A Region Directly Following the Second Transmembrane Domain in γENaC Is Required for Normal Channel Gating
2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês
10.1074/jbc.m305400200
ISSN1083-351X
AutoresRachell E. Booth, Qiusheng Tong, Jorge Medina, Peter M. Snyder, Pravina Patel, James D. Stockand,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoWe used a yeast one-hybrid complementation screen to identify regions within the cytosolic tails of the mouse α, β, and γ epithelial Na+ channel (ENaC) important to protein-protein and/or protein-lipid interactions at the plasma membrane. The cytosolic COOH terminus of αENaC contained a strongly interactive domain just distal to the second transmembrane region (TM2) between Met610 and Val632. Likewise, γENaC contained such a domain just distal to TM2 spanning Gln573–Pro600. Interactive domains were also localized within Met1–Gln54 and the last 17 residues of α- and βENaC, respectively. Confocal images of Chinese hamster ovary cells transfected with enhanced green fluorescent fusion proteins of the cytosolic tails of mENaC subunits were consistent with results in yeast. Fusion proteins of the NH2 terminus of αENaC and the COOH termini of all three subunits co-localized with a plasma membrane marker. The functional importance of the membrane interactive domain in the COOH terminus of γENaC was established with whole-cell patch clamp experiments of wild type (α, β, and γ) and mutant (α, β, and γΔQ573-P600) mENaC reconstituted in Chinese hamster ovary cells. Mutant channels had about 13% of the activity of wild type channels with 0.33 ± 0.14 versus 2.5 ± 0.80 nA of amiloridesensitive inward current at –80 mV. Single channel analysis of recombinant channels demonstrated that mutant channels had a decrease in P o with 0.16 ± 0.03 versus 0.67 ± 0.07 for wild type. Mutant γENaC associated normally with the other two subunits in co-immunoprecipitation studies and localized to the plasma membrane in membrane labeling experiments and when visualized with evanescent-field fluorescence microscopy. Similar to deletion of Gln573–Pro600, deletion of Gln573–Arg583 but not Thr584–Pro600 decreased ENaC activity. The current results demonstrate that residues within Gln573–Arg583 of γENaC are necessary for normal channel gating. We used a yeast one-hybrid complementation screen to identify regions within the cytosolic tails of the mouse α, β, and γ epithelial Na+ channel (ENaC) important to protein-protein and/or protein-lipid interactions at the plasma membrane. The cytosolic COOH terminus of αENaC contained a strongly interactive domain just distal to the second transmembrane region (TM2) between Met610 and Val632. Likewise, γENaC contained such a domain just distal to TM2 spanning Gln573–Pro600. Interactive domains were also localized within Met1–Gln54 and the last 17 residues of α- and βENaC, respectively. Confocal images of Chinese hamster ovary cells transfected with enhanced green fluorescent fusion proteins of the cytosolic tails of mENaC subunits were consistent with results in yeast. Fusion proteins of the NH2 terminus of αENaC and the COOH termini of all three subunits co-localized with a plasma membrane marker. The functional importance of the membrane interactive domain in the COOH terminus of γENaC was established with whole-cell patch clamp experiments of wild type (α, β, and γ) and mutant (α, β, and γΔQ573-P600) mENaC reconstituted in Chinese hamster ovary cells. Mutant channels had about 13% of the activity of wild type channels with 0.33 ± 0.14 versus 2.5 ± 0.80 nA of amiloridesensitive inward current at –80 mV. Single channel analysis of recombinant channels demonstrated that mutant channels had a decrease in P o with 0.16 ± 0.03 versus 0.67 ± 0.07 for wild type. Mutant γENaC associated normally with the other two subunits in co-immunoprecipitation studies and localized to the plasma membrane in membrane labeling experiments and when visualized with evanescent-field fluorescence microscopy. Similar to deletion of Gln573–Pro600, deletion of Gln573–Arg583 but not Thr584–Pro600 decreased ENaC activity. The current results demonstrate that residues within