-in cell lines expressing chimera were grown in 25- or 35-mm dishes (6- or 12-well clusters). Cells were washed once with 1 ml of Dulbecco'ns modified Eagle'ns medium lacking methionine and cysteine (ICN, Costa Mesa, CA) and starved for Met and Cys in 1 ml of the same medium for 1 h before addition of 50–100 μCi of [35S]Met/Cys (MP Biomedicals Inc., Irvine, CA) for 15 min as described previously (39Altschuler Y. Kinlough C.L. Poland P.A. Bruns J.B. Apodaca G. Weisz O.A. Hughey R.P. Mol. Biol. Cell. 2000; 11: 819-831Crossref PubMed Scopus (146) Google Scholar). Labeled cells were chased in DMEM/Ham'ns F-12 medium (with 3% fetal bovine serum) containing excess Met (2.5 mm) for the times indicated. Cells were washed with 1 ml of phosphate-buffered saline (137 mm NaCl, 2.6 mm KCl, 15.2 mm Na2HPO4, 1.47 mm KH2PO4, 0.5 mm MgCl2, and 0.7 mm CaCl2) and extracted into 0.15 ml (12-well) or 0.3 ml (6-well) of lysis buffer (0.4% sodium deoxycholate, 1% Nonidet P-40, 63 mm EDTA, and 50 mm Tris-HCl, pH 8) for 20 min at room temperature (10Hughey R.P. Mueller G.M. Bruns J.B. Kinlough C.L. Poland P.A. Harkleroad K.L. Carattino M.D. Kleyman T.R. J. Biol. Chem. 2003; 278: 37073-37082Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Protease inhibitor mixture set III from Calbiochem was added to the lysis buffer as directed by the manufacturer. Extracts were centrifuged for 7 min at 14,000 rpm in a centrifuge at 4 °C to remove insoluble material. Supernatants were incubated overnight at 4 °C on a rotating wheel with either mouse monoclonal anti-Tac antibody (anti-CD25, Ancell, Bayport, MN) or goat anti-GGT antibody (40Joyce-Brady M. Jean J.-C. Hughey R.P. J. Biol. Chem. 2001; 276: 9468-9477Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and protein G conjugated to Sepharose beads (Zymed Laboratories Inc., San Francisco, CA). MDCK cells expressing αβγENaC (using either double tagged αWT, α624, α613, αHRF, αRSRYW, or αSPG) were starved for Met and Cys for 30 min, pulse-labeled with [35S]Met/Cys for 20 min, and chased in DMEM (with 10% fetal bovine serum) containing excess Met (2.5 mm) for varying times. After extraction in lysis buffer and removal of insoluble material by centrifugation, 10% SDS was added to a final concentration of 2.5% to dissociate the channel into individual subunits. Samples were incubated at room temperature for 20 min and diluted with lysis buffer to reduce the SDS concentration to 0.73% prior to immunoprecipitation of the α subunit (95-kDa uncleaved and 65-kDa cleaved forms) with mouse monoclonal anti-V5 antibodies (Serotec, Oxford, UK) and protein A conjugated to Sepharose beads (Zymed Laboratories Inc.). Immunoprecipitates were recovered by brief centrifugation and washed once with 1 ml each of 1% Triton X-100 (Roche Applied Science) in HEPES-buffered saline (Ref. 10Hughey R.P. Mueller G.M. Bruns J.B. Kinlough C.L. Poland P.A. Harkleroad K.L. Carattino M.D. Kleyman T.R. J. Biol. Chem. 2003; 278: 37073-37082Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar; 150 mm NaCl and 10 mm HEPES, pH 7.4), 0.01% SDS in HEPES-buffered saline, and finally HEPES-buffered saline, before elution into SDS sample buffer. In some cases, samples were treated overnight with or without Endo H (0.04 unit, Roche Diagnostics) or peptide N-glycanase-F (500 units, New England Biolabs, Beverly, MA). Proteins were recovered by heating the immunoprecipitates for 3.5 min at 95 °C in 30 μl of SDS sample buffer containing fresh 0.14 m β-mercaptoethanol. Samples were subjected to SDS-PAGE (Criterion Tris-HCl precast 4–15% acrylamide gradient gels from Bio-Rad) and transferred to nitrocellulose (0.45-μm pore size Immobilon-NC membrane from Millipore, Bedford, MA). Radioactive protein bands were imaged and quantitated using a Molecular Imager and Quantity One software (Bio-Rad). Immunoblotting of proteins on nitrocellulose was carried out with anti-V5 antibodies from Serotec as described previously (10Hughey R.P. Mueller G.M. Bruns J.B. Kinlough C.L. Poland P.A. Harkleroad K.L. Carattino M.D. Kleyman T.R. J. Biol. Chem. 2003; 278: 37073-37082Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Measurement of ENaC Exocytosis and Endocytosis in Xenopus Oocytes—ENaC exocytosis and endocytosis rates were determined as described previously (41Carattino M.D. Hill W.G. Kleyman T.R. J. Biol. Chem. 2003; 278: 36202-36213Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 42Snyder P.M. Olson D.R. Bucher D.B. J. Biol. Chem. 1999; 274: 28484-28490Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) from oocytes injected with cRNAs for wild type β, mutant γG542C, and α with (αWT) or without (α613) its carboxyl cytoplasmic tail. None of the constructs had epitope tags. 24–36 h after injection, channels at the cell surface were blocked with 1 mm MTSET for 4 min, resulting in a covalent modification of the channel that causes an ∼80% reduction of current. After