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• W2022689385 abstract "Proton-gated Na+ channels (ASIC) are new members of the epithelial sodium channel/degenerin gene family. ASIC3 mRNA has been detected in the homogenate of pulmonary tissues. However, whether ASIC3 is expressed in the apical membranes of lung epithelial cells and whether it regulates cystic fibrosis transmembrane conductance regulator (CFTR) function are not known at the present time. Using reverse transcription-PCR, we found that the ASIC3 mRNA was expressed in the human airway mucosal gland (Calu-3) and human airway epithelial (16HBE14o) cells. Indirect immunofluorescence microscopy revealed that ASIC3 was co-segregated with CFTR in the apical membranes of Calu-3 cells. Proton-gated, amiloride-sensitive short circuit Na+ currents were recorded across Calu-3 monolayers mounted in an Ussing chamber. In whole-cell patch clamp studies, activation of CFTR channels with cAMP reduced proton-gated Na+ current in Calu-3 cells from -154 ± 28 to -33 ± 16 pA (n = 5, p < 0.05) at -100 mV. On the other hand, cAMP-activated CFTR activity was significantly inhibited following constitutive activation of putative ASIC3 at pH 6.0. Immunoassays showed that both ASIC3 and CFTR proteins were expressed and co-immunoprecipitated mutually in Calu-3 cells. Similar results were obtained in human embryonic kidney 293T cells following transient co-transfection of ASIC3 and CFTR. Our results indicate that putative CFTR and ASIC3 channels functionally interact with each other, possibly via an intermolecular association. Because acidic luminal fluid in the cystic fibrosis airway and lung tends to stimulate ASIC3 channel expression and activity, the interaction of ASIC3 and CFTR may contribute to defective salt and fluid transepithelial transport in the cystic fibrotic pulmonary system. Proton-gated Na+ channels (ASIC) are new members of the epithelial sodium channel/degenerin gene family. ASIC3 mRNA has been detected in the homogenate of pulmonary tissues. However, whether ASIC3 is expressed in the apical membranes of lung epithelial cells and whether it regulates cystic fibrosis transmembrane conductance regulator (CFTR) function are not known at the present time. Using reverse transcription-PCR, we found that the ASIC3 mRNA was expressed in the human airway mucosal gland (Calu-3) and human airway epithelial (16HBE14o) cells. Indirect immunofluorescence microscopy revealed that ASIC3 was co-segregated with CFTR in the apical membranes of Calu-3 cells. Proton-gated, amiloride-sensitive short circuit Na+ currents were recorded across Calu-3 monolayers mounted in an Ussing chamber. In whole-cell patch clamp studies, activation of CFTR channels with cAMP reduced proton-gated Na+ current in Calu-3 cells from -154 ± 28 to -33 ± 16 pA (n = 5, p < 0.05) at -100 mV. On the other hand, cAMP-activated CFTR activity was significantly inhibited following constitutive activation of putative ASIC3 at pH 6.0. Immunoassays showed that both ASIC3 and CFTR proteins were expressed and co-immunoprecipitated mutually in Calu-3 cells. Similar results were obtained in human embryonic kidney 293T cells following transient co-transfection of ASIC3 and CFTR. Our results indicate that putative CFTR and ASIC3 channels functionally interact with each other, possibly via an intermolecular association. Because acidic luminal fluid in the cystic fibrosis airway and lung tends to stimulate ASIC3 channel expression and activity, the interaction of ASIC3 and CFTR may contribute to defective salt and fluid transepithelial transport in the cystic fibrotic pulmonary system. Increased reabsorption of Na+ ions via apically located Na+ channels is a major pathophysiological feature in cystic fibrosis (CF) 2The abbreviations used are: CF, cystic fibrosis; ENaC, epithelial sodium channel; CFTR, cystic fibrosis transmembrane conductance regulator; ASIC, proton-gated sodium channel; RT, reverse transcription; MES, 4-morpholineethanesulfonic acid; HEK, human embryonic kidney. epithelia (1Kunzelmann K. Schreiber R. Nitschke R. Mall M. Pfluegers Arch. 2000; 440: 193-201PubMed Google Scholar, 2Mall M. Bleich M. Greger R. Schreiber R. Kunzelmann K. J. Clin. Investig. 