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Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine

  • Ion channels, receptors and transporters
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Abstract

Intestinal epithelial electrolyte secretion is activated by increase in intracellular cAMP or Ca2+ and opening of apical Cl− channels. In infants and young animals, but not in adults, Ca2+-activated chloride channels may cause secretory diarrhea during rotavirus infection. While detailed knowledge exists concerning the contribution of cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) channels, analysis of the role of Ca2+-dependent Cl− channels became possible through identification of the anoctamin (TMEM16) family of proteins. We demonstrate expression of several anoctamin paralogues in mouse small and large intestines. Using intestinal-specific mouse knockout models for anoctamin 1 (Ano1) and anoctamin 10 (Ano10) and a conventional knockout model for anoctamin 6 (Ano6), we demonstrate the role of anoctamins for Ca2+-dependent Cl− secretion induced by the muscarinic agonist carbachol (CCH). Ano1 is preferentially expressed in the ileum and large intestine, where it supports Ca2+-activated Cl− secretion. In contrast, Ano10 is essential for Ca2+-dependent Cl− secretion in jejunum, where expression of Ano1 was not detected. Although broadly expressed, Ano6 has no role in intestinal cholinergic Cl− secretion. Ano1 is located in a basolateral compartment/membrane rather than in the apical membrane, where it supports CCH-induced Ca2+ increase, while the essential and possibly only apical Cl− channel is CFTR. These results define a new role of Ano1 for intestinal Ca2+-dependent Cl− secretion and demonstrate for the first time a contribution of Ano10 to intestinal transport.

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References

  1. Almaca J, Tian Y, AlDehni F, Ousingsawat J, Kongsuphol P, Rock JR, Harfe BD, Schreiber R, Kunzelmann K (2009) TMEM16 proteins produce volume regulated chloride currents that are reduced in mice lacking TMEM16A. J Biol Chem 284:28571–28578

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Ball JM, Tian P, Zeng CQ, Morris AP, Estes MK (1996) Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science 272:101–104

    Article  CAS  PubMed  Google Scholar 

  3. Barro Soria R, AlDehni F, Almaca J, Witzgall R, Schreiber R, Kunzelmann K (2009) ER localized bestrophin1 acts as a counter-ion channel to activate Ca2+ dependent ion channels TMEM16A and SK4. Pflugers Arch 459:485–497

    Article  PubMed  Google Scholar 

  4. Billet A, Hanrahan JW (2013) The secret life of CFTR as a calcium-activated chloride channel. J Physiol 591(21):5273–5278

  5. Billig GM, Pál B, Fidzinski P, Jentsch TJ (2011) Ca2+−activated Cl− currents are dispensable for olfaction. Nat Neurosci 14:763–769

    Article  CAS  PubMed  Google Scholar 

  6. Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, Sondo E, Pfeffer U, Ravazzolo R, Zegarra-Moran O, Galietta LJ (2008) TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science 322:590–594

    Article  CAS  PubMed  Google Scholar 

  7. Chamova T, Florez L, Guergueltcheva V, Raycheva M, Kaneva R, Lochmuller H, Kalaydjieva L, Tournev I (2012) ANO10 c.1150_1151del is a founder mutation causing autosomal recessive cerebellar ataxia in Roma/Gypsies. J Neurol 259:906–911

    Article  PubMed  Google Scholar 

  8. Dekkers JF, Wiegerinck CL, De Jonge HR, Bronsveld I, Janssens HM, de Winter-de Groot KM, Brandsma AM, de Jong NW, Bijvelds MJ, Scholte BJ, Nieuwenhuis EE, van den Brink S, Clevers H, van der Ent CK, Middendorp S, Beekman JM (2013) A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med 19:939–945

    Article  CAS  PubMed  Google Scholar 

  9. Dong Y, Zeng CQ, Ball JM, Estes MK, Morris AP (1997) The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1,4,5- trisphosphate production. Proc Natl Acad Sci U S A 94:3960–3965

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Ehlen HW, Chinenkova M, Moser M, Munter HM, Krause Y, Gross S, Brachvogel B, Wuelling M, Kornak U, Vortkamp A (2012) Inactivation of Anoctamin-6/Tmem16f, a regulator of phosphatidylserine scrambling in osteoblasts, leads to decreased mineral deposition in skeletal tissues. J Bone Miner Res 28:246–259

