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. 2012 Sep 17;590(Pt 23):6141–6155. doi: 10.1113/jphysiol.2012.240838

Association of TMEM16A chloride channel overexpression with airway goblet cell metaplasia

Paolo Scudieri 1, Emanuela Caci 1, Silvia Bruno 2, Loretta Ferrera 1, Marco Schiavon 3, Elvira Sondo 1, Valeria Tomati 1, Ambra Gianotti 1, Olga Zegarra-Moran 1, Nicoletta Pedemonte 1, Federico Rea 3, Roberto Ravazzolo 1,4, Luis J V Galietta 1
PMCID: PMC3530122  PMID: 22988141

Abstract

The TMEM16A protein has a potential role as a Ca2+-activated Cl− channel (CaCC) in airway epithelia where it may be important in the homeostasis of the airway surface fluid. We investigated the function and expression of TMEM16A in primary human bronchial epithelial cells and in a bronchial cell line (CFBE41o–). Under resting conditions, TMEM16A protein expression was relatively low. However, TMEM16A silencing with short-interfering RNAs caused a marked inhibition of CaCC activity, thus demonstrating that a low TMEM16A expression is sufficient to support Ca2+-dependent Cl− transport. Following treatment for 24–72 h with interleukin-4 (IL-4), a cytokine that induces mucous cell metaplasia, TMEM16A protein expression was strongly increased in approximately 50% of primary bronchial epithelial cells, with a specific localization in the apical membrane. IL-4 treatment also increased the percentage of cells expressing MUC5AC, a marker of goblet cells. Interestingly, MUC5AC was detected specifically in cells expressing TMEM16A. In particular, MUC5AC was found in 15 and 60% of TMEM16A-positive cells when epithelia were treated with IL-4 for 24 or 72 h, respectively. In contrast, ciliated cells showed expression of the cystic fibrosis transmembrane conductance regulator Cl− channel but not of TMEM16A. Our results indicate that TMEM16A protein is responsible for CaCC activity in airway epithelial cells, particularly in cells treated with IL-4, and that TMEM16A upregulation by IL-4 appears as an early event of goblet cell differentiation. These findings suggest that TMEM16A expression is particularly required under conditions of mucus hypersecretion to ensure adequate secretion of electrolytes and water.

Key points

  • Chloride channels are important for proper hydration of the airway surface.

  • TMEM16A protein is an important component of calcium-activated chloride channels.

  • Interleukin-4, a cytokine that induces mucous cell metaplasia, also upregulates calcium-dependent chloride secretion in human bronchial epithelial cells.

  • In bronchial epithelial cells treated with interleukin-4, we found that TMEM16A protein becomes highly expressed in goblet but not in ciliated cells.

  • Upregulation of TMEM16A by interleukin-4 may be important for secretion and proper expansion of mucins.

Introduction

The transport of Cl− and other anions across the airway epithelium plays a major role in the defence mechanisms against microbial pathogens. Secretion of Cl−, paralleled by Na+ and water, allows hydration of the airway surface and therefore maintenance of mucociliary clearance (Boucher, 2004). Airway epithelia also secrete bicarbonate. Besides contributing to regulation of airway surface liquid (ASL) pH, bicarbonate may be critical for the proper expansion of mucin granules upon exocytic secretion (Garcia et al. 2009). Another important anion secreted in the ASL is thiocyanate (SCN−). In the presence of H2O2, produced by dual oxidases, SCN− is converted by lactoperoxidase to hypothiocyanite (OSCN−), an antimicrobial molecule (Gerson et al. 2000; Moskwa et al. 2007).

Secretion of anions in the ASL is mediated by a variety of channels and transporters localized in the apical membrane of epithelial cells (Boucher, 2004). Other types of transporters and channels in the basolateral membrane are also involved as they provide the driving force needed for anion secretion on the apical surface (Boucher, 2004). Among the apical membrane proteins, the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel plays a particularly important role. Mutations in the CFTR gene impair cAMP-dependent Cl− secretion thus causing cystic fibrosis (CF), an autosomal recessive genetic disease characterized by bacterial infection and chronic inflammation in the lung (Boucher, 2004; Riordan, 2008). Besides CFTR, there is at least one other Cl− channel in the airway epithelium (Tarran et al. 2002). This channel is activated by an increase in the cytosolic free Ca2+ concentration as that caused by ATP and UTP binding to purinergic receptors on the epithelial surface (Mason et al. 1991; Tarran et al. 2002). Ca2+-activated Cl− channels (CaCCs) are also expressed in several other epithelial and non-epithelial cells (Ferrera et al. 2010). The molecular identity of CaCCs was elusive until the TMEM16A protein was identified as the most probable candidate by three independent research teams (Caputo et al. 2008; Schroeder et al. 2008; Yang et al. 2008). In one of these studies, TMEM16A (also known as anoctamin-1, ANO1) was cloned from bronchial epithelial cells stimulated for 24 h with interleukin-4 (IL-4) (Caputo et al. 2008). Indeed, the Th-2 cytokines IL-4 and IL-13 upregulate Ca2+-dependent Cl− secretion (Danahay et al. 2002; Galietta et al. 2002). Global gene expression analysis with microarrays followed by gene silencing with short-interfering RNAs (siRNAs) identified TMEM16A as the protein responsible for increased Cl− secretion in IL-treated cells (Caputo et al. 2008). However, the role of TMEM16A protein as a CaCC in the airway epithelium has been recently questioned given the results obtained with a TMEM16A small molecule inhibitor (Namkung et al. 2011). This compound, T16inh-A01, was identified by high-throughput screening of a chemical library using a TMEM16A-expressing cell line (Namkung et al. 2011). When tested on cultured bronchial epithelial cells, T16inh-A01 caused only partial inhibition of the Cl− secretion triggered by Ca2+-elevating agents. Therefore, it was postulated that another type of CaCC exists in addition to TMEM16A. This conclusion contrasts with the inhibition of CaCC activity previously observed when bronchial epithelial cells, either treated with IL-4 or untreated, are transfected with siRNA against TMEM16A (Caputo et al. 2008). Furthermore, TMEM16A knockout mice show a significantly reduced Ca2+-dependent Cl− secretion and altered mucociliary transport (Ousingsawat et al. 2009; Rock et al. 2009). However, it has been argued that the role of TMEM16A may be more relevant in mouse than in human airways (Namkung et al. 2011).