removal of MTSET from the bath, Na+ current was measured by two-electrode voltage clamp at -100 mV every 30 s for 10 min to monitor current recovery. The initial rates of reappearance of amiloride-sensitive currents were determined from the linear portion of the current recovery curve (0–2 min). ENaC endocytosis rates were determined from oocytes injected with cRNA for wild type β, mutant γG542C, and α with (αWT) or without (α613) its carboxyl cytoplasmic tail. 24–36 h after injection, amiloride-sensitive currents were determined by two-electrode voltage clamp before and after 2, 4, and 6 h of incubation with 5 μm brefeldin A. Amiloride-sensitive currents were expressed relative to the initial amiloride-sensitive current, and data for the current declines were compared. Identification of an ER Exit Signal in the Carboxyl Cytoplasmic Tail of αENaC—To identify potential ER retention or exit signals present in the cytoplasmic domains of ENaC subunits, chimeras were designed to individually characterize functional sequences within the Nt or Ct cytoplasmic tails of the α, β, and γ subunits. To maintain the correct orientation of the tails with respect to the ER membrane, Nt and Ct were attached to the transmembrane and ectodomains of the type 2 transmembrane glycoprotein γ-glutamyltranspeptidase (Nt-GGT) and the type 1 transmembrane glycoprotein interleukin receptor α subunit referred to as Tac (Tac-Ct), respectively. Because both GGT and Tac ectodomains exhibit Asn-linked glycosylation (35Kinlough C.L. Poland P.A. Bruns J.B. Hughey R.P. Methods Enzymol. 2005; 401: 426-449Crossref PubMed Scopus (38) Google Scholar, 36Marks M.S. Roche P.A. van Donselaar E. Woodruff L. Peters P.J. Bonifacino J.S. J. Cell Biol. 1995; 131: 351-369Crossref PubMed Scopus (178) Google Scholar), the rate of chimera exit from the ER was measured in pulse-chase experiments by following the acquisition of Endo H resistance. Glycoproteins with Asn-linked glycans are sensitive to cleavage by Endo H prior to reaching the medial Golgi complex, and most glycans become resistant to cleavage after Asn-linked glycans are trimmed by mannosidase II in that compartment (for reviews, see Refs. 43Ellgaard L. Helenius A. Nat. Rev. Mol. Cell Biol. 2003; 4: 181-191Crossref PubMed Scopus (1674) Google Scholar and 44Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1984) Google Scholar). To measure ER exit of the proteins, Chinese hamster ovary flip-in cell lines stably expressing GGT (residues 1–568), Tac (residues 1–266), or one of the six chimeras were pulse-labeled for 15 min with [35S]Met/Cys and chased for 10–30 min. GGT, Tac, and the six chimeras were immunoprecipitated from cell extracts with anti-ectodomain antibodies, and duplicate samples were treated with or without Endo H and subjected to SDS-PAGE. Radioactive bands with altered mobility after Endo H treatment due to removal of Asn-linked glycans represented immature chimera (from markings ◂ to ◃), whereas bands that did not shift after Endo H treatment represented mature chimera with processed Asn-linked glycans (arrow) (see Fig. 1, A–D and F–I). The mature forms of the chimera also exhibited slower mobility than the immature forms consistent with processing of Asn-linked glycans to more complex types, and their levels increased with time of chase consistent with a precursor-product relationship. We observed a significant increase in maturation (ER exit) of Tac-αCt when compared with Tac (20 and 30 min of chase, p < 0.001) and a small but significant increase in maturation of Tac-γCt when compared with Tac (30 min, p < 0.05) (Fig. 1E). The increase in maturation of Tac-βCt when compared with Tac was not statistically significant. There was no apparent difference in the rate of maturation between GGT and any of the Nt-GGT chimeras (Fig. 1J). Therefore, there appeared to be a strong ER exit signal present in the cytoplasmic carboxyl-terminal tail of αENaC but no ER retention signal in any of the six cytoplasmic tails. Only a Small Fraction of the α Subunit Is Assembled into a Channel That Transits the Golgi Complex and TGN—We previously showed that the α and γ subunits of αβγENaC complex are cleaved by the resident TGN protease furin during transit through the Golgi complex where Asn-linked glycans are terminally processed to complex type (28Hughey R.P. Bruns J.B. Kinlough C.L. Harkleroad K.L. Tong Q. Carattino M.D. Johnson J.P. Stockand J.D. Kleyman T.R. J. Biol. Chem. 2004; 279: 18111-18114Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Processing of subunits within an individual channel is an all-or-none event, and only the cleaved forms of the α (65-kDa) and γ (75-kDa) subunits exhibit resistance to Endo H cleavage (28Hughey R.P. Bruns J.B. Kinlough C.L. Harkleroad K.L. Tong Q. Carattino M.D. Johnson J.P. Stockand J.D. Kleyman T.R. J. Biol. Chem. 2004; 279:
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