1998; 102: 15-21Crossref PubMed Scopus (185) Google Scholar). The molecular basis of the hyperactive amiloride-sensitive Na+ channels is not clear. Several cation channels, including epithelial Na+ channels (ENaC), cyclic nucleotide-gated Na+ channels, non-selective cation channels, and acid-sensitive ion channels (ASIC), are detected in lung epithelia. Amiloride inhibits these apically located Na+ channels with varying efficacies. CFTR down-regulates amiloride-sensitive αβγ ENaC activity in Xenopus oocytes (3Schreiber R. Hopf A. Mall M. Greger R. Kunzelmann K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5310-5315Crossref PubMed Scopus (83) Google Scholar, 11Boucherot A. Schreiber R. Kunzelmann K. Biochim. Biophys. Acta. 2001; 1515: 64-71Crossref PubMed Scopus (22) Google Scholar). These results are in agreement with observations from normal and CF tissues (12Knowles M. Gatzy J. Boucher R. N. Engl. J. Med. 1981; 305: 1489-1495Crossref PubMed Scopus (458) Google Scholar, 17Ecke D. Bleich M. Greger R. Pfluegers Arch. 1996; 431: 984-986Crossref PubMed Scopus (42) Google Scholar). On the other hand, ENaC up-regulates the CFTR current by up to 6-fold, and this process is not Na+-dependent (7Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 8Ji H.L. Chalfant M.L. Jovov B. Lockhart J.P. Parker S.B. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2000; 275: 27947-27956Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 18Chabot H. Vives M.F. Dagenais A. Grygorczyk C. Berthiaume Y. Grygorczyk R. J. Membr. Biol. 1999; 169: 175-188Crossref PubMed Scopus (46) Google Scholar, 19Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Mutagenesis studies have identified the first nucleotide binding domain of CFTR and Cl- transport as being required for CFTR-mediated down-regulation of αβγ ENaC (3Schreiber R. Hopf A. Mall M. Greger R. Kunzelmann K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5310-5315Crossref PubMed Scopus (83) Google Scholar, 11Boucherot A. Schreiber R. Kunzelmann K. Biochim. Biophys. Acta. 2001; 1515: 64-71Crossref PubMed Scopus (22) Google Scholar), whereas the cytosolic C termini of β and γ ENaC subunits are involved in the intermolecular regulation between CFTR and αβγ ENaC (7Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 8Ji H.L. Chalfant M.L. Jovov B. Lockhart J.P. Parker S.B. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2000; 275: 27947-27956Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In contrast to the extensive studies of the ENaC channels and their interactions with CFTR in the last three decades, little is known about the contribution of other apical cation channels of normal and CF epithelia on CFTR function. A new branch of the ENaC/degenerin family, the proton-gated Na+ channels (ASIC1a, -1b, -2a, -2b, -3, and -4), has been cloned from neural tissue (20Waldmann R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N. Y. Acad. Sci. 1999; 868: 67-76Crossref PubMed Scopus (180) Google Scholar, 22Krishtal O. Trends Neurosci. 2003; 26: 477-483Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). ASIC3 activity and expression are regulated by low extracellular pH, divalent cations, inflammation, and ischemia, all of which are commonly featured in CF lungs (20Waldmann R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N. Y. Acad. Sci. 1999; 868: 67-76Crossref PubMed Scopus (180) Google Scholar, 22Krishtal O. Trends Neurosci. 2003; 26: 477-483Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 23Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1036) Google Scholar). Distribution of ASIC and ENaC overlaps in both epithelial and neural tissue. For example, in the human testis, expression of CFTR, ASIC3, and all four ENaC subunits have been detected (24Leung G.P. Gong X.D. Cheung K.H. Cheng-Chew S.B. Wong P.Y. Biol. Reprod. 2001; 64: 1509-1515Crossref PubMed Scopus (19) Google Scholar, 26Waldmann R. Champigny G. Bassilana F. Voilley N. Lazdunski M. J. Biol. Chem. 1995; 270: 27411-27414Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Analysis of CF fluid collected from epithelial organs (e.g. airway, pancreas, intestine, and testis) confirmed that CF epithelial secretions, in addition to being thicker and having less volume, have an abnormal ionic composition and an acidic pH (27Wong P.Y. Reprod. Fertil. Dev. 1990; 2: 115-127Crossref PubMed Scopus (17) Google Scholar, 32Song Y. Salinas D. Nielson D.W. Verkman A.S. Am. J. Physiol. 