    Article  Google Scholar 

  11. Elvers M, Stegner D, Hagedorn I, Kleinschnitz C, Braun A, Kuijpers ME, Boesl M, Chen Q, Heemskerk JW, Stoll G, Frohman MA, Nieswandt B (2010) Impaired alpha(IIb)beta(3) integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1. Sci Signal 3:ra1

    Article  PubMed Central  PubMed  Google Scholar 

  12. Faria D, Schlatter E, Witzgall R, Grahammer F, Bandulik S, Schweda F, Bierer S, Rock JR, Heitzmann D, Kunzelmann K, Schreiber R (2013) The calcium activated chloride channel Anoctamin 1 contributes to the regulation of renal function. Kindey Int 85(6):1369–1381

  13. Faria D, Schreiber R, Kunzelmann K (2009) CFTR is activated through stimulation of purinergic P2Y2 receptors. Pflugers Arch 457:1373–1380

    Article  CAS  PubMed  Google Scholar 

  14. Frizzell RA, Hanrahan JW (2012) Physiology of epithelial chloride and fluid secretion. Cold Spring Harb Perspect Med 2:a009563

    Article  PubMed Central  PubMed  Google Scholar 

  15. Grubb S, Poulsen KA, Juul CA, Kyed T, Klausen TK, Larsen EH, Hoffmann EK (2013) TMEM16F (Anoctamin 6), an anion channel of delayed Ca2+ activation. J Gen Physiol 141:585–600

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. He Q, Halm ST, Zhang J, Halm DR (2011) Activation of the basolateral membrane Cl conductance essential for electrogenic K secretion suppresses electrogenic Cl secretion. Exp Physiol 96:305–316

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Heemskerk JW, Bevers EM, Lindhout T (2002) Platelet activation and blood coagulation. Thromb Haemost 88:186–193

    CAS  PubMed  Google Scholar 

  18. Hennig B, Schultheiss G, Kunzelmann K, Diener M (2008) Ca(2+ )-induced Cl (−) efflux at rat distal colonic epithelium. J Membr Biol 221:61–72

    Article  CAS  PubMed  Google Scholar 

  19. Hogan DL, Crombie DL, Isenberg JI, Svendsen P, Schaffalitzky de Muckadell OB, Ainsworth MA (1997) CFTR mediates cAMP- and Ca2+−activated duodenal epithelial HCO3- secretion. Am J Physiol 272:G872–G878

    CAS  PubMed  Google Scholar 

  20. Juul CA, Grubb S, Poulsen KA, Kyed T, Hashem N, Lambert IH, Larsen EH, Hoffmann EK (2014) Anoctamin 6 differs from VRAC and VSOAC but is involved in apoptosis and supports volume regulation in the presence of Ca. Pflugers Arch [Epub ahead of print]

  21. Kmit A, van Kruchten R, Ousingsawat J, Mattheij NJ, Senden-Gijsbers B, Heemskerk JW, Bevers EM, Kunzelmann K (2013) Calcium-activated and apoptotic phospholipid scrambling induced by Ano6 can occur independently of Ano6 ion currents. Cell Death Dis 4:e611

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Ko EA, Jin BJ, Namkung W, Ma T, Thiagarajah JR, and Verkman AS (2013) Chloride channel inhibition by a red wine extract and a synthetic small molecule prevents rotaviral secretory diarrhoea in neonatal mice. Gut 63(7):1120–1129

  23. Kunzelmann K, Kongsuphol P, AlDehni F, Tian Y, Ousingsawat J, Warth R, Schreiber R (2009) Bestrophin and TMEM16—Ca2+ activated Cl− channels with different functions. Cell Calcium 46:233–241

    Article  CAS  PubMed  Google Scholar 

  24. Kunzelmann K, Mall M (2002) Electrolyte transport in the colon: mechanisms and implications for disease. Physiol Rev 82:245–289

    CAS  PubMed  Google Scholar 

  25. Kunzelmann K, Mehta A (2013) CFTR: a hub for kinases and cross-talk of cAMP and Ca. FEBS J 280:4417–4429

    Article  CAS  PubMed  Google Scholar 

  26. Kunzelmann K, Nilius B, Owsianik G, Schreiber R, Ousingsawat J, Sirianant L, Wanitchakool P, Bevers EM, Heemskerk JW (2013) Molecular functions of anoctamin 6 (TMEM16F): A chloride channel, cation channel or phospholipid scramblase? Pflügers Arch 466(3):407–14

  27. Kunzelmann K, Tian Y, Martins JR, Faria D, Kongsuphol P, Ousingsawat J, Thevenod F, Roussa E, Rock JR, Schreiber R (2011) Anoctamins. Pflugers Arch 462:195–208