Gln573–Arg583 of γENaC are necessary for normal channel gating. Activity of integral membrane proteins is regulated, in general, by the following two means: post-translational modification, and discretionary interaction with accessory, regulatory proteins and/or lipids. These two modalities of regulation are not necessarily mutually exclusive and impact function by influencing several parameters, including protein localization and kinetics. Ion channels are integral membrane proteins that play fundamental roles in many diverse cellular processes. Similar to other membrane proteins, ion channel activity is, in part, a manifestation of channel kinetics and cellular locale. The amiloride-sensitive epithelial Na+ channel (ENaC) 1The abbreviations used are: ENaC, epithelial Na+ channel; mENaC, mouse epithelial Na+ channel; CHO, Chinese hamster ovary; TM, transmembrane region; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; wt, wild type; ECFP, enhanced cyan fluorescent protein; EF, evanescent-field; TIRF, total internal reflection fluorescence; FRT, Fischer rat thyroid; PBS, phosphate-buffered saline; mt, mutant. is an ion channel localized to the luminal plasma membrane of epithelial cells (1Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1036) Google Scholar, 2Harvey K.F. Shearwin-Whyatt L.M. Fotia A. Parton R.G. Kumar S. J. Biol. Chem. 2002; 277: 9307-9317Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 3Alvarez D.L.R. Canessa C.M. Fyfe G.K. Zhang P. Annu. Rev. Physiol. 2000; 62: 573-594Crossref PubMed Scopus (289) Google Scholar). Activity of this channel is the rate-limiting step in Na+ transport across electrically tight epithelium. Thus, ENaC plays a pivotal role in Na+ and concomitant water (re)absorption across many epithelial tissues. This channel, consequently, is centrally positioned as an effector for systemic hormones and other factors that modulate blood pressure. Gain and loss of function mutations in ENaC and its regulatory pathways, indeed, cause blood pressure disorders in humans associated with aberrant Na+ and water metabolism (4Lifton R.P. Gharavi A.G. Geller D.S. Cell. 2001; 104: 545-556Abstract Full Text Full Text PDF PubMed Scopus (1368) Google Scholar). Although it is accepted that ENaC activity is dynamically modulated by regulation of channel localization to the luminal membrane, little is actually known about the cellular control points and queues impinging upon this modulation. In addition, the specific residues and domains within the channel itself important to localization and control of channel activity remain obscure. ENaC is a member of the Deg/ENaC superfamily of ion channels (3Alvarez D.L.R. Canessa C.M. Fyfe G.K. Zhang P. Annu. Rev. Physiol. 2000; 62: 573-594Crossref PubMed Scopus (289) Google Scholar, 5Benos D.J. Stanton B.A. J. Physiol. (Lond.). 1999; 520: 631-644Crossref Scopus (153) Google Scholar). This superfamily contains a functionally diverse array of channels that all share a common tertiary structure with members having two-transmembrane spanning regions, a large extracellular ectodomain and two short cytosolic tails. Channels within this superfamily play important roles in sensory perception, including taste, touch, hearing, nociception, and neurotransmission, as well as vectorial Na+ transport across epithelia. In native epithelia, ENaC is composed of three homologous but distinct subunits: α, β, and γ. Canessa et al. (6Canessa C.M. Horisberger J.D. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (827) Google Scholar, 7Canessa 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) and Lingueglia et al. (8Lingueglia E. Voilley N. Waldmann R. Lazdunski M. Barbry P. FEBS Lett. 1993; 318: 95-99Crossref PubMed Scopus (317) Google Scholar) were the first to identify the molecular correlates of ENaC. Most results suggest that the functional channel has a stoichiometry of two α and one β and γ subunit (9Firsov D. Gautschi I. Merillat A.M. Rossier B.C. Schild L. EMBO J. 1998; 17: 344-352Crossref PubMed Scopus (369) Google Scholar, 10Kosari F. Sheng S. Li J. Mak D.O. Foskett J.K. Kleyman T.R. J. Biol. Chem. 1998; 273: 