To clarify the role of TMEM16A, we have studied its expression and function in primary human bronchial epithelial cells and in a human bronchial epithelial cell line. Our data favour a major role of TMEM16A as a CaCC in the airway epithelium following treatment with IL-4. The identity of the CaCC in resting cells is less clear, although some indications suggest the involvement of TMEM16A also under this condition. Interestingly, stimulation with IL-4 causes a strong upregulation of TMEM16A in non-ciliated and mucus-expressing cells. In fact, the percentage of cells expressing mucin MUC5AC and TMEM16A increases with time during stimulation with the cytokine. These findings may suggest a particular role of TMEM16A protein under inflammatory conditions characterized by mucus hypersecretion and mucous cell metaplasia.

Methods

Cell culture

The CF bronchial epithelial cells, CFBE41o–, with stable expression of the halide-sensitive yellow fluorescent protein (HS-YFP), were cultured in minimal essential (MEM) medium. HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 (1:1). Both media were supplemented with 10% fetal calf serum (Sigma-Aldrich, St Louis, MO, USA), 2 mm l-glutamine, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin.

To isolate primary airway epithelial cells, mainstem human bronchi, derived from CF and non-CF individuals undergoing lung transplant (Department of Thoracic Surgery, University of Padova), were dissected and extensively washed with physiological saline solution (0.9% NaCl). Bronchi were then transferred in saline solution at 4°C to the laboratory (Gaslini Institute, Genova) within 4–5 h (the entire procedure, also involving informed consent from patients, was approved by the Ethical Committee of Gaslini Institute under the supervision of the Italian Ministry of Health). Bronchi were further washed 3–5 times in Falcon tubes containing saline solution and then placed overnight at 4°C in Hanks’ saline solution (Euroclone, Milan, Italy) containing 0.25% (w/v) Protease XIV (Sigma-Aldrich, Milan, Italy). The bronchi were then gently removed from the protease solution, and epithelial cells were collected by flushing energically the bronchial lumen with Hank's solution. Detached epithelial cell layers were pelleted by centrifugation and resuspended in 5–10 ml of Ca2+/Mg2+-free PBS containing 0.05% trypsin and 0.02% EDTA (Euroclone). After 5–10 min at 37°C, cells were dissociated by repeated pipetting. After this step, trypsin was neutralized with an equal volume of DMEM/Ham's F12 medium containing 10% bovine serum. The cell suspension was diluted by addition of Hanks’ solution up to a final volume of 30–40 ml and then centrifuged. The pellet was resuspended in a serum-free medium containing a 1:1 mixture of RPMI 1640 and LHC basal medium (Life Technologies, Monza, Italy) supplemented with 40.6 μm CaCl2, 2.5 μg ml−1 bovine insulin, 5 μg ml−1 human transferrin, 2.5 ng ml−1 human epidermal growth factor, 202 nm phosphoethanolamine, 247.5 nm ethanolamine, 1.365 μm adrenaline, 1.665 nm retinoic acid, 0.483 nm triiodothyronine (T3), 99.3 nm hydrocortisone, 25 μg ml−1 bovine pituitary extract, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. This medium also contained other salts (Stock 4, Stock 11, and trace element solutions, see Supplemental Material). When cells were from a CF patient, the medium in the first 4 days also contained additional antibiotics to eradicate bacterial contamination. For this, the mixture of antibiotics (usually colistin, piperacillin and tazobactam) and dosage were designed on the basis of the antibiogram of bacteria isolated from the most recent expectorate of the patient. Cells were cultured in 75 cm2 flasks coated with rat tail collagen (Sigma-Aldrich) for 2–5 passages in serum-free medium. When needed, cells were plated on 12 mm Snapwell (Corning, code 3801; Sigma-Aldrich) or 24 mm Transwell (Corning, code 3450; Sigma-Aldrich) permeable supports (500,000 and 2500,000 cells, respectively). After 24 h, the serum-free medium was replaced with DMEM/Ham's F12 containing 2% New Zealand fetal bovine serum (Life Technologies), 2.5 μg ml−1 bovine insulin, 5 μg ml−1 human transferrin, 202 nm phosphoethanolamine, 247.5 nm ethanolamine, 3.3 nm retinoic acid, 0.966 nm T3, 198.6 nm hydrocortisone, 25 μg ml−1 bovine pituitary extract, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. All special supplements used for bronchial cells were from Sigma-Aldrich except the epidermal growth factor (Merck, Darmstadt, Germany) and bovine pituitary extract (Life Technologies). Differentiation of cells into a tight epithelium was checked by measuring transepithelial electrical resistance and potential difference with an epithelial voltohmmeter (EVOM1; World Precision Instruments, Sarasota, FL, USA). The medium was replaced daily on both sides of permeable supports up to 8–10 days (liquid–liquid culture). Subsequently the apical medium was totally removed and the cells received nutrients only from the basolateral side (air–liquid culture). This condition favoured a further differentiation of the epithelium. Cells were maintained under air–liquid culture for 2–3 weeks. All experiments were performed on non-CF cells. Where specifically indicated, experiments were also performed on CF cells (with ΔF508/ΔF508 genotype).

Short-circuit current recordings

Snapwell supports carrying differentiated bronchial epithelia were mounted in a vertical diffusion chamber resembling a Ussing chamber with internal fluid circulation. Both apical and basolateral hemichambers were filled with 5 ml of a Kreb's bicarbonate solution (in mm): 126 NaCl, 0.38 KH2PO4, 2.13 K2HPO4, 1 MgSO4, 1 CaCl2, 24 NaHCO3 and 10 glucose. Both sides were continuously bubbled with a gas mixture containing 5% CO2–95% air and the temperature of the solution was kept at 37°C. The transepithelial voltage was short-circuited with a voltage-clamp (DVC-1000; World Precision Instruments) connected to the apical and basolateral chambers via Ag/AgCl electrodes and agar bridges (1 m KCl in 1% agar). The offset between voltage electrodes and the fluid resistance were cancelled before experiments. The short-circuit current was recorded with a PowerLab 4/25 (ADInstruments, Colorado Springs, CO, USA) analog-to-digital converter connected to a Macintosh computer.

Detection of TMEM16A protein by Western blot

CFPAC, CFBE41o– and HEK-293 cells were grown to confluence on 60 mm-diameter dishes. To express the TMEM16A protein, subconfluent monolayers of HEK-293 cells were transiently transfected with 4 μg of mammalian expression plasmid pcDNA 3.1 encoding the TMEM16A(abc) isoform, or the yellow fluorescent protein plasmid, using 10 μl lipofectamine 2000. Primary bronchial epithelial cells were instead seeded on 24.5 mm-diameter Transwell permeable supports (Corning, code 3450) and cultured as described above.