2006; 290: C741-C749Crossref PubMed Scopus (131) Google Scholar). The condensate from the exhaled breath of CF patients is also more acidic (pH 5.3 versus 6.1) (33Ojoo J.C. Mulrennan S.A. Kastelik J.A. Morice A.H. Redington A.E. Thorax. 2005; 60: 22-26Crossref PubMed Scopus (140) Google Scholar). Although ASIC3 mRNA has been detected in a number of epithelial organs (34Waldmann R. Bassilana F. de Weille J. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 36Gitterman D.P. Wilson J. Randall A.D. J. Physiol. (Lond.). 2005; 562: 759-769Crossref Scopus (39) Google Scholar), its cellular expression and physiological role in the lung have not been studied. To test the hypothesis that ASIC3 mRNA and proteins are expressed in the apical membrane of pulmonary epithelial cells and contribute to elevated Na+ channel activity in CF epithelia, we studied the expression of ASIC3 channels and possible interaction with CFTR in Calu-3 cells. Our results confirm that ASIC3 and ASIC2 mRNAs were expressed in Calu-3 cells, human airway, pancreatic, and colonic epithelial cells. ASIC3 and CFTR co-segregated in the apical membrane of polarized Calu-3 monolayers. Co-immunoprecipitation of native and heterologously expressed CFTR and ASIC3 proteins suggests that these two proteins may interregulate each other via a physical association. ASIC3 channels functionally contribute to transepithelial Na+ transport, which is down-regulated by CFTR in Calu-3 cells. Reverse Transcription (RT)-PCR—Total RNA was isolated from Calu-3 (submucosal cells), 16HBE14o (bronchial cells), CFPAC (pancreatic cells), and T84 (colonic cells) using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA concentration was assessed by spectrophotometry at 260 nm. RNA quality was confirmed by running 5 μg of total RNA from each preparation on a 1.2% denatured agarose gel. The first strand cDNAs were synthesized using the Superscript™ first strand synthesis system for RT-PCR kit (Invitrogen). For RT-PCR analysis of ASIC3 expression, the forward and reverse primers were 5′-TAT GAG ACC GTG GAG CAG-3′ and 5′-TGT GTG ACA AGG TAG CAG G-3′, which correspond to nucleotides 1279-1296 and 1610-1592, respectively, of ASIC3 (GenBank™ accession number AF057711) (37Berdiev B.K. Xia J. McLean L.A. Markert J.M. Gillespie G.Y. Mapstone T.B. Naren A.P. Jovov B. Bubien J.K. Ji H.L. Fuller C.M. Kirk K.L. Benos D.J. J. Biol. Chem. 2003; 278: 15023-15034Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). PCR was performed in a Mastercycler gradient thermocycler (Eppendorf Scientific). Two microliters of the first strand product was used as a template in each 50-μl PCR reaction, and PCR conditions were optimized using the PCR Optimizer™ kit (Invitrogen). The cycling parameters consisted of one cycle of 95 °C for 3 min and then 30 cycles of 95 °C for 0.5 min, 58 °C for 0.5 min, and 72 °C for 1.5 min followed by a single 10-min cycle at 72 °C for extension. RT-PCR products were electrophoresed on a 2% agarose gel using 100-bp PCR markers (Promega) as a standard to determine the molecular size. Products of the predicted molecular size were excised and purified from the gel using the QIAquick gel extraction kit (Qiagen) and subcloned into the pCR-2.1 vector (Invitrogen). Positive clones were selected by blue/white screening and followed by digestion with EcoRI (Promega) to verify incorporation of the insert of the correct size. PCR products in the pCR2.1 vector were verified by automated DNA sequencing (DNA Sequencing Facility, Iowa State University). Immunofluorescent Staining—Calu-3 cells were grown as polarized monolayers on 12-mm tissue culture inserts (Costar). Three percent formaldehyde-fixed cells were stained by incubating with the primary antibodies (rabbit polyclonal anti-ASIC3 antibody from Neuromics diluted 1:50 in phosphate-buffered saline with 5% bovine serum albumin (blocking buffer) and mouse monoclonal