    Article  CAS  PubMed  Google Scholar 

  28. Kunzelmann K, Tian Y, Martins JR, Faria D, Kongsuphol P, Ousingsawat J, Wolf L, Schreiber R (2012) Cells in focus: airway epithelial cells-Functional links between CFTR and anoctamin dependent Cl(−) secretion. Int J Biochem Cell Biol 44:1897–1900

    Article  CAS  PubMed  Google Scholar 

  29. Mall M, Bleich M, Greger R, Schürlein M, Kühr J, Seydewitz HH, Brandis M, Kunzelmann K (1998) Cholinergic ion secretion in human colon requires co-activation by cAMP. Am J Physiol 275:G1274–G1281

    CAS  PubMed  Google Scholar 

  30. Malvezzi M, Chalat M, Janjusevic R, Picollo A, Terashima H, Menon AK, Accardi A (2013) Ca(2+)-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel. Nat Commun 4:2367

    Article  PubMed Central  PubMed  Google Scholar 

  31. Martins JR, Faria D, Kongsuphol P, Reisch B, Schreiber R, Kunzelmann K (2011) Anoctamin 6 is an essential component of the outwardly rectifying chloride channel. Proc Natl Acad Sci U S A 108:18168–18172

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Maruyama H, Morino H, Miyamoto R, Murakami N, Hamano T, Kawakami H (2013) Exome sequencing reveals a novel ANO10 mutation in a Japanese patient with autosomal recessive spinocerebellar ataxia. Clin Genet 85(3):296–7

  33. Murek M, Kopic S, Geibel J (2010) Evidence for intestinal chloride secretion. Exp Physiol 95:471–478

    Article  CAS  PubMed  Google Scholar 

  34. Namkung W, Finkbeiner WE, Verkman AS (2010) CFTR-Adenylyl Cyclase I association is responsible for UTP activation of CFTR in well-differentiated primary human bronchial cell cultures. Mol Biol Cell 21:2639–2648

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Neussert R, Muller C, Milenkovic VM, Strauss O (2010) The presence of bestrophin-1 modulates the Ca(2+) recruitment from Ca (2+) stores in the ER. Pflugers Arch 460:163–175

    Article  CAS  PubMed  Google Scholar 

  36. Ousingsawat J, Kongsuphol P, Schreiber R, Kunzelmann K (2011) CFTR and TMEM16A are separate but functionally related Cl channels. Cell Physiol Biochem 28:715–724

    Article  CAS  PubMed  Google Scholar 

  37. 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 284:28698–28703

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Ousingsawat J, Tian Y, AlDehni F, Roussa E, Schreiber R, Mirza M, Cook DI, Kunzelmann K (2011) Rotavirus toxin NSP4 activates the calcium dependent chloride channel TMEM16A and inhibits absorptive Na+transport. Pflugers Arch 461:579–589

    Article  CAS  PubMed  Google Scholar 

  39. Pifferi S, Dibattista M, Menini A (2009) TMEM16B induces chloride currents activated by calcium in mammalian cells. Pflugers Arch 458:1023–1038

    Article  CAS  PubMed  Google Scholar 

  40. Puntheeranurak S, Schreiber R, Spitzner M, Ousingsawat J, Krishnamra N, Kunzelmann K (2007) Control of ion transport in mouse proximal and distal colon by prolactin. Cell Physiol Biochem 19:77–88

    Article  CAS  PubMed  Google Scholar 

  41. Sailer A, Houlden H (2012) Recent advances in the genetics of cerebellar ataxias. Curr Neurol Neurosci Rep 12:227–236

    Article  CAS  PubMed  Google Scholar 

  42. Schreiber R, Kunzelmann K (2005) Purinergic P2Y6 receptors induce Ca2+ and CFTR dependent Cl− secretion in mouse trachea. Cell Physiol Biochem 16:99–108

    Article  CAS  PubMed  Google Scholar 

  43. Schreiber R, Uliyakina I, Kongsuphol P, Warth R, Mirza M, Martins JR, Kunzelmann K (2010) Expression and function of epithelial anoctamins. J Biol Chem 285:7838–7845

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Schroeder BC, Cheng T, Jan YN, Jan LY (2008) Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell 134:1019–1029

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Seidler U, Blumenstein I, Kretz A, Viellard-Baron D, Rossmann H, Colledge WH, Evans M, Ratcliff R, Gregor M (1997) A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca(2+)-dependent HCO3- secretion. J Physiol 505:411–423