Cell lines and primary cells were lysed in lysis buffer (20 mm Hepes pH 7, 150 mm NaCl, 1 mm EGTA, 1% Igepal) containing Complete Protease Inhibitor Cocktail (Roche, NJ, USA). The concentration of lysates was quantified using the Bradford Reagent (Sigma-Aldrich). Twenty micrograms of total lysates was separated onto a NuPAGE Novex Bis-Tris 4–12% gel (Life Technologies) and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) for Western blotting. TMEM16A protein was immunodetected by a rabbit monoclonal antibody (SP31, Abcam, Cambridge, MA, USA) 1:1000, followed by anti-rabbit HRP (Millipore, Billerica, MA, USA) 1:10,000. Membranes were also stripped with the Restore Western Blot Stripping Buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and incubated with anti-actin goat polyclonal IgG (I19, Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by anti-goat HRP-conjugated secondary antibody (Santa Cruz).

All antibodies were dissolved in 5% skimmed-milk in Tris-buffered saline–Tween 20. Protein bands were visualized using the ECL Advanced Western Blotting Detection Kit (GE Healthcare Europe, Little Chalfont, UK). Direct recording of the chemiluminescence was performed using the Molecular Imager ChemiDoc XRS System (Bio-Rad).

Immunofluorescence

Primary human bronchial epithelial cells on Snapwell permeable supports were rinsed twice with PBS and fixed by adding 200 μl of Bouin's solution (Sigma-Aldrich) to the apical side for 10 min at room temperature. After washing, cells were blocked with 1% BSA in PBS for 2 h and then incubated overnight at 4°C with 200 μl of primary antibodies diluted in PBS-BSA 1%-Triton X-100 0.3%. To detect TMEM16A protein, the following commercially available primary antibodies and dilutions were used: ab64085 rabbit monoclonal (SP31) (Abcam) at 1:200, ab53212 rabbit polyclonal (Abcam) at 1:200, ab66170 rabbit monoclonal (BV10) (Abcam, no longer available) at 1:1, NBP1-49559 mouse monoclonal (DOG 1.1) (Novus Biologicals, Littleton, CO, USA) at 1:1 and 516-16711 rabbit polyclonal (DOG1) (Zytomed, Berlin, Germany) at 1:1. Comparative tests with these antibodies showed qualitatively similar results. However, the SP31 antibody was the best in terms of sensitivity and specificity. Therefore, all data reported in the figures and text were obtained with this antibody. To distinguish the apical from the basolateral membrane we used a mouse IgG1 anti-ZO-1 antibody (33-9100, Zymed Laboratories) at 15 μg ml−1. Ciliated cells were identified with a mouse IgG2B anti-acetylated tubulin antibody (7451, Sigma-Aldrich) diluted 1:300. Following incubation with primary antibody, cells were rinsed three times in PBS and incubated with 200 μl of a solution of secondary goat anti-rabbit 488 and goat anti-mouse Alexa Fluor 555 antibodies (Invitrogen, Carlsbad, CA, USA) diluted in PBS-BSA 1% for 1 h in the dark. After three further washes in PBS, the porous membrane carrying the cells was cut from the plastic support of the Snapwell, placed on microscope slides and mounted with Fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) to stain cell nuclei.

To detect MUC5AC, a marker of goblet cells, and CFTR together with TMEM16A a different protocol was used. Cultures were fixed in 10% neutral buffered formalin for 10 min and then processed for antigen retrieval with 10 mm citrate buffer, pH 6 (or Tris 10 mm– EDTA 1 mm, pH 9), heated to 95°C in a microwave for 5 min. Samples were then cooled to room temperature in PBS and permeabilized with PBS-Triton X-100 0.3% for 5 min. Blocking and subsequent steps were made as described above. The following primary antibodies and dilutions were used: NCL-HGM-45M1 mouse IgG1 anti-MUC5AC (Novocastra, Newcaste Upon Tyne, UK) at 1:50, ab64085 rabbit monoclonal (SP31) (Abcam) at 1:200 and ab570 mouse IgG1 anti-CFTR (J. R. Riordan, University of North Carolina at Chapel Hill, and Cystic Fibrosis Foundation Therapeutics, Bethesda, MD, USA) at 1:300 (Cui et al. 2007). For CFTR and acetylated tubulin co-immunostaining, monospecific secondary antibodies were used (Alexa Fluor 546 goat anti-mouse IgG1 and Alexa Fluor 633 goat anti-mouse IgG2B).

Immunofluorescent detection of TMEM16A protein in FRT cells and CFBE41o– cells was done as described for primary bronchial epithelial cells, using Bouin's fixative, SP31 antibody and DAPI staining.

Confocal microscopy was performed using a laser scanning spectral confocal microscope (TCS SP2-AOBS; Leica Microsystems, Heidelberg, Germany). Image analysis was performed using Leica and ImageJ software. TMEM16A-positive cells, ciliated cells, goblet cells and total number of cells (nuclei) were manually counted in xy fields of 375 × 375 μm. Each field contained 904 ± 23 cells (at least 4000–5000 cells were counted per each data point).

Transfection with siRNA

To maximize siRNA transfer to CFBE41o– cells we used a reverse transfection protocol, in which cells were transfected at the time of plating. For HS-YFP assays, CFBE41o– cells were plated on clear-bottomed black-wall 96-well microplates (Corning, code 3603; Sigma-Aldrich). Each well received 50,000 cells, resuspended in 100 μl of MEM without antibiotics, plus 50 μl of OPTI-MEM medium (Life Technologies) containing pre-formed complexes of siRNA (20 nm final concentration) and 0.25 μl of Lipofectamine 2000 (Life Technologies). siRNA complexes were removed after 24 h and CaCC activity was assayed after an additional 24 h. For patch-clamp experiments, CFBE41o– cells were plated in 35 mm Petri dishes together with siRNA/Lipofectamine 2000 complexes.