anti-CFTR antibody from Chemicon 1:100 in blocking buffer) for 2 h at room temperature. The samples were rinsed in phosphate-buffered saline following blocking buffer incubation and exposed to the secondary antibodies (1:1000; Molecular Probes, Eugene, OR) for 2 h at room temperature. The samples were mounted and imaged using an Olympus 1 × 70 epifluorescence microscope. Negative controls consisted of substituting non-immune IgG for the primary antibody. For imaging side views of the Calu-3 monolayers, the filters were folded sharply, and the cells at the folded edge were photographed. Oocyte Expression and Voltage Clamp Assay— cRNA for ASIC3 and CFTR were prepared as previously described (8Ji H.L. Chalfant M.L. Jovov B. Lockhart J.P. Parker S.B. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2000; 275: 27947-27956Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Defolliculated oocytes were cytosolically injected with a 1:1 ratio of ASIC3 and CFTR cRNA (25 ng of cRNA in 50 nl of RNase-free water/oocyte) and incubated in half-strength L-15 medium. Whole-cell ASIC3 and CFTR currents were measured by the two-electrode voltage clamp technique 2 days after injection (8Ji H.L. Chalfant M.L. Jovov B. Lockhart J.P. Parker S.B. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2000; 275: 27947-27956Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Deparaffinized human lung tissue sections were provided by University of Alabama at Birmingham Human Tissue Procurement. Sections from both CF patients and healthy controls were incubated with anti-ASIC3 antibody and fluorescein isothiocyanate-conjugated IgG using similar protocols as described above for Calu-3 monolayers. Patch Clamp Experiments—Calu-3 cells were grown on coverslips and cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (38Bebok Z. Varga K. Hicks J.K. Venglarik C.J. Kovacs T. Chen L. Hardiman K.M. Collawn J.F. Sorscher E.J. Matalon S. J. Biol. Chem. 2002; 277: 43041-43049Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In brief, patch clamp experiments were performed at room temperature (20-24 °C) 2-4 days after seeding (39Ji H.L. Jovov B. Fu J. Bishop L.R. Mebane H.C. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2002; 277: 8395-8405Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The whole-cell mode was used to detect the channel activity of ASIC3 and CFTR in Calu-3 cells. The reference electrode was an Ag/AgCl pellet in the bath (in mm: Na+ 130, Cl- 148, Ca2+ 2, Mg2+ 2, Ba2+ 5, HEPES 20, pH 7.4/NaOH). HEPES was substituted by MES to prepare the bath solutions at a pH below 6.0. The patch pipettes were pulled from fire-polished borosilicate glass (WPI Instruments) using a multistepped micropipette puller (model P97, Flaming/Brown) followed by fire polishing. An Axopatch 1B amplifier (Axon Instruments, Union City, CA) was used to record the current. Resistance of the electrode was 3-6 MΩ when filled with pipette solution (in mm: Cs+ 120, triethanolamine+ 20, Na+ 5, Cl- 144, Mg2+ 2, HEPES 10, EDTA 2.0, Ca2+ 0.1 (free Ca2+ 10 nm), pH 7.2/CsOH). Currents were digitized at 2-5 kHz by DigiData 1200, which was driven by CLAMPEX version 7.0 (Axon). The data were filtered at 1-2 kHz. Delivery of superfusates at various pH values were controlled by the SF-77B perfusion fast step system (Warner) and CLAMPEX version 7.1. Solutions from the square perfusion tubing with a size of 1 × 1 mm covered the clamped cell completely. Approximately 1 ms was measured to switch perfusate tubing from one to another in a chamber of 0.1 ml. Ussing Chamber Studies—Calu-3 monolayers were mounted into Ussing chambers (Jim's Instrument Manufacturing, Inc., Iowa City, IA) and bathed with solution consisting of (in mm): Na+ 145, K+ 5, Ca2+ 1.2, Mg2+ 1.2, gluconic acid 125, HEPES 10, glucose 10 (pH 7.4). Bath solution in the apical chamber contained 10 mm mannitol in place of glucose to minimize the contribution of the Na+-glucose co-transporter to the Na+ transport. Bath solutions were continuously bubbled with 100% N2, combined with both HPO4- -free and HCO3- -free medium to eliminate pH buffering and the activity of the HCO3- -dependent transporter/exchangers (pH 7.4). Bath solutions