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  46. Shimizu T, Lehara T, Sato K, Fujii T, Sakai H, Okada Y (2013) TMEM16F is a component of a Ca2+−activated Cl− channel but not a volume-sensitive outwardly rectifying Cl− channel. Am J Physiol Cell Physiol 304:C748–C759

    Article  CAS  PubMed  Google Scholar 

  47. Stohr H, Heisig JB, Benz PM, Schoberl S, Milenkovic VM, Strauss O, Aartsen WM, Wijnholds J, Weber BH, Schulz HL (2009) TMEM16B, a novel protein with calcium-dependent chloride channel activity, associates with a presynaptic protein complex in photoreceptor terminals. J Neurosci 29:6809–6818

    Article  PubMed  Google Scholar 

  48. Strauss O, Muller C, Reichhart N, Tamm ER, Gomez NM (2014) The role of bestrophin-1 in intracellular ca(2+) signaling. Adv Exp Med Biol 801:113–119

    Article  PubMed  Google Scholar 

  49. Tian Y, Schreiber R, Kunzelmann K (2012) Anoctamins are a family of Ca2+ activated Cl− channels. J Cell Sci 125:4991–4998

    Article  CAS  PubMed  Google Scholar 

  50. Vermeer S, Hoischen A, Meijer RP, Gilissen C, Neveling K, Wieskamp N, de Brouwer A, Koenig M, Anheim M, Assoum M, Drouot N, Todorovic S, Milic-Rasic V, Lochmuller H, Stevanin G, Goizet C, David A, Durr A, Brice A, Kremer B, van de Warrenburg BP, Schijvenaars MM, Heister A, Kwint M, Arts P, van der Wijst J, Veltman J, Kamsteeg EJ, Scheffer H, Knoers N (2010) Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia. Am J Hum Genet 87:813–819

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  51. Viitanen T, Sukumaran P, Lof C, Tornquist K (2012) Functional coupling of TRPC2 cation channels and the calcium-activated anion channels in rat thyroid cells: implications for iodide homeostasis. J Cell Physiol 228(4):814–823

  52. Watt SA, Kular G, Fleming IN, Downes CP, Lucocq JM (2002) Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1. Biochem J 363(Pt.3):657–666

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  53. Wolf W, Kilic A, Schrul B, Lorenz H, Schwappach B, Seedorf M (2012) Yeast Ist2 recruits the endoplasmic reticulum to the plasma membrane and creates a ribosome-free membrane microcompartment. PLoS ONE 7:e39703

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  54. Yang H, Kim A, David T, Palmer D, Jin T, Tien J, Huang F, Cheng T, Coughlin SR, Jan YN, Jan LY (2012) TMEM16F forms a Ca(2+)-activated cation channel required for lipid scrambling in platelets during blood coagulation. Cell 151:111–122

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  55. Yang YD, Cho H, Koo JY, Tak MH, Cho Y, 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 455:1210–1215

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study was supported by DFG SFB699A7 and Wilhelm-Sander Stiftung Ano6 and Deutsche Krebshilfe Projekt 109438. We gratefully acknowledge the generous supply of the Ano6−/− mice by Prof. Dr. A. Vortkamp (Department Entwicklungsbiologie, University of Essen, Essen, Germany) and Ano-1 antibodies by Prof. Dr. Brian Harfe (University of Florida at Gainesville, Gainesville, USA).

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The authors declare no conflict of interest.

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Authors and Affiliations

  1. Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany

    Rainer Schreiber, Diana Faria, Podchanart Wanitchakool & Karl Kunzelmann

  2. Institut für Experimentelle Pathologie (ZMBE), Westfälischen Wilhelms-Universität Münster, 48149, Münster, Germany

    Boris V. Skryabin

  3. Department of Anatomy, University California, San Francisco, San Francisco, CA, USA

    Jason R. Rock

  4. Department of Medicine, University California, San Francisco, San Francisco, CA, USA

    Jason R. Rock

  5. Cardiovascular Research Institute, University California, San Francisco, San Francisco, CA, USA

    Jason R. Rock

  6. Interdisciplinary Center for Clinical Research (IZKF), University of Münster, Münster, Germany

    Boris V. Skryabin

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  1. Rainer Schreiber
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Correspondence to Karl Kunzelmann.

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Schreiber, R., Faria, D., Skryabin, B.V. et al. Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine. Pflugers Arch - Eur J Physiol 467, 1203–1213 (2015). https://doi.org/10.1007/s00424-014-1559-2

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