For HS-YFP assays we used siRNA targeting human TMEM16A from different vendors. Stealth siRNA against human TMEM16A (code HSS182856, HSS182857 and HSS123904) and corresponding negative controls (non-targeting siRNAs having a guanine cytosine content matching that of the anti-TMEM16A duplex; code 12935-200) were from Life Technologies. Four siRNAs (TMEM16 nos. 1, 2, 3 and 4) and the corresponding negative control (P1, IBONI Firefly Luciferase) were purchased from Riboxx (Euroclone). A pool of four siRNAs against TMEM16A was from Dharmacon (Lafayette, CO, USA). An additional three siRNA molecules (Hs01_00231162, Hs01_00231163 and Hs01_00231164) were purchased from Sigma-Aldrich, along with the corresponding negative control (non-targeting siRNA S01). For patch-clamp experiments, cells were transfected with a pool of two siRNAs against TMEM16A (HSS182857 and HSS123904, Life Technologies) or with appropriate non-targeting siRNAs. Real-time RT-PCR assays confirmed that TMEM16A mRNA levels were downregulated (60–80%) by siRNA relative to controls.

HS-YFP assay

CFBE41o– cells expressing the HS-YFP (Galietta et al. 2001a) were plated in clear-bottomed black-wall 96-well microplates (50,000 cells per well) with or withour siRNA/Lipofectamine complexes. After 48 h, cells were washed twice with 150 μl PBS (containing 137 mm NaCl, 2.7 mm KCl, 8.1 mm Na2HPO4, 1.5 mm KH2PO4, 1 mm CaCl2 and 0.5 mm MgCl2) and then incubated for 30 min with 60 μl PBS alone or with PBS plus TMEM16A/CaCC inhibitors at different concentrations. Cells were then transferred to a microplate reader (FluoStar Galaxy; BMG Labtech, Ortenberg, Germany) for CaCC activity determination. The plate reader was equipped with high-quality excitation (HQ500/20X: 500 ± 10 nm) and emission (HQ535/30M: 535 ± 15 nm) filters for YFP (Chroma Technology Corp., Brattleboro, VT, USA). Each assay consisted of a continuous 14 s fluorescence reading with 2 s before and 12 s after injection of 165 μl modified PBS (total Cl− replaced by I−; final I− concentration in the well: 100 mm) also containing 100 μm UTP. Data were normalized to the initial, background-subtracted, fluorescence. To determine fluorescence quenching rate associated with I− influx, the final 11 s of the data for each well were fitted with an exponential function to extrapolate the initial slope (dF/dt).

Patch-clamp experiments

Whole-cell membrane currents were recorded in CFBE41o– cells after transfection with siRNAs against TMEM16A or with control siRNAs. The extracellular solution had the following composition: 150 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, 10 mm glucose, 10 mm mannitol and 10 mm Na-Hepes (pH 7.4). To determine ion selectivity, we used two modified extracellular solutions in which NaCl was replaced by 150 mm CsCl (high CsCl) or by 30 mm CsCl plus 230 mm mannitol (low CsCl). The pipette (intracellular) solution contained: 130 mm CsCl, 10 mm EGTA, 1 mm MgCl2, 10 mm Hepes, 1 mm ATP (pH 7.4) plus CaCl2 to obtain the desired free Ca2+ concentration: 1 mm for 8 nm, and 8 mm for 305 nm (calculated with Patcher's Power Tool developed by Dr Francisco Mendes and Franz Wurriehausen, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany). During experiments, the membrane capacitance and series resistance were compensated for using the built-in circuitry of the EPC7 patch-clamp amplifier. The usual protocol for stimulation consisted of 600 ms-long voltage steps from –100 to 100 mV in 20 mV increments starting from a holding potential of –60 mV. The waiting time between steps was 4 s. Membrane currents were filtered at 1 kHz and digitized at 5 kHz with an ITC-16 (Instrutech, Port Washingon, NY, USA) AD/DA converter. Data were analysed using the Igor Pro software (Wavemetrics, Lake Oswego, OR, USA) supplemented by custom software kindly provided by Dr Oscar Moran (Institute of Biophysics, CNR, Genova, Italy).

Chloride channel inhibitors

The 6-t-butyl-2-(furan-2-carboxamido)-4,5,6,7-tetrahy-drobenzo[b] thiophene-3-carboxylic acid (CaCCinh-A01; De La Fuente et al. 2008) and the 2-[(5-ethyl-1,6-dihydro-4-methyl-6-oxo-2-pyrimidinyl)thio]-N-[4-(4-methoxyphenyl)-2-thiazolyl]acetamide (T16Ainh-A01; Namkung et al. 2011) were obtained from Dr Derek Paisley (Novartis, Horsham, UK).

Statistics

Data are presented as representative traces/images or as mean ± SEM. Statistical analysis was done with the InStat software (GraphPad, La Jolla, CA, USA). Significant differences between data were calculated with Student's t test.

Results

We and others have previously reported that treatment of primary bronchial epithelial cells with IL-4 or IL-13 leads to a markedly increased response of CaCCs to Ca2+-elevating agents (Danahay et al. 2002; Galietta et al. 2002; Pedemonte et al. 2007). This is demonstrated by the 6- to 10-fold increase in the amplitude of the current elicited by UTP in short-circuit recordings (Fig. 1A). The upregulation of Ca2+-dependent Cl− secretion was similar in non-CF and CF cells. To investigate the correlation between CaCC activity and TMEM16A protein expression, we have used Western blot and immunofluorescence experiments. In lysates from HEK-293 cells transfected with the TMEM16A coding sequence, the SP31 antibody revealed a strong signal corresponding to a protein migrating in the polyacrylamide gel with an apparent molecular weight of 120 kDa, in agreement with a glycosylated TMEM16A protein (Fig. 1B, top). A comparable strong signal was also present in CFPAC-1, a cell line with high endogenous activity of CaCCs (Caputo et al. 2008). The band detected by the SP31 antibody was instead absent in non-transfected HEK-293 cells. When western blots were performed on lysates from untreated primary bronchial epithelial cells, we observed a faint band at 120 kDa thus indicating a low TMEM16A expression (Fig. 1B). We found a similar result with CFBE41o– (Kunzelmann et al. 1993), a bronchial epithelial cell line with endogenous TMEM16A expression, as shown previously by real-time RT-PCR (Caputo et al. 2008). In contrast, primary bronchial epithelial cells treated for 24 h with IL-4 showed a marked increase in TMEM16A protein expression, consistent with the upregulation of Ca2+-dependent Cl− secretion. After cytokine treatment, the expression level became comparable with that of CFPAC-1 cells (Fig. 1B). We found no significant differences in terms of TMEM16A expression between cultured bronchial epithelial cells isolated from CF patients and those from control individuals (Fig. 1B).

