Expression and function of Anoctamin 1/TMEM16A calcium-activated chloride channels in airways of in vivo mouse models for cystic fibrosis research
- 531 Downloads
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.
KeywordsAirway epithelium Chloride secretion Cystic fibrosis Anoctamin TMEM16A Mouse models
We thank Dr. Alexei Diakov for kindly providing the βENaC antibody.
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.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.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
- 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
- 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
- 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
- 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.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
- 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