were maintained at 37 °C. The monolayers were voltage-clamped at 0 mV. Transepithelial short circuit current (Isc) and resistance (RTE) were simultaneously measured using 5-mV pulses every 20 s. Data were recorded for off-line analysis using the Acquire and Analyze program combined with an epithelial voltage clamp amplifier VCC MC8 (Physiologic Instruments, Inc., San Diego, CA). After stabilization of the basal Isc, the pH of the apical or basolateral solution was adjusted to 6.8 by 1 m HCl. A pH of 6.8 was chosen, because more acidic pH values would have altered gap junction and possibly the intracellular pH (40Peracchia C. Biochim. Biophys. Acta. 2004; 1662: 61-80Crossref PubMed Scopus (204) Google Scholar). Clinically, pH 6.8 is within the range of acidic CF luminal fluid (29Freedman S.D. Kern H.F. Scheele G.A. Gastroenterology. 2001; 121: 950-957Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 41Thiagarajah J.R. Song Y. Haggie P.M. Verkman A.S. FASEB J. 2004; 18: 875-877Crossref PubMed Scopus (97) Google Scholar, 42von Cooper T.G. Eckardstein S. Rutscha K. Meschede D. Horst J. Nieschlag E. Fertil. Steril. 2000; 73: 1226-1231Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Moreover, Verkman and co-workers (32Song Y. Salinas D. Nielson D.W. Verkman A.S. Am. J. Physiol. 2006; 290: C741-C749Crossref PubMed Scopus (131) Google Scholar) have recently reported that the pH of mucus stimulated with pilocarpine is from ∼6.6 to ∼6.9. The pH values of bath solutions were concurrently monitored by a digital pH meter during Ussing chamber experiments (Corning). Western Blotting and Co-immunoprecipitation—Calu-3 cells were split and plated, per 75-mm flask, with minimum essential medium supplemented with 10% fetal bovine serum (Cellgro), 1% glutamine, and 1% strep and penicillin for 48 h. The cells were lysed in 0.7 ml of lysis buffer (1% Triton X-100, 50 mm Tris buffer pH 7.5) with a freshly added protein inhibitor mixture tablet (44Hardiman K.M. McNicholas-Bevensee C.M. Fortenberry J. Myles C.T. Malik B. Eaton D.C. Matalon S. Am. J. Respir. Cell Mol. Biol. 2004; 30: 720-728Crossref PubMed Scopus (52) Google Scholar). Immunoprecipitation and Western blotting assays were performed as described previously (44Hardiman K.M. McNicholas-Bevensee C.M. Fortenberry J. Myles C.T. Malik B. Eaton D.C. Matalon S. Am. J. Respir. Cell Mol. Biol. 2004; 30: 720-728Crossref PubMed Scopus (52) Google Scholar, 45Ji H.L. Su X.F. Kedar S. Li J. Barbry P. Smith P.R. Matalon S. Benos D.J. J. Biol. Chem. 2006; 281: 8233-8241Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Equivalency of loading for Western blot analysis and immunoprecipitation was ensured by performing protein assays (BCA; Pierce) and using equal amounts of protein for each group. Briefly, anti-CFTR monoclonal antibody (University of Alabama at Birmingham) or anti-ASIC3 polyclonal antibody (Chemicon) was added into the cell lysates of the protein samples and rotated at 4 °C overnight. For co-immunoprecipitation, the protein complexes were immunoprecipitated by protein G-Sepharose beads (Amersham Biosciences), and the samples were loaded and run in either 6 or 8% SDS-PAGE gel. The precipitates were transferred to a polyvinylidene difluoride membrane and immunoblotted by different antibodies. All blots were developed by the Enhanced Chemiluminescence kit (Pierce). For co-immunoprecipitation in human embryonic kidney 293T (HEK293T) cells, Lipofectamine 2000 (Invitrogen) was used to transiently transfect ASIC3 and CFTR following the manufacturer's instructions. Cell protein was purified for CFTR and ASIC3 expression 2 days post-transfection. The amount of plasmid DNA used per 35-mm dish was 2 μg for CFTR in pcDNA3 and 1 μg for rat ASIC3 in pCI per 35-mm dish. Cells were lysed in 1% Triton X-100 in phosphate-buffered saline and a mixture of protease inhibitors (Roche Applied Science). CFTR was immunoprecipitated using 1 μg of anti-C-terminal CFTR monoclonal antibody cross-linked to protein A/G-agarose beads (Santa Cruz Biotechnology). After incubating the lysates with cross-linked antibody, the beads were pelleted and washed three times in phosphate-buffered saline containing 1% Triton X-100, and the samples were processed for Western blotting using the monoclonal anti-CFTR antibody (Chemicon) or polyclonal anti-ASIC3 antibody (Neuromics). Data Analysis—Proton-activated Na+ current was calculated as the difference between the basal current and sustained current at acidic pH. The EC50 value of external pH was computed by fitting the dose-response curve with the Hill equation INa = Imax · pHon/(pHon + EC50n) (39Ji H.L. Jovov B. Fu J. Bishop L.R. Mebane H.C. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2002; 277: 8395-8405Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), where INa represents the proton-activated current, Imax the maximal proton-activated current, pHo the extraoocyte pH, EC50 the value of pHo that results in half of the maximal current of the ASIC3 channel, and n the Hill coefficient. All average results are presented as mean ± S.E. One-way analysis of variance computation combined with the Bonferroni test was used to analyze data with unequal variance between each group. A probability level of 0.05 was considered significant. ASIC3 mRNA in CFTR-expressing Human Epithelial Cells— The expression of ASIC3 in airway and other epithelial cells has not been examined previously. Our RT-PCR results revealed that ASIC3 mRNA was present in CFTR-expressing human epithelial cell lines Calu-3 (submucosal cells), 16HBE14o (bronchial cells), CFPAC (pancreatic cells), and T84 (colonic cells) (Fig. 1). However, ASIC1 (585 bp) was not detected in Calu-3 cells. Interestingly, expression of ASIC2 mRNA was also detected in these epithelial cells. The RT-PCR products (585 bp for ASIC1, 503 bp for ASIC2, and 351 bp for ASIC3) from each cell line were ligated into the pCR2.1 vector and sequenced. BLAST analysis revealed that sequences of the RT-PCR products were identical to corresponding human ASIC1, -2, and -3. ASIC3 Protein Expression in Human CF Lung Tissues—Because ASIC channels are regulated by protons and inflammatory mediators (46Mamet J. Baron A. Lazdunski M. Voilley N. J. Neurosci. 2002; 22: 10662-10670Crossref PubMed Google Scholar, 47Voilley N. de Weille J. Mamet J. Lazdunski M. J. Neurosci. 2001; 21: 8026-8033Crossref PubMed Google Scholar), the expression of ASIC3 in CF lung may be enhanced by acidic luminal fluid and infection. To examine the expression of ASIC3 in normal and CF lung tissues, we immunolabeled human lung tissue sections with a specific anti-ASIC3 antibody. As shown in Fig. 2, ASIC3 was expressed in the pneumocytes of human lung alveoli. In CF lung, the normal alveolar structure was altered by the aggregation of inflammatory cells and propagation of non-flat abnormal alveolar cells (Fig. 2, E and F). ASIC3 expression, as shown by the brightness of the green color, is greater in CF lung sections due to inflammation than that in non-CF tissues. Immunolabeling of ASIC3 was not detected when the anti-ASIC3 antibody was neutralized with ASIC3 immunopeptide (Fig. 2, A and D). Co-segregation of ASIC3 and CFTR in the Apical Membrane of Calu-3 Monolayers—To examine the subcellular location of ASIC3 and its possible co-expression with CFTR, Calu-3 monolayers grown on filters and cultured with an air-liquid interface were incubated with specific anti-ASIC3 (green channel) and anti-CFTR (red channel) antibodies. Lateral views of the monolayers show that ASIC3 is located in the apical membrane of most Calu-3 cells (Fig. 2, A and D). In agreement with previous observations (38Bebok Z. Varga K. Hicks J.K. Venglarik C.J. Kovacs T. Chen L. Hardiman K.M. Collawn J.F. Sorscher E.J. Matalon S. J. Biol. Chem. 2002; 277: 43041-43049Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), CFTR was also observed in the apical membrane of Calu-3 cells (Fig. 2, B and E). Merged images indicate that ASIC3 and CFTR are co-segregated with each other within the apical membrane domain of Calu-3 cells (Fig. 2, C and F). Apical labeling of the monolayers was not observed when non-immune rabbit and mouse IgG were substituted for the anti-ASIC3 and -CFTR antibodies, respectively. To verify these immunostaining results, Western blot assay was used to determine the ASIC3 and CFTR protein in Calu-3 cells (Fig. 3, B and C). A 75-kDa