Figure 1. TMEM16A protein expression in cultured human bronchial epithelial cells.

Figure 1

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A, representative short-circuit current recordings (top) and summary of data (bottom) showing upregulation of Ca2+-dependent Cl− secretion by IL-4 (10 ng ml−1 for 24 h). Before stimulation with apical UTP (100 μm), cells were previously exposed to amiloride and CFTRinh-172 (10 μm in the apical solution for both compounds) to inhibit the epithelial sodium channel (ENaC) and CFTR activity, respectively. Bars report the amplitude of the current induced by UTP (n= 6–10, **P < 0.01). B, detection of TMEM16A protein by Western blot in lysates from HEK-293 (with and without TMEM16A transfection), CFPAC-1, CFBE41o– and primary cultured bronchial epithelial cells from non-CF and CF individuals (N-BE and CF-BE, with and without IL-4 treatment). The bar graph shows a summary of the densitometric analysis performed for experiments as above. Each bar reports the relative density of TMEM16A band normalized to actin band for each sample (n= 5, *P < 0.05). C, representative xz images taken with a confocal microscope to show immunofluorescence for TMEM1A (green) and ZO-1 (red) in bronchial epithelial cells with and without IL-4 treatment. Nuclei are also stained with DAPI (blue). The blue signal below the cells arises from the porous membrane autofluorescence. The images were taken with a 40× or a 63× (bottom two images) objective. Arrows show untreated cells with apical TMEM16A localization. Scale bars: 10 μm. Data are representative of four preparations with non-CF and three preparations from CF cells.

TMEM16A protein expression was also investigated in cultured bronchial epithelia by immunofluorescence combined with confocal microscopy (see xz images in Fig. 1C and xy images in Fig. S1). To distinguish the apical from the basolateral membrane, we used an antibody against the ZO-1 tight junction marker. In untreated epithelia, we found a punctate pattern of TMEM16A expression that appeared in part intracellular although more concentrated in the apical pole of the cell. In some cells, the signal was stronger and more clearly localized in the membrane (Fig. 1C, bottom). In control conditions, the proportion of non-CF cells with a TMEM16A signal was 9.7 ± 3.2% (n= 12; Fig. S1). If the analysis was restricted to the most apical part of the cells, this value decreased to 1.9 ± 0.6%. Treatment with IL-4 not only increased the percentage of TMEM16A-expressing cells (47.2 ± 4.8%, n= 12, P < 0.01) but also the extent of expression (Fig. 1C and Fig. S1). Indeed, in cytokine-treated cells, many cells displayed a strong immunoreactivity with a clear localization in the apical membrane as indicated by the position of the ZO1 marker (Fig. 1C). We found similar results in CF epithelia (data not shown). Interestingly, the upregulation of TMEM16A by IL-4 was not uniform as many cells remained with a low or negligible TMEM16A protein expression. We analysed the subcellular localization of TMEM16A also in FRT cells as they have been widely used to study the activity and pharmacology of CFTR and TMEM16A (Galietta et al. 2001b; Ferrera et al. 2009; Namkung et al. 2011). Expression of TMEM16A in FRT cells was in part basolateral (Fig. S1).

We applied immunofluorescence also to CFBE41o– cells. These experiments showed a low level of TMEM16A expression (Fig. S1), in agreement with Western blot results. Such findings seem to contrast with previous functional data, which revealed an endogenous CaCC activity in CFBE41o– cells (Caputo et al. 2008). Because of the low expression of TMEM16A protein, as indicated by Western blots and immunofluorescence, we investigated whether CaCC activity is actually due to another type of Cl− channel. Therefore, CFBE41o– cells were transfected with siRNAs against TMEM16A and CaCC function was assessed by different techniques. In the first set of experiments, we used the assay based on the HS-YFP. With this technique, the anion transport through Cl− channels is measured as the HS-YFP fluorescence quenching caused by I− influx. In all experiments, the transfection of siRNAs against TMEM16A, obtained from different sources (11 different siRNAs), caused a significant inhibition of Ca2+-activated anion transport relative to cells transfected with non-silencing siRNAs or with siRNAs against CFTR or against other proteins of the TMEM16 family (Fig. 2A–C and Fig. S2).

Figure 2. Inhibition of CaCC activity by anti-TMEM16A siRNA and channel inhibitors.

Figure 2

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A–C, CaCC activity measured as normalized quenching rate (QR) of HS-YFP fluorescence in CFBE41o– cells. Cells were transfected with siRNAs against indicated targets obtained from Invitrogen (A), Riboxx (B) and Dharmacon (C). **P < 0.01. D, inhibition of CaCC activity (QR) in CFBE41o– cells by various concentrations of T16inh-A01 and CaCCinh-A01. Graphs show data from four separate experiments.

Our experiments on CFBE41o– cells indicated that the endogenous Ca2+-dependent Cl− conductance is strongly dependent on TMEM16A expression. Therefore, we expected that this conductance had to be sensitive to the T16inh-A01 inhibitor as well. When CaCC activity was measured with the HS-YFP technique, we found that T16inh-A01 is only partially effective with a maximal inhibition near to 40% at a concentration higher than 5 μm (Fig. 2D). In contrast, the CaCCinh-A01 compound, also identified by high-throughput screening (De La Fuente et al. 2008), almost totally inhibited CaCC activity at 10 μm with an apparent Kd of 2.6 μm.

We also evaluated CaCC activity in CFBE41o– cells with the whole-cell configuration of the patch-clamp technique. Cells transfected with control siRNA showed typical Ca2+-dependent Cl− currents. At a free Ca2+ concentration of 305 nm in the intracellular (pipette) solution, there were voltage-dependent currents, showing activation and deactivation at voltages more positive and more negative than the resting potential, respectively (Fig. 3A). The maximum current amplitude measured at the end of voltage pulses at +100 mV was 1758 ± 53 pA (n= 17). This value decreased significantly to 220 ± 20 pA (n= 11; P < 0.01) in cells transfected with siRNAs against TMEM16A (Fig. 3A). In such cells, the currents showed no relaxations, thus indicating the loss of the voltage-dependent component. Interestingly, in experiments performed with a very low intracellular Ca2+ concentration (8 nm), the cells transfected with control siRNA showed small currents indistinguishable from those recorded in TMEM16A-silenced cells, either with high or with low Ca2+ (Fig. 3B). These results demonstrate that the Ca2+-dependent conductance in CFBE41o– cells is completely abolished by TMEM16A knockdown. To demonstrate that the TMEM16A-dependent currents are Cl−-selective we lowered the extracellular Cl− concentration from 154 to 34 mm. Under these conditions, the voltage-dependent outward currents measured at positive membrane potentials in control-transfected cells were strongly decreased (by 87% at +100 mV) and the reversal potential was shifted by nearly 40 mV in the positive direction, as expected for a current mainly carried by Cl− ions with respect to cations (Fig. 3C). Therefore, these results are consistent with the presence of a large Ca2+-dependent Cl− conductance in non-silenced cells. In TMEM16A-silenced cells, the lowering of extracellular Cl− also decreased the outward currents but the extent of this effect was small in absolute and relative terms if compared with that of control-transfected cells (∼30% decrease at +100 mV). Furthermore, there was a shift in reversal potential (∼25 mV in the positive direction; Fig. 3D). Such results indicate the presence of a small Ca2+-independent Cl− current remaining after TMEM16A knockdown. The molecular identity of this current is at present unknown.

