Pflügers Archiv - European Journal of Physiology

, Volume 470, Issue 9, pp 1335–1348 | Cite as

Expression and function of Anoctamin 1/TMEM16A calcium-activated chloride channels in airways of in vivo mouse models for cystic fibrosis research

  • Anne Hahn
  • Johanna J. Salomon
  • Dominik Leitz
  • Dennis Feigenbutz
  • Lisa Korsch
  • Ina Lisewski
  • Katrin Schrimpf
  • Pamela Millar-Büchner
  • Marcus A. Mall
  • Stephan FringsEmail author
  • Frank Möhrlen
Molecular and cellular mechanisms of disease
Part of the following topical collections:
  1. Topical Collection: Molecular and cellular mechanisms of disease


Physiological processes of vital importance are often safeguarded by compensatory systems that substitute for primary processes in case these are damaged by gene mutation. Ca2+-dependent Cl secretion in airway epithelial cells may provide such a compensatory mechanism for impaired Cl secretion via cystic fibrosis transmembrane conductance regulator (CFTR) channels in cystic fibrosis (CF). Anoctamin 1 (ANO1) Ca2+-gated Cl channels are known to contribute to calcium-dependent Cl secretion in tracheal and bronchial epithelia. In the present study, two mouse models of CF were examined to assess a potential protective function of Ca2+-dependent Cl secretion, a CFTR deletion model (cftr−/−), and a CF pathology model that overexpresses the epithelial Na+ channel β-subunit (βENaC), which is encoded by the Scnn1b gene, specifically in airway epithelia (Scnn1b-Tg). The expression levels of ANO1 were examined by mRNA and protein content, and the channel protein distribution between ciliated and non-ciliated epithelial cells was analyzed. Moreover, Ussing chamber experiments were conducted to compare Ca2+-dependent Cl secretion between wild-type animals and the two mouse models. Our results demonstrate that CFTR and ANO1 channels were co-expressed with ENaC in non-ciliated cells of mouse tracheal and bronchial epithelia. Ciliated cells did not express these proteins. Despite co-localization of CFTR and ANO1 in the same cell type, cells in cftr−/− mice displayed no altered expression of ANO1. Similarly, ANO1 expression was unaffected by βENaC overexpression in the Scnn1b-Tg line. These results suggest that the CF-related environment in the two mouse models did not induce ANO1 overexpression as a compensatory system.


Airway epithelium Chloride secretion Cystic fibrosis Anoctamin TMEM16A Mouse models 



We thank Dr. Alexei Diakov for kindly providing the βENaC antibody.

Funding information

This project was supported through a grant to AH by the Studienstiftung des deutschen Volkes and in part by the German Ministry for Education and Research (82DZL00401, 82DZL0040A1) to MAM.

Compliance with ethical standards

All experiments were approved by the Regierungspräsidium Karlsruhe and were conducted in agreement with national and international guidelines.