13469-13474Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar); however, the alternative that the functional channel is composed of three copies of each of the three subunits has also been proposed (11Eskandari S. Snyder P.M. Kreman M. Zampighi G.A. Welsh M.J. Wright E.M. J. Biol. Chem. 1999; 274: 27281-27286Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Heterologously expressed αENaC alone and together with either β- or γENaC also forms homomeric and heterodimeric channels, although with much decreased activity and slightly different biophysical characteristics from the endogenous channel in native epithelia (6Canessa C.M. Horisberger J.D. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (827) Google Scholar, 7Canessa 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, 12Awayda M.S. Tousson A. Benos D.J. Am. J. Physiol. 1997; 273: C1889-C1899Crossref PubMed Google Scholar, 13McNicholas C.M. Canessa C.M. J. Gen. Physiol. 1997; 109: 681-692Crossref PubMed Scopus (154) Google Scholar, 14Fyfe G.K. Canessa C.M. J. Gen. Physiol. 1998; 112: 423-432Crossref PubMed Scopus (94) Google Scholar). The cytosolic tails of ENaC are believed to be regulatory domains and/or effector sites that impinge on channel gating and locale. Recent findings from our laboratory showing that the NH2 terminus of αENaC and the COOH termini of all three subunits contain domains involved in protein-protein and/or protein-lipid interactions localized to the plasma membrane are consistent with such a possibility (15Hendron E. Patel P. Hausenfluke M. Gamper N. Shapiro M.S. Booth R.E. Stockand J.D. J. Biol. Chem. 2002; 277: 34480-34488Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Moreover, deletion of the entire NH2 terminus from any subunit inactivates ENaC (16Chalfant M.L. Denton J.S. Langloh A.L. Karlson K.H. Loffing J. Benos D.J. Stanton B.A. J. Biol. Chem. 1999; 274: 32889-32896Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Conversely, deletion of the complete COOH tail of β- and γ- but not αENaC activates the channel (17Snyder 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, 18Schild L. Canessa C.M. Shimkets R.A. Gautschi I. Lifton R.P. Rossier B.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5699-5703Crossref PubMed Scopus (293) Google Scholar). Deletion of the latter half of the COOH tail of αENaC, however, does increase activity (19Volk K.A. Snyder P.M. Stokes J.B. J. Biol. Chem. 2001; 276: 43887-43893Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). These results, as well as others, suggest that the cytosolic tails of ENaC are involved in both positive and negative regulation of channel activity. Interestingly, the COOH terminus of ENaC subunits are the least well conserved portions of the channel. It has been hypothesized that these regions may impart the well described tissue- and species-specific regulation of ENaC by allowing differential interaction with site-specific intermediary effector/accessory proteins (5Benos D.J. Stanton B.A. J. Physiol. (Lond.). 1999; 520: 631-644Crossref Scopus (153) Google Scholar). The most well described regulation of ENaC involving the cytosolic tails of the channel is down-regulation of activity upon binding of the ubiquitin ligase Nedd4. The WW domains within Nedd4 target this protein and similar ligases to PY motifs (XPPXY) in the distal portions of the ENaC COOH termini promoting ubiquitination of the NH2 terminus of α- and γENaC subunits and subsequent internalization of the channel (20Abriel 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, 21Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar). The cytosolic COOH tails of ENaC also contain a tyrosine-based endocytic tag overlapping the PY motif (YXXL) (22Shimkets 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) that in some instances is functionally independent of the PY motif at least in γENaC (15Hendron E. Patel P. Hausenfluke M. Gamper N. Shapiro M.S. Booth R.E. Stockand J.D. J. Biol. Chem. 2002; 277: 34480-34488Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Moreover, COOH tails contain SH3 binding domains (23Rotin 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 (218) Google Scholar). Such