Figure 3. Inhibition of CaCC currents by anti-TMEM16A siRNA.

Figure 3

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Whole-cell membrane currents recorded with the patch-clamp technique in CFBE41o– cells transfected with control and anti-TMEM16A siRNAs. Experiments were performed with a free Ca2+ concentration of 305 nm (A) and 8 nm (B) in the pipette (intracellular) solution. Each panel shows representative currents recorded from a cell transfected with control (non-targeting, NT) siRNA and from a cell transfected with anti-TMEM16A siRNA. Current–voltage relationships summarizing the results obtained from similar experiments (mean ± SEM, n= 11–17) are depicted below corresponding traces. TMEM16A silencing caused a significant decrease of currents at all positive membrane potentials (P < 0.01) in experiments performed with high Ca2+ in the pipette solution (A). With low intracellular Ca2+, the effect of TMEM16A knockdown was not detectable (B). C, whole-cell membrane currents (left) and current–voltage relationship (right) from a representative experiment (out of three total experiments) performed on a cell transfected with a non-targeting siRNA. Currents were recorded with high and low Cl− concentration in the extracellular solution. D, as C but from a cell transfected with anti-TMEM16A siRNA (n= 3).

We tested T16inh-A01 also on primary bronchial epithelial cells. In untreated cells, the application of apical UTP (100 μm) elicited a maximal current of 3.8 ± 1.0 μA cm−2. In the presence of T16inh-A01 (10 μm), the UTP-dependent current was significantly reduced to 1.2 ± 0.4 μA cm−2 (n= 6 per condition, P < 0.05), with a nearly 70% inhibition (Fig. 4A). In cells treated with IL-4, the UTP-dependent current was 24.4 ± 1.6 and 18.7 ± 1.3 μA cm−2 (n= 6 per condition) in the absence and presence of T16inh-A01, respectively (Fig. 4A). The difference, corresponding to ∼25% inhibition, was statistically significant (P < 0.05). CaCCinh-A01 was more effective than T16inh-A01 also in primary cells. At 10 μm, the UTP-dependent current was inhibited by 65% in both untreated cells and IL-4 treated cells (data not shown).

Figure 4. Inhibition of Ca2+-dependent Cl− secretion by TMEM16A inhibitor.

Figure 4

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Short-circuit currents recordings from human bronchial epithelial cells under control conditions (A) or after treatment with IL-4 (B). Cells were stimulated with UTP (100 μm) with and without T16inh-A01 (10 μm). The figure shows representative traces and the summary of the peak of UTP-dependent current. Experiments were carried out in the presence of amiloride and CFTRinh-172 (10 μm each). In the presence of T16inh-A01, the peak of UTP current was significantly decreased (n= 5 per condition, *P < 0.05).

Bronchial epithelia are characterized by different types of cells that may coexist in the same area (Rock & Hogan, 2011). Therefore, we investigated whether TMEM16A expression is associated with a specific cell type. To identify ciliated cells by microscopy, we used an antibody against acetylated tubulin. As previously reported by others for IL-13 (Turner et al. 2011), we found that IL-4 caused a decrease in the percentage of ciliated cells (Figs 5A and 6C), from 14.9 ± 2.3 to 8.8 ± 1.3% in non-CF cells (n= 6 per condition, P < 0.05). Interestingly, ciliated cells appeared to lack TMEM16A expression, either with or without treatment with IL-4 (Fig. 5A and B). Indeed, cells with a strong TMEM16A staining, appearing after treatment with the cytokine, were clearly separated from those showing cilia (Fig. 5B). Only in a few ciliated cells (<0.1%) did we detect expression of TMEM16A. The separate localization of TMEM16A and cilia was also observed in CF cells (not shown).

Figure 5. Expression of TMEM16A in non-ciliated cells.

Figure 5

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A, confocal microscope xy images (375 × 375 μm) of human bronchial epithelial cells with and without IL-4 treatment (10 ng ml−1, 24 h) showing immunoreactivity for TMEM16A (green) and tubulin (red). B, xz and xy images of untreated and treated cells at higher magnification. Nuclei are also stained with DAPI. Scale bars: 10 μm. Size of xy image: 65 × 65 μm.

Figure 6. Expression profile of TMEM16A and MUC5AC.

Figure 6

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A and B, confocal microscope xy and xz images of human bronchial epithelial cells with and without IL-4 treatment (10 ng ml−1, 24 h) showing immunoreactivity for TMEM16A (green) and MUC5AC (red). Scale bars: 10 μm. Size of xy images: 375 × 375 μm. Nuclei are also stained with DAPI. The arrow indicates cells where MUC5AC appears to be localized in the apical membrane. C, percentage of cells expressing TMEM16A, MUC5AC and cilia with and without IL-4 for 24 h (n= 6–12, **P < 0.01, *P < 0.05).

We also looked for expression of MUC5AC, which is a classical marker for mucus-secreting goblet cells. Treatment of non-CF cells with IL-4 for 24 h clearly increased the percentage of MUC5AC-positive cells (Fig. 6), from 2.7 ± 0.6 to 7.2 ± 0.6% of total cells (n= 6–9, P < 0.01), in agreement with the induction of mucus metaplasia by Th2 cytokines (Dabbagh et al. 1999; Curran & Cohn, 2010; Turner et al. 2011). Interestingly, the MUC5AC-expressing cells were also strongly positive for TMEM16A, with the latter protein appearing in the apical membrane and the mucin residing instead more intracellularly, although close to the membrane (Fig. 6B). Close inspection of cells expressing both proteins showed that in some cases the MUC5AC signal was apparently localized on the apical surface (Fig. 6B, arrow) whereas TMEM16A immunoreactivity resided on the side of the mucin signal. These images may be interpreted as the result of MUC5AC exocytic secretion, caused by fusion of mucin-containing vesicles to the apical membrane and, consequently, displacement of TMEM16A towards the periphery of the cell. However, we also found cells with a partial overlap between TMEM16A and MUC5AC signals.