  1. 1.
    Anagnostopoulou P, Riederer B, Duerr J, Michel S, Binia A, Agrawal R, Liu X, Kalitzki K, Xiao F, Chen M, Schatterny J, Hartmann D, Thum T, Kabesch M, Soleimani M, Seidler U, Mall MA (2012) SLC26A9-mediated chloride secretion prevents mucus obstruction in airway inflammation. J Clin Invest 122:3629–3634CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Bartoszewski R, Matalon S, Collawn JF (2017) Ion channels of the lung and their role in disease pathogenesis. Am J Phys 313:L859–L872Google Scholar
  3. 3.
    Benedetto R, Ousingsawat J, Wanitchakool P, Zhang Y, Holtzman MJ, Amaral M, Rock JR, Schreiber R, Kunzelmann K (2017) Epithelial chloride transport by CFTR requires TMEM16A. Sci Rep 7:12397CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Button B, Cai LH, Ehre C, Kesimer M, Hill DB, Sheehan JK, Boucher RC, Rubinstein M (2012) A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia. Science 337:937–941CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Button B, Okada SF, Frederick CB, Thelin WR, Boucher RC (2013) Mechanosensitive ATP release maintains proper mucus hydration of airways. Sci Signal 6:ra46PubMedPubMedCentralGoogle Scholar
  6. 6.
    Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, Sondo E, Pfeffer U, Ravazzolo R, Zegarra-Moran O, Galietta LJV (2008) TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science 322:590–594CrossRefPubMedGoogle Scholar
  7. 7.
    Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159CrossRefPubMedGoogle Scholar
  8. 8.
    Clarke LL, Boucher RC (1992) Chloride secretory response to extracellular ATP in human normal and cystic fibrosis nasal epithelia. Am J Phys 263:C348–C356CrossRefGoogle Scholar
  9. 9.
    Clarke LL, Grubb BR, Yankaskas JR, Cotton CU, McKenzie A, Boucher RC (1994) Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(-/-) mice. Proc Natl Acad Sci U S A 91:479–483CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Dauner K, Lissmann J, Jeridi S, Frings S, Mohrlen F (2012) Expression patterns of anoctamin 1 and anoctamin 2 chloride channels in the mammalian nose. Cell Tiss Res 347:327–341CrossRefGoogle Scholar
  11. 11.
    Esther CR Jr, Lazaar AL, Bordonali E, Qaqish B, Boucher RC (2011) Elevated airway purines in COPD. Chest 140:954–960CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Evans JH, Sanderson MJ (1999) Intracellular calcium oscillations regulate ciliary beat frequency of airway epithelial cells. Cell Calcium 26:103–110CrossRefPubMedGoogle Scholar
  13. 13.
    Ferrera L, Caputo A, Galietta LJV (2010) TMEM16A protein: a new identity for Ca2+-dependent Cl- channels. Physiology 25:357–363CrossRefPubMedGoogle Scholar
  14. 14.
    Gabriel SE, Makhlina M, Martsen E, Thomas EJ, Lethem MI, Boucher RC (2000) Permeabilization via the P2X7 purinoreceptor reveals the presence of a Ca2+-activated Cl- conductance in the apical membrane of murine tracheal epithelial cells. J Biol Chem 275:35028–35033CrossRefPubMedGoogle Scholar
  15. 15.
    Galietta LJ, Pagesy P, Folli C, Caci E, Romio L, Costes B, Nicolis E, Cabrini G, Goossens M, Ravazzolo R, Zegarra-Moran O (2002) IL-4 is a potent modulator of ion transport in the human bronchial epithelium in vitro. J Immunol 168:839–845CrossRefPubMedGoogle Scholar
  16. 16.
    Genovese F, Thews M, Mohrlen F, Frings S (2016) Properties of an optogenetic model for olfactory stimulation. J Physiol 594:3501–3516CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Gentzsch M, Dang H, Dang Y, Garcia-Caballero A, Suchindran H, Boucher RC, Stutts MJ (2010) The cystic fibrosis transmembrane conductance regulator impedes proteolytic stimulation of the epithelial Na+ channel. J Biol Chem 285:32227–32232CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Gianotti A, Ferrera L, Philp AR, Caci E, Zegarra-Moran O, Galietta LJ, Flores CA (2016) Pharmacological analysis of epithelial chloride secretion mechanisms in adult murine airways. Eur J Pharmacol 781:100–108CrossRefPubMedGoogle Scholar
  19. 19.