a domain in αENaC binds the SH3 domain within α-spectrin and has been implicated in localizing the channel to the luminal membrane in epithelia. In addition, the COOH termini of β- and γENaC may impact ENaC open probability by promoting channel closing (24Copeland 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, 25Ji H.L. Fuller C.M. Benos D.J. J. Biol. Chem. 1999; 274: 37693-37704Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). All of these COOH-terminal domains, described previously (15Hendron E. Patel P. Hausenfluke M. Gamper N. Shapiro M.S. Booth R.E. Stockand J.D. J. Biol. Chem. 2002; 277: 34480-34488Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar), are more distal than the membrane reactive domain we recently identified in the COOH tail of γENaC. The NH2 terminus of αENaC is required for normal channel function (16Chalfant M.L. Denton J.S. Langloh A.L. Karlson K.H. Loffing J. Benos D.J. Stanton B.A. J. Biol. Chem. 1999; 274: 32889-32896Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Overexpression of a peptide containing this region of αENaC acts as a competitive inhibitor of wild type channels. Channels missing the first 109 residues of αENaC, in addition, have decreased activity; however, they localize to the plasma membrane (16Chalfant M.L. Denton J.S. Langloh A.L. Karlson K.H. Loffing J. Benos D.J. Stanton B.A. J. Biol. Chem. 1999; 274: 32889-32896Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 26Berdiev B.K. Karlson K.H. Jovov B. Ripoll P.J. Morris R. Loffing-Cueni D. Halpin P. Stanton B.A. Kleyman T.R. Ismailov I.I. Biophys. J. 1998; 75: 2292-2301Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). This region also contains another possible endocytic tag (KGDK) (16Chalfant M.L. Denton J.S. Langloh A.L. Karlson K.H. Loffing J. Benos D.J. Stanton B.A. J. Biol. Chem. 1999; 274: 32889-32896Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and a well conserved 5-amino acid tract containing a glycine (Gly95) crucial to normal channel gating (27Grunder S. Jaeger N.F. Gautschi I. Schild L. Rossier B.C. Pfluegers Arch. 1999; 438: 709-715Crossref PubMed Scopus (69) Google Scholar). Interestingly, α-rENaC is differentially spliced to produce αENaC subunits with unique NH2 termini (28Thomas C.P. Auerbach S. Stokes J.B. Volk K.A. Am. J. Physiol. 1998; 274: C1312-C1323Crossref PubMed Google Scholar). The functional ramifications of this have yet to be determined. Similar to the NH2 terminus and to the COOH terminus of the other subunits, the COOH terminus of αENaC plays a role in modulating channel activity. Binding of actin to the COOH-tail of α-rENaC increases channel open probability but decreases conductance (24Copeland 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 COOH terminus of αENaC also contains a region that supports channel activity and is involved in kinase regulation of the channel (19Volk K.A. Snyder P.M. Stokes J.B. J. Biol. Chem. 2001; 276: 43887-43893Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In particular, residues Pro595 and Gly596 in αENaC are critical to normal localization of the channel to the plasma membrane. Thus, there is convincing evidence that the cytosolic tails of ENaC subunits affect channel activity by impacting both channel locale and gating. However, only a few residues and specific domains within these regions of ENaC have been identified in detail and linked to function. Gupta and Canessa (29Gupta S.S. Canessa C.M. FEBS Lett. 2000; 481: 77-80Crossref PubMed Scopus (10) Google Scholar) reported previously that heterologous expression of α- and β-rENaC results in yeast becoming salt- and amiloride-sensitive, demonstrating that this recombinant channel is active in this background. In the current study, we built on this earlier work by using a simple yeast one-hybrid complementation screen to define regions within the cytosolic tails of ENaC important to protein-protein and/or protein-lipid interactions at the plasma membrane. Importantly, we determined that a region within the COOH-terminal tail of γENaC identified with our yeast screen had functional ramifications in a mammalian system. Materials—All