It is important to note that the high expression of TMEM16A and MUC5AC within the same cells was rarely seen in the absence of IL-4 treatment. Co-expression was instead evident following treatment with IL-4. More precisely, the MUC5AC-expressing cells at 24 h of IL-4 treatment represented a fraction (∼15%) of those expressing TMEM16A (Fig. 7A, right). We investigated whether this situation could change with longer IL-4 treatments. Interestingly, after 72 h the number of TMEM16A-expressing cells remained the same but the percentage of TMEM16A-positive cells also expressing MUC5AC increased to ∼60% (Fig. 7A).

Figure 7. Differential expression of TMEM16A and CFTR in goblet and ciliated cells.

Figure 7

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A, representative image (left) showing a high level of TMEM16A and MUC5AC co-expression in primary bronchial epithelial cells treated with IL-4 for 72 h (compare with 24 h effect in Fig. 6B). Panel A also shows a bar graph (right) demonstrating the increase in cells co-expressing MUC5AC and TMEM16A at 72 h vs. 24 h (n= 6, **P < 0.01). B, representative images indicating differential expression of TMEM16A (green) and CFTR (red). Cells were treated with IL-4 for 72 h. The image on the left shows that the two proteins are expressed in different cells with little overlap. The image on the right, taken from the same field, shows also the staining for cilia (acetylated tubulin, magenta). Comparison of the two images shows perfect overlap between CFTR and cilia staining. This result is also evident from the xz image shown in the inset. Size of xy images: 375 × 375 μm.

We also looked for CFTR protein localization. The images in Fig. 7B show that, in most cases, TMEM16A and CFTR were not co-expressed within the same cells. In fact, staining for acetylated tubulin revealed that CFTR was predominantly expressed in ciliated cells (Fig. 7B).

Discussion

TMEM16A protein appears as the main constituent of Ca2+-dependent Cl− channels in many cell types and tissues (Caputo et al. 2008; Schroeder et al. 2008; Yang et al. 2008; Huang et al. 2009; Hwang et al. 2009; Romanenko et al. 2010; Dutta et al. 2011; Xiao et al. 2011). A close paralogue, TMEM16B (ANO2), also works as a Ca2+-dependent Cl− channel but with a pattern of expression different from that of TMEM16A (Pifferi et al. 2009; Stephan et al. 2009; Stöhr et al. 2009; Billig et al. 2011; Huang et al. 2012). The role of the other TMEM16 proteins (from TMEM16C to TMEM16K) is unclear. Most of these proteins may be localized in intracellular organelles (Duran et al. 2012). However, TMEM16F (also known as ANO6) has been recently found as a Ca2+-independent Cl− channel activated by pro-apoptotic stimuli (Martins et al. 2011).

In airway epithelial cells, the CaCC represents an alternative route for Cl− secretion with respect to CFTR (Tarran et al. 2002). Therefore, it is a potential target for drugs aiming at increasing the thickness of the ASL, a beneficial effect in CF (Matsui et al. 1998) and possibly other chronic respiratory diseases. However, the importance of TMEM16A as a CaCC in the airway epithelium has been recently questioned. Indeed, a TMEM16A inhibitor, T16inh-A01, was found to inhibit CaCC-dependent Cl− secretion only poorly (Namkung et al. 2011). This result would imply that another CaCC protein, different from TMEM16A, is expressed in the airways. Due to these findings, we have re-examined the role of TMEM16A by comparing its expression to CaCC activity in primary bronchial epithelial cells and in a bronchial cell line. Our study shows that TMEM16A expression correlates reasonably with CaCC function in primary bronchial epithelia as demonstrated by Western blot/immunofluorescence and short-circuit recordings. Indeed, the marked increase in TMEM16A protein expression caused by chronic treatment with IL-4 is paralleled by a large increase in Ca2+-activated Cl− secretion. The relationship between TMEM16A and Ca2+-dependent Cl− secretion in the bronchial epithelium was also established by us previously using siRNA-based gene knockdown (Caputo et al. 2008). To further investigate the relationship between TMEM16A and epithelial CaCC, we have also used the bronchial cell line CFBE41o– because of its endogenous CaCC activity and its suitability for a series of assays, including patch-clamp recordings. According to our Western blot and immunofluorescence experiments, this cell line has a low TMEM16A expression, similar to that of untreated primary bronchial epithelial cells. We have previously shown, using the YFP-based assay, that TMEM16A silencing with siRNAs causes a significant inhibition of endogenous CaCC activity in CFBE41o– cells (Caputo et al. 2008). This result has now been confirmed by using a larger set of siRNAs from different sources and by using the patch-clamp technique in addition to YFP assay. In this respect, whole-cell patch-clamp recordings have shown that the Cl− currents silenced by TMEM16A knockdown in CFBE41o– cells have the typical biophysical appearance of CaCCs, i.e. activation at positive membrane potentials (Xiao et al. 2011). Despite the clear link between TMEM16A and CaCC activity, demonstrated by siRNA experiments, the T16inh-A01 compound was poorly effective in CFBE41o– cells. A similar low efficacy was also found in primary bronchial epithelial cells. In particular, we observed that the inhibition of Ca2+-activated Cl− secretion by T16inh-A01 appeared larger in untreated primary cells than in cells treated with IL-4. This finding is actually the opposite of what one would expect given the TMEM16A upregulation by the cytokine. Such results would suggest that the suitability of T16inh-A01 as a selective TMEM16A probe is affected by cell background and/or experimental conditions. In contrast, CaCCinh-A01, another inhibitor of CaCCs (De La Fuente et al. 2008), strongly inhibited Ca2+-dependent anion transport in both primary and CFBE41o– cells.

Summarizing, our results, based on gene knockdown and protein expression, are in agreement with a significant contribution of TMEM16A protein to anion transport in airway epithelial cells, particularly after stimulation with IL-4, a condition that mimics allergic and asthmatic inflammation. It is intriguing that a relatively low TMEM16A protein expression is sufficient to account for a significant Cl− secretion in untreated primary bronchial epithelia and for large Cl− currents in CFBE41o– cells. However, these results may be explained by a relatively low sensitivity of available TMEM16A antibodies.