protein was shown with an identical size as in dorsal root ganglia (Fig. 3B). As previously reported (38Bebok Z. Varga K. Hicks J.K. Venglarik C.J. Kovacs T. Chen L. Hardiman K.M. Collawn J.F. Sorscher E.J. Matalon S. J. Biol. Chem. 2002; 277: 43041-43049Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), CFTR protein is expressed in Calu-3 cells abundantly (Fig. 3C). ASIC3-type Channel Activity in Calu-3 Monolayers—If ASIC3 functions as a Na+ channel, the channel should be activated by lowering apical pH. To test this hypothesis, we bathed Calu-3 cells with HPO3- -free, HCO3- -free, Cl--free medium on both sides and bubbled them with pure gaseous nitrogen. Lowering the apical pH from 7.5 to 6.8 resulted in a transient followed by a sustained activation of an apical Na+ current (Fig. 4). The proton activation was repeatable. In the presence of amiloride (100 μm), an increase of apical proton concentration did not elicit acid-sensitive sustained Isc (Fig. 4). Putative Proton-gated Whole-cell Na+ Current in Calu-3 Cells—To further characterize the putative ASIC3 channels in individual Calu-3 cells, we applied a series of external pH values from 4.0 to 8.5 after the whole-cell configuration of the patch clamp assay was established (Fig. 5). In 27 of 32 cells, a transiently activated current and a sustained current were activated by protons, whereas alkali pH (8.5) inhibited basal current slightly (5A). An EC50 value of 5.2 for proton activation was determined (5.2 ± 0.1, n = 6) by fitting the dose-response curve with the Hill equation (Fig. 5B). Multiple sites were required for proton activation (Hill coefficient, 12 ± 3), which supports previous publications on this topic (20Waldmann R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N. Y. Acad. Sci. 1999; 868: 67-76Crossref PubMed Scopus (180) Google Scholar, 22Krishtal O. Trends Neurosci. 2003; 26: 477-483Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). Seventy percent of this proton-gated current was abolished in Na+-free medium. The gating pattern of proton-dependent Na+ current and the EC50 value for proton activation in Calu-3 cells were identical to those of cloned ASIC3 but not ASIC1a and -2a channels (48Hesselager M. Timmermann D.B. Ahring P.K. J. Biol. Chem. 2004; 279: 11006-11015Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The large sustained current, which tends to become stable, is a hallmark of homo- and heteromeric ASIC3, which is not observed in other ASIC channels. Down-regulation of Putative ASIC3 Na+ Channels by cAMP-activated CFTR in Calu-3 Cells—To investigate the effect of CFTR activation on the proton-gated ASIC3-type Na+ current in Calu-3 cells, proton-gated Na+ currents from a group of cells in the absence of forskolin were compared with those obtained from a parallel group in the presence of 10 μm forskolin. After CFTR activity reached its maximum in whole-cell patch clamp mode and was stabilized at neutral pH (7.5), pH 6.0 was applied to activate native ASIC3 (Fig. 5C). The proton-gated sustained Na+ current in Calu-3 cells in the presence of forskolin was -33 ± 16 pA (n = 5), which was markedly less than the controls in the absence of forskolin (-154 ± 28 pA, n = 5, p < 0.01). To exclude the direct effect of forskolin on ASIC3 activity, we expressed ASIC3 in oocytes and measured whole-cell currents using the two-electrode voltage clamp technique before and after the addition of forskolin at pH 6.0. The ASIC3 current at -60 mV was not altered by forskolin (Fig. 5D). These results were consistent with our previous observation that elevation of cAMP does not have an effect on αβγ ENaC and ASIC1a/2a channels expressed in oocytes (8Ji H.L. Chalfant M.L. Jovov B. Lockhart J.P. Parker S.B. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2000; 275: 27947-27956Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 39Ji H.L. Jovov B. Fu J. Bishop L.R. Mebane H.C. Fuller C.M. Stanton B.A. Benos D.J" @default.
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• W2022689385 title "Interregulation of Proton-gated Na+ Channel 3 and Cystic Fibrosis Transmembrane Conductance Regulator" @default.
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