    Grubb BR, Vick RN, Boucher RC (1994) Hyperabsorption of Na+ and raised Ca(2+)-mediated Cl- secretion in nasal epithelia of CF mice. Am J Phys 266:C1478–C1483CrossRefGoogle Scholar
  20. 20.
    Hahn A, Faulhaber J, Srisawang L, Stortz A, Salomon JJ, Mall MA, Frings S, Mohrlen F (2017) Cellular distribution and function of ion channels involved in transport processes in rat tracheal epithelium. Physiol Rep 5:e13290CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Huang T, You Y, Spoor MS, Richer EJ, Kudva VV, Paige RC, Seiler MP, Liebler JM, Zabner J, Plopper CG, Brody SL (2003) Foxj1 is required for apical localization of ezrin in airway epithelial cells. J Cell Sci 116:4935–4945CrossRefPubMedGoogle Scholar
  22. 22.
    Johannesson B, Hirtz S, Schatterny J, Schultz C, Mall MA (2012) CFTR regulates early pathogenesis of chronic obstructive lung disease in betaENaC-overexpressing mice. PLoS One 7:e44059CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Kreda SM, Mall M, Mengos A, Rochelle L, Yankaskas J, Riordan JR, Boucher RC (2005) Characterization of wild-type and deltaF508 cystic fibrosis transmembrane regulator in human respiratory epithelia. Mol Biol Cell 16:2154–2167CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Krueger B, Haerteis S, Yang L, Hartner A, Rauh R, Korbmacher C, Diakov A (2009) Cholesterol depletion of the plasma membrane prevents activation of the epithelial sodium channel (ENaC) by SGK1. Cell Physiol Biochem 24:605–618CrossRefPubMedGoogle Scholar
  25. 25.
    Kunzelmann K, Schreiber R, Boucherot A (2001) Mechanisms of the inhibition of epithelial Na(+) channels by CFTR and purinergic stimulation. Kidney Int 60:455–461CrossRefPubMedGoogle Scholar
  26. 26.
    Kunzelmann K, Tian Y, Martins JR, Faria D, Kongsuphol P, Ousingsawat J, Wolf L, Schreiber R (2012) Airway epithelial cells—functional links between CFTR and anoctamin dependent Cl(-) secretion. Int J Biochem Cell Biol 44:1897–1900CrossRefPubMedGoogle Scholar
  27. 27.
    Lazarowski ER, Tarran R, Grubb BR, van Heusden CA, Okada S, Boucher RC (2004) Nucleotide release provides a mechanism for airway surface liquid homeostasis. J Biol Chem 279:36855–36864CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Li H, Salomon JJ, Sheppard DN, Mall MA, Galietta LJ (2017) Bypassing CFTR dysfunction in cystic fibrosis with alternative pathways for anion transport. Curr Opin Pharmacol 34:91–97CrossRefPubMedGoogle Scholar
  29. 29.
    Lin J, Jiang Y, Li L, Liu Y, Tang H, Jiang D (2015) TMEM16A mediates the hypersecretion of mucus induced by Interleukin-13. Exp Cell Res 334:260–269CrossRefPubMedGoogle Scholar
  30. 30.
    Lommatzsch M, Cicko S, Muller T, Lucattelli M, Bratke K, Stoll P, Grimm M, Durk T, Zissel G, Ferrari D, Di Virgilio F, Sorichter S, Lungarella G, Virchow JC, Idzko M (2010) Extracellular adenosine triphosphate and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 181:928–934CrossRefPubMedGoogle Scholar
  31. 31.
    Mall MA (2008) Role of cilia, mucus, and airway surface liquid in mucociliary dysfunction: lessons from mouse models. J Aerosol Med Pulm Drug Deliv 21:13–24CrossRefPubMedGoogle Scholar
  32. 32.
    Mall MA, Galietta LJ (2015) Targeting ion channels in cystic fibrosis. J Cyst Fibros 14:561–570CrossRefPubMedGoogle Scholar
  33. 33.
    Mall M, Bleich M, Greger R, Schreiber R, Kunzelmann K (1998) The amiloride-inhibitable Na+ conductance is reduced by the cystic fibrosis transmembrane conductance regulator in normal but not in cystic fibrosis airways. J Clin Invest 102:15–21CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Mall M, Wissner A, Gonska T, Calenborn D, Kuehr J, Brandis M, Kunzelmann K (2000) Inhibition of amiloride-sensitive epithelial Na(+) absorption by extracellular nucleotides in human normal and cystic fibrosis airways. Am J Respir Cell Mol Biol 23:755–761CrossRefPubMedGoogle Scholar
  35. 35.
    Mall M, Grubb BR, Harkema JR, O’Neal WK, Boucher RC (2004) Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. Nat Med 10:487–493CrossRefPubMedGoogle Scholar
