Molecular functions of anoctamin 6 (TMEM16F): a chloride channel, cation channel, or phospholipid scramblase?

  • Karl KunzelmannEmail author
  • Bernd Nilius
  • Grzegorz Owsianik
  • Rainer Schreiber
  • Jiraporn Ousingsawat
  • Lalida Sirianant
  • Podchanart Wanitchakool
  • Edouard M. Bevers
  • Johan W. M. Heemskerk
Invited Review


Anoctamin 6 (Ano6; TMEM16F gene) is a ubiquitous protein; the expression of which is defective in patients with Scott syndrome, an inherited bleeding disorder based on defective scrambling of plasma membrane phospholipids. For Ano6, quite diverse functions have been described: (1) it can form an outwardly rectifying, Ca2+-dependent and a volume-regulated Cl channel; (2) it was claimed to be a Ca2+-regulated nonselective cation channel permeable for Ca2+; (3) it was shown to be essential for Ca2+-mediated scrambling of membrane phospholipids; and (4) it can regulate cell blebbing and microparticle shedding. Deficiency of Ano6 in blood cells from Scott patients or Ano6 null mice appears to affect all of these cell responses. Furthermore, Ano6 deficiency in mice impairs the mineralization of osteoblasts, resulting in reduced skeletal development. These diverse results have been obtained under different experimental conditions, which may explain some of the contradictions. This review therefore aims to summarize the currently available information on the diverse roles of Ano6 and tries to clear up some of the existing controversies.


TMEM16F Anoctamin 6 Ano6 Scott syndrome Cation channels Chloride channels Phospholipid scrambling 



This work was supported by DFG SFB699 (KK, JO, RS, LS, PW) and the Cardiovascular Center of Maastricht University (EB, JH) and by grants from the Belgian Federal Government (IUAP P6/28 and P7/13), the Research Foundation-Flanders (F.W.O., G.0