chemicals were of reagent grade and purchased from either Sigma or Fisher unless noted otherwise. The BCA Protein Assay was from Pierce. All materials used in Western blot analysis were from Bio-Rad. The monoclonal anti-Myc antibody was from Clontech (Palo Alto, CA), and the anti-HA was from Roche Applied Science. Antimouse horseradish peroxidase-conjugated 2o antibody was from Kirkegaard & Perry Laboratories (Gaithersburg, MD). ECL reagents were from PerkinElmer Life Sciences. All DNA sequencing was performed by the molecular biology core facility at the University of Texas Health Science Center, San Antonio. The Saccharomyces cerevisiae cdc25H yeast strain (cdc25H: MATα ura3-52 his3-200 ade2-101 lys2-801 trp1-90 leu2-3,112 cdc25-2 (ts) Gal +) and the pSOS plasmid were from Stratagene (La Jolla, CA). The pECFP-M, pEGFP-F, pDsRed2-N, pCMV-Myc, and pCMV-HA plasmids were from Clontech. The plasmids encoding mouse ENaC subunit cDNAs have been described previously (30Ahn Y.J. Brooker D.R. Kosari F. Harte B.J. Li J. Mackler S.A. Kleyman T.R. Am. J. Physiol. 1999; 277: F121-F129Crossref PubMed Google Scholar) and were the gift from Dr. T. R. Kleyman. Plasmids—Full-length mouse α-, β-, and γENaC were ligated inframe behind the epitope tag into pCMV-Myc and pCMV-HA by using XhoI and NotI. Initially, channel subunits were amplified from the original pBluescript (SK–) plasmids described by Ahn and colleagues (30Ahn Y.J. Brooker D.R. Kosari F. Harte B.J. Li J. Mackler S.A. Kleyman T.R. Am. J. Physiol. 1999; 277: F121-F129Crossref PubMed Google Scholar) with standard PCRs. For α-, β-, and γ-mENaC, the upstream and downstream primers were 5′-CGAACTCGAGTTATGCTGGACCACACCAGAGC and 5′-GCAAGCGGCCGCTCAGAGTGCCATGGCCGGAGC; 5′-CGAACTCGAGTTATGCCAGTGAAGAAGTACC and 5′-GCAAGCGGCCGCCTAGATGGCCTCCACCTCACTG; and 5′-CGAACTCGAGTTATGGCGCCTGGAGAGAAG and 5′-GCAAGCGGCCGCTTAGAACTCATTGGTCAACTG, respectively. These primer sets engineered XhoI and NotI sites in each of the respective subunit cDNAs. The plasmids encoding fusion proteins of the full mENaC cytosolic tails and EGFP, as well as hSOS, have been described previously (15Hendron E. Patel P. Hausenfluke M. Gamper N. Shapiro M.S. Booth R.E. Stockand J.D. J. Biol. Chem. 2002; 277: 34480-34488Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Mutagenesis of pSOS-ENaC and pMyc-γENaC Constructs—Mutagenesis of pSOS-ENaC and its derivatives was completed using QuikChange (Stratagene) site-directed mutagenesis per the manufacturer's instructions. The primers and templates used to create the deletion and truncation mutations used in the current study are listed in Table I. The pMyc-γENaCΔQ573-P600 deletion mutant was generated with 5′-GCCGCCAGTGGGCCCTGGATACGG and 5′-CCGTATCCAGGGCCCACTGGCGGC upstream and downstream primers, respectively, in conjunction with full-length pCMV-myc-γENaC. The pMyc-γENaCΔT584-P600 and -γENaCΔQ573-R583 deletion mutants were generated with the 5′-CCCGTAGGCGGGCCCTGGATACG and 5′-CGTATCCAGGGCCCGCCTACGGG upstream and downstream primers, respectively, and the 5′-GCCGCCAGTGGACACCACCCTCC and 5′-GGAGGGTGGTGTCCACTGGCGGC upstream and downstream primers, respectively, in conjunction with full-length pCMV-myc-γENaC. All constructs were sequenced to ensure proper mutagenesis and to confirm orientation, reading frame, and sequence identity.Table IMutagenesis primers for pSOS-ENaC constructsENaC subunitaThe primers used to generate pSOS-αM1-A113, -αM610-end, -βM1-W52, -βA555-end, -γM1-W56, and -γA568-end, as well as the pSOS-γENaC mutants -γA568-P600, -γA601-end, and -γT584-600 have been described previously (15).SequencePrimerTemplateαMet1—Gln545′-GCGTGAAGAACAGTAATTAATTAATTAACCGCGGCC-3′pSOS-αM1-A1135′-GGCCGCGGTTAATTAATTAATTACTGTTCTTCACGC-3′Ala55—Ala1135′-GTAGGATCCCCCAGGCGCTGGG-3′pSOS-αM1-A1135′-CCCAGCGCCTGGGGGATCCTAC-3′Met1—Ser255′-CCGAAGGGATCCTAATTAATTAATTAACC-3′pSOS-αM1-Q545′-GGTTAATTAATTAATTAGGATCCCTTCGG-3′Met26—Gen545′-ATCCCCATGGCCATGAAGGGCAAC-3′pSOS-αM1-Q545′-GTTGCCCTTCATGGCCATGGGGAT-3′Met610—Pro6665′-CCTTCTTTGCCCTGAGAGAGGGCG-3′pSOS-αM610-End5′-CGCCCTCTCTCAGGGCAAAGAAGG-3′Gen667—End5′-GTAGGATCCCCCAGCAGGGCACG-3′pSOS-αM610-End5′-CGTGCCCTGCTGGGGGATCCTAC-3′Met610—Val6325′-GCCAGGGAGGTGTGAGAGAGGGCG-3′pSOS-αM610-P6665′-CGCCCTCTCTCACACCTCCCTGGC-3′Ala633—Pro6665′-CCATGTTACTGGCCTCTACCCC-3′pSOS-αM610-P6665′-GGGGTAGAGGCCAGTAACATGG-3′βAla555—Ser5945′-CCTGACACAACCAGCTAGATCCGCGG-3′PSOS-βA555-End5′-CCGCGGATCTAGCTGGTTGTGTCAGG-5′Cya595—End5′-GTAGGATCCTCTGCAGGCCCCACG-3′PSOS-βA555-End5′-CGTGGGGCCTGCAGAGGATCCTAC-3′Cya595—Thr6135′-ATCCCGGGGACTTAGATCCGCGG-3′PSOS-βC595-End5′-CCGCGGATCTAAGTCCCCGGGAT-5′Arg622—End5′-AGTAGGATCCTCAGGCTGCAGCCG-3′PSOS-βC595-End5′-CGGCTGCAGCCTGAGGATCCTACT-3′γAla568—Arg5835′-CGTAGGCGGTGACTGGGAAAACC-3′pSOS-γA568-P6005′-GGTTTTCCCAGTCACCGCCTACG-3′a The primers used to generate pSOS-αM1-A113, -αM610-end, -βM1-W52, -βA555-end, -γM1-W56, and -γA568-end, as well as the pSOS-γENaC mutants -γA568-P600, -γA601-end, and -γT584-600 have been described previously (15Hendron E. Patel P. Hausenfluke M. Gamper N. Shapiro M.S. Booth R.E. Stockand J.D. J. Biol. Chem. 2002; 277: 34480-34488Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Open table in a new tab Yeast One-hybrid Complementation Screen—The one-hybrid screen used in the current study has been described previously (15Hendron E. Patel P. Hausenfluke M. Gamper N. Shapiro M.S. Booth R.E. Stockand J.D. J. Biol. Chem. 2002; 277: 34480-34488Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). In brief, S. cerevisiae cdc25H yeast transformed with pSOS-ENaC constructs were initially plated on SD/glucose (–Leu) agar at 24 °C. Colonies were allowed to form over a 4-day period. Colonies were then patched from the source plate in duplicate onto SD/glucose (–Leu) plates to create two identical arrays. Arrays were grown in parallel at permissive (24 °C) and restrictive (37 °C) temperatures. Colony formation (growth) was quantified after 2 days (refer to Fig. 1). Cdc25 is the yeast homologue of hSOS and is required for Ras-dependent growth. The yeast strain used in the current study has temperature-sensitive Cdc25 that is not functional at restrictive temperatures (37 °C). Because pSOS encodes a truncated hSOS incapable of membrane localization, hSOS-ENaC fusion proteins only activate Ras signaling and thus initiate yeast growth when information in the ENaC portion of the hybrid protein enables the full fusion protein to localize at or near the plasma membrane. Digital images of yeast plates were captured with a DC4800 Zoom Digital Camera (Eastman Kodak Co.) interfaced with a personal computer running Kodak IFS Core software. ENaC Expression in CHO Cells—CHO cells were maintained in culture with Dulbecco's modified Eagle's medium supplemented with 10% FBS and antibiotics (penicillin and streptomycin) by using standard methods (31Gamper N. Stockand J.D. Shapiro M.S. J. Neurosci. 2003; 23: 84-95Crossref PubMed Google Scholar). For patch clamp analysis and confocal imaging, cells were plated on coverglass chips treated with 0.01% polylysine. Plated cells were transfected with four plasmids encoding α-, β-, and γENaC and GFP using the PolyFect reagent (Qiagen, Valencia, CA) per the manufacturer's recommendations. In brief, cells ∼60% confluent in a 35-mm dish were treated with 2.5 μg of total plasmid cDNA for 24–48 h. Cells were used for patch clamp analysis up to 96 h after transfection and were maintained in culture in the presence of 10 μm amiloride replenished daily. Cells used for protein analysis were grown in 100-mm dishes, transfected with 4 μg of total plasmid cDNA, and extracted 24–48 h after transfection. Confocal Imaging—Transfected CHO cells were grown on number 0 coverglass, fixed in 4% paraformaldehyde, and mounted using Vectashield (Vector Laboratories, Burlingame, CA). Confocal images were collected using a ×60 (1.3 NA) oil-immersion lens on a Nikon Eclipse TE2000 (Nikon Instruments, Melville, NY) inverted microscope fitted with a Cascade Photometric CCD camera (Roper Scientific, Tucson, AZ), the CARV confocal fluorescence imaging unit (Kinetic Imagine, Weston, Ontario, Canada), and a Lambda 10-2 filter wheel (Sutter Instruments, Novato, CA). This unit is driven by the Metamorph program suite (Universal Imaging Corp., Downingtown, PA) and interfaced with a piezosystem (Piezosystem Jena, Hopedale, MA) for Z-series imaging. For DsRed2 a triple pass polychroic emission filter (D/F/TXRD 62002; Chroma Technology Corp., Brattleboro, VT) was used in conjunction with a single pass excitation filter. For ECFP, images were collected using the yellow fluorescent protein/cyan fluorescent protein dual pass polychroic emission filter (cyan fluorescent protein/yellow fluorescent protein 51017v2; Chroma Technology Corp.) in conjunction with a single pass excitation filter. EGFP was visualized with a single pass dichroic emission filter (endow GFP 41017; Chroma Technology Corp.) in conjunction with a single pass excitation filter. With these filter sets, fluorophores were easily discriminated with no bleed through (see Fig. 2). Evanescent-field (EF) Fluorescence Microscopy—To selectively illuminate the plasma membrane and its associated channel subunits, we used EF microscopy. Cells used for EF microscopy were plated on glass coverslips and fixed as above for confocal imaging. Methods followed closely those described previously by Almers and colleagues (32Merrifield C.J. Feldman M.E. Wan L. Almers W. Nat. Cell Biol. 2002; 4: 691-698Crossref PubMed Scopus (561) Google Scholar, 33Taraska J.W. Perrais D. Ohara-Imaizumi M. Nagamatsu S. Almers W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2070-2075Crossref PubMed Scopus (302) Google Scholar). In brief, EF microscopy was performed using an inverted TE2000 microscope with through-the-lens fluorescence imaging. EF illumination was generated by total internal reflection fluorescence (TIRF) after the light beam struck the interface between the glass coverslip and cellular plasma membrane at a glancing angle (34Steyer J.A. Almers W. Nat. Rev. Mol. Cell. Biol. 2001; 2: 268-275Crossref PubMed Scopus (340) Google Scholar). Samples were viewed through a Plan Apo TIRF ×60 oil-immersion, high resolution (1.45 NA) objective (Nikon). TIRF generates an EF that declines exponentially with increasing distance from the interface between the cover glass and plasma membrane illuminating only a small optical slice of the cell (∼200 nm) including the plasma membrane. Thus, with TIRF only fluorophores in the plasma membrane and its immediate vicinity contribute to emission, whereas those deeper in the cell do not (see Fig. 8A). DsRed2 and EGFP-F were excited with green HeNe and argon lasers, respectively, with emissions subsequently passing through 543- and 488-nm single pass filters, respectively. This system was also interfaced with a mercury lamp with appropriate dichroic excitation and emissions filter sets enabling wide field epifluorescence imaging of DsRed2 and EGFP-F. Images were collected and processed as above with a CCD camera interfaced to a PC running Metamorph software. Patch Clamp Recording and Single Channel Analysis—Whole-cell macroscopic current recordings of ENaC reconstituted in CHO cells were made under voltage clamp conditions using standard methods (31Gamper N. Stockand J.D. Shapiro M.S. J. Neurosci. 2003; 23: 84-95Crossref PubMed Google Scholar). Prior to patch clamp analysis, cells were rinsed of culture media and amiloride and patched at room temperature under constant perfusion in a bath solution of (in mm) 160 NaCl, 1 CaCl2, 2 MgCl2, and 10 HEPES (pH 7.4, 320 mOsm). Pipette solution was (in mm) 145 KCl, 5 NaCl, 2 MgCl2, 0.5 CaCl2, 10 EGTA, 10 HEPES (pH 7.4), 3.0 ATP, and 0.1 GTP (330 mOsm). Current recordings were acquired with a PC-505B patch clamp amplifier (Warner Instruments; Hamden, CT) interfaced via a Digidata 1320A (Axon Instruments, Union City, CA) with a PC running the pClamp 8.1 suite of software. All currents were filtered at 1 kHz. Both a family of test pulses stepping by 20-mV increments (500 ms each separated by 400 ms) form –120 to +100 mV, and voltage ramps (100 ms) over the same range were used to generate current-voltage (I-V) relations. The whole-cell capacitance was routinely compensated and was approximately ∼12 picofarads for CHO cells. Series resistances, on average 2–5 megohms, were also compensated. Currents, however, were not leak-corrected. For all experiments, holding potential was 30–50 mV. For v
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