  36. 36.
    Mall MA, Harkema JR, Trojanek JB, Treis D, Livraghi A, Schubert S, Zhou Z, Kreda SM, Tilley SL, Hudson EJ, O’Neal WK, Boucher RC (2008) Development of chronic bronchitis and emphysema in beta-epithelial Na+ channel-overexpressing mice. Am J Resp Crit Care Med 177:730–742CrossRefPubMedGoogle Scholar
  37. 37.
    Mall MA, Button B, Johannesson B, Zhou Z, Livraghi A, Caldwell RA, Schubert SC, Schultz C, O’Neal WK, Pradervand S, Hummler E, Rossier BC, Grubb BR, Boucher RC (2010) Airway surface liquid volume regulation determines different airway phenotypes in liddle compared with betaENaC-overexpressing mice. J Biol Chem 285:26945–26955CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Marino A, Rodrig Y, Metioui M, Lagneaux L, Alzola E, Fernandez M, Fogarty DJ, Matute C, Moran A, Dehaye JP (1999) Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea. Biochim Biophys Acta 1439:395–405CrossRefPubMedGoogle Scholar
  39. 39.
    Okada SF, Nicholas RA, Kreda SM, Lazarowski ER, Boucher RC (2006) Physiological regulation of ATP release at the apical surface of human airway epithelia. J Biol Chem 281:22992–23002CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Okada SF, Ribeiro CM, Sesma JI, Seminario-Vidal L, Abdullah LH, van Heusden C, Lazarowski ER, Boucher RC (2013) Inflammation promotes airway epithelial ATP release via calcium-dependent vesicular pathways. Am J Respir Cell Mol Biol 49:814–820CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Ousingsawat J, Martins JR, Schreiber R, Rock JR, Harfe BD, Kunzelmann K (2009) Loss of TMEM16A causes a defect in epithelial Ca2+-dependent chloride transport. J Biol Chem 284:28698–28703CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Ousingsawat J, Kongsuphol P, Schreiber R, Kunzelmann K (2011) CFTR and TMEM16A are separate but functionally related Cl- channels. Cell Physiol Biochem 28:715–724CrossRefPubMedGoogle Scholar
  43. 43.
    Paisley D, Gosling M, Danahay H (2010) Regulation of airway mucosal hydration. Expert Rev Clin Pharmacol 3:361–369CrossRefPubMedGoogle Scholar
  44. 44.
    Pedemonte N, Galietta LJ (2014) Structure and function of TMEM16 proteins (anoctamins). Physiol Rev 94:419–459CrossRefPubMedGoogle Scholar
  45. 45.
    Perez-Cornejo P, Gokhale A, Duran C, Cui Y, Xiao Q, Hartzell HC, Faundez V (2012) Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin-radixin-moesin network. Proc Natl Acad Sci U S A 109:10376–10381CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Qin Y, Jiang Y, Sheikh AS, Shen S, Liu J, Jiang D (2016) Interleukin-13 stimulates MUC5AC expression via a STAT6-TMEM16A-ERK1/2 pathway in human airway epithelial cells. Int Immunopharmacol 40:106–114CrossRefPubMedGoogle Scholar
  48. 48.
    Rock JR, O'Neal WK, Gabriel SE, Randell SH, Harfe BD, Boucher RC, Grubb BR (2009) Transmembrane protein 16A (TMEM16A) is a Ca2+-regulated Cl- secretory channel in mouse airways. J Biol Chem 284:14875–14880CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Rokicki W, Rokicki M, Wojtacha J, Dzeljijli A (2016) The role and importance of club cells (Clara cells) in the pathogenesis of some respiratory diseases. Kardiochir Torakochirurgia Pol 13:26–30PubMedPubMedCentralGoogle Scholar
  50. 50.
    Ruffin M, Voland M, Marie S, Bonora M, Blanchard E, Blouquit-Laye S, Naline E, Puyo P, Le Rouzic P, Guillot L, Corvol H, Clement A, Tabary O (2013) Anoctamin 1 dysregulation alters bronchial epithelial repair in cystic fibrosis. Biochim Biophys Acta 1832:2340–2351CrossRefPubMedGoogle Scholar
  51. 51.
    Schroeder BC, Cheng T, Jan YN, Jan LY (2008) Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell 134:1019–1029CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Scudieri P, Caci E, Bruno S, Ferrera L, Schiavon M, Sondo E, Tomati V, Gianotti A, Zegarra-Moran O, Pedemonte N, Rea F, Ravazzolo R, Galietta LJ (2012) Association of TMEM16A chloride channel overexpression with airway goblet cell metaplasia. J Physiol 590:6141–6155CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Short DB, Trotter KW, Reczek D, Kreda SM, Bretscher A, Boucher RC, Stutts MJ, Milgram SL (1998) An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J Biol Chem 273:19797–19801CrossRefPubMedGoogle Scholar
  54. 54.
    Snouwaert JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, Smithies O, Koller BH (1992) An animal model for cystic fibrosis made by gene targeting. Science 257:1083–1088CrossRefPubMedGoogle Scholar
  55. 55.
    Sondo E, Caci E, Galietta LJ (2014) The TMEM16A chloride channel as an alternative therapeutic target in cystic fibrosis. Int J Biochem Cell Biol 52:73–76CrossRefPubMedGoogle Scholar
  56. 56.
    Tarran R, Boucher RC (2002) Thin-film measurements of airway surface liquid volume/composition and mucus transport rates in vitro. Meth Mol Med 70:479–492Google Scholar
  57. 57.
    Tarran R, Button B, Picher M, Paradiso AM, Ribeiro CM, Lazarowski ER, Zhang L, Collins PL, Pickles RJ, Fredberg JJ, Boucher RC (2005) Normal and cystic fibrosis airway surface liquid homeostasis. The effects of phasic shear stress and viral infections. J Biol Chem 280:35751–35759CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Thomas EJ, Gabriel SE, Makhlina M, Hardy SP, Lethem MI (2000) Expression of nucleotide-regulated Cl(-) currents in CF and normal mouse tracheal epithelial cell lines. Am J Phys 279:C1578–C1586CrossRefGoogle Scholar
  59. 59.
    Veit G, Bossard F, Goepp J, Verkman AS, Galietta LJ, Hanrahan JW, Lukacs GL (2012) Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia. Mol Biol Cell 23:4188–4202CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Warren NJ, Tawhai MH, Crampin EJ (2010) The effect of intracellular calcium oscillations on fluid secretion in airway epithelium. J Theor Biol 265:270–277CrossRefPubMedGoogle Scholar
  61. 61.
    Wei L, Vankeerberghen A, Cuppens H, Eggermont J, Cassiman JJ, Droogmans G, Nilius B (1999) Interaction between calcium-activated chloride channels and the cystic fibrosis transmembrane conductance regulator. Pflugers Arch Eur J Physiol 438:635–641CrossRefGoogle Scholar
  62. 62.
    Wei L, Vankeerberghen A, Cuppens H, Cassiman JJ, Droogmans G, Nilius B (2001) The C-terminal part of the R-domain, but not the PDZ binding motif, of CFTR is involved in interaction with Ca(2+)-activated Cl- channels. Pflug Arch: Eur J Physiol 442:280–285Google Scholar
  63. 63.
    Xu Z, Gupta V, Lei D, Holmes A, Carlson E, Gruenert DC (1998) In-frame elimination of exon 10 in Cftrtm1Unc CF mice. Gene 211:117–123CrossRefPubMedGoogle Scholar
  64. 64.
    Yang YD, Cho HW, Koo JY, Tak MH, Cho YY, Shim WS, Park SP, Lee J, Lee B, Kim BM, Raouf R, Shin YK, Oh U (2008) TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 455:1210–1215CrossRefPubMedGoogle Scholar
  65. 65.
    Zhou L, Dey CR, Wert SE, DuVall MD, Frizzell RA, Whitsett JA (1994) Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR. Science 266:1705–1708CrossRefPubMedGoogle Scholar
  66. 66.
    Zhou Z, Duerr J, Johannesson B, Schubert SC, Treis D, Harm M, Graeber SY, Dalpke A, Schultz C, Mall MA (2011) The ENaC-overexpressing mouse as a model of cystic fibrosis lung disease. J Cyst Fibros 10(Suppl 2):S172–S182CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Anne Hahn
    • 1
  • Johanna J. Salomon
    • 2
  • Dominik Leitz
    • 2
  • Dennis Feigenbutz
    • 1
  • Lisa Korsch
    • 1
  • Ina Lisewski
    • 1
  • Katrin Schrimpf
    • 1
  • Pamela Millar-Büchner
    • 2
  • Marcus A. Mall
    • 2
    • 3
    • 4
  • Stephan Frings
    • 1
    Email author
  • Frank Möhrlen
    • 1
  1. 1.Department of Animal Molecular Physiology, Centre of Organismal StudiesUniversity of HeidelbergHeidelbergGermany
  2. 2.Department of Translational Pulmonology, Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL)University of HeidelbergHeidelbergGermany
  3. 3.Department of Pediatric Pulmonology and ImmunologyCharité-Universitätsmedizin BerlinBerlinGermany
  4. 4.Berlin Institute of Health (BIH)BerlinGermany

Personalised recommendations