Using immunofluorescence, we have found that TMEM16A protein is expressed on the apical membrane of bronchial epithelial cells as one would expect from a channel involved directly in anion secretion. There is no evidence of TMEM16A expression in the basolateral membrane as instead found in intestinal epithelial cells (He et al. 2011) and in transfected FRT cells (this study). Interestingly, CFTR was also found to be expressed in the basolateral membrane of FRT cells (Sheppard et al. 1994). The apical localization of TMEM16A in bronchial epithelial cells is more evident after treatment with IL-4, as in untreated conditions there are cells showing a more intracellular signal. Interestingly, immunofluorescence experiments have also revealed that TMEM16A expression is not homogeneous. Even after IL-4 treatment, there are ∼50% cells with low or negligible TMEM16A expression. Staining for tubulin shows that these TMEM16A-negative cells are in part ciliated. On the other hand, after IL-4 treatment, most cells expressing the mucin MUC5AC, a marker of goblet cells, also express TMEM16A. One may assume that TMEM16A is already highly expressed in goblet cells under resting conditions and that overall upregulation of TMEM16A by IL-4 is simply a direct consequence of the increase in goblet cell number. However, this is not the case as MUC5AC-positive cells in untreated epithelia had low or null TMEM16A expression. It is interesting to note that after 24 h of IL-4 treatment, only 16% of TMEM16A-expressing cells showed MUC5AC expression. However, this percentage increased markedly to 60% after 72 h. To explain these results we can postulate that IL-4 induces TMEM16A expression in a subpopulation of bronchial epithelial cells that are without cilia and MUC5AC expression. During treatment with the cytokine, cells showing TMEM16A upreguation also acquire, with a more delayed response, MUC5AC expression, thus becoming goblet cells. Therefore, the cells that are responsive to IL-4 may represent a specific cell type that is able to differentiate into goblet cells. If our hypothesis is correct, TMEM16A upregulation would be an early event of differentiation towards goblet cells during induction of mucous cell metaplasia. The identity of the putative goblet cell precursor in our experiments is unclear. However, other studies have shown that goblet cells may derive from Clara cells (Curran & Cohn, 2010; Park et al. 2007).

In conclusion, our study has shown a strong TMEM16A protein expression in bronchial epithelial cells treated with IL-4. This upregulation explains the large increase in Ca2+-dependent Cl− secretion occurring after cytokine treatment. The role of TMEM16A under resting conditions (i.e. without IL-4) is less clear. We cannot completely exclude that another Cl− channel is also involved in Ca2+-dependent Cl− secretion. However, the results obtained with gene silencing in resting primary cells (Caputo et al. 2008) and in CFBE41o– cells (this study) indicate an important role of TMEM16A despite a relatively low protein expression level. Interestingly, TMEM16A overexpression by IL-4 is not global in the cultured epithelium but involves a specific cellular set corresponding to precursors of goblet cells. The upregulation of TMEM16A by Th2 cytokines, namely IL-4 and IL-13, would suggest that it is involved in the secretion and/or hydration of mucus. In fact, the secretion of anions, particularly bicarbonate, is considered important for the expansion of mucin granules (Garcia et al. 2009). In the absence of proper bicarbonate secretion, mucus remains densely packed. We previously reported that pendrin (SLC26A4), an electroneutral transporter for various anions including Cl− and bicarbonate (Garnett et al. 2011), is strongly upregulated by IL-4 in bronchial epithelial cells (Pedemonte et al. 2007). The hyperexpression of TMEM16A and pendrin by Th2 cytokines may be part of a co-ordinated programme that boosts the capacity for anion secretion in the airways.

It is interesting to note that vesicles containing mucins in goblet cells have also a high content of ATP and other nucleotides (Kreda et al. 2007). Therefore, mucin secretion is paralleled by a release of nucleotides that, by interacting with purinergic receptors on the cell surface, would cause intracellular Ca2+ increase and CaCC activation. The expression of TMEM16A protein within and close to goblet cells would ensure that a co-ordinated spike of anion secretion occurs close to mucin release.

In future studies, it would be particularly interesting to understand the effect of direct pharmacological activation of TMEM16A activity on mucus secretion and mucociliary clearance. This may have a particular relevance for the treatment of lung diseases characterized by mucus hypersecretion including asthma, CF and chronic obstructive pulmonary disease (Rogers, 2007). In this respect, it would be important to determine whether TMEM16A upregulation also occurs in CF patients, as a consequence of bacterial infection and inflammation, or is a phenomenon specifically linked to the Th2 signalling cascade. Furthermore, it would also be interesting to investigate possible differences in TMEM16A expression among CF patients which could explain the heterogeneity of lung phenotype (Riordan, 2008). This type of information is important to assess the suitability of TMEM16A as a drug target in CF. Indeed, stimulation of TMEM16A with pharmacological activators could circumvent defective CFTR activity in CF airways. However, this approach needs to take into account our finding that CFTR and TMEM16A show separate expression (in ciliated and goblet cells, respectively). This may indicate that the two channels actually have different functions. Alternatively, it is possible that both proteins contribute to anion secretion and ASL regulation but through different cell types.

Acknowledgments

We thank Dr Derek Paisley (Novartis, Horsham, UK) for providing T16Ainh-A01 and CaCCinh-01. This work was supported by grants from the Telethon Foundation (GGP10026) and from the Italian Cystic Fibrosis Foundation (FFC no. 2/2009 with the contribution of ‘Delegazione FFC di Vicenza’). N.P. is a recipient of a grant to young investigators from the Italian Ministry of Health (GR-2008-1141326).

Glossary

ASL

airway surface liquid

CaCC

Ca2+-activated Cl− channel

CF

cystic fibrosis

CFTR

cystic fibrosis transmembrane conductance regulator

HS-YFP

halide-sensitive yellow fluorescent protein

IL

interleukin

siRNAs

short-interfering RNAs

Author contributions

P.S., E.C., S.B., L.F., E.S., V.T., A.G., O.Z.M., and N.P. performed experiments. M.S., F.R., R.R., and L.J.V.G. planned the study. P.S., E.C., L.F., and L.J.V.G analyzed data. L.J.V.G wrote the manuscript.

Supplementary material

Fig. S1

Fig. S2

tjp0590-6141-SD1.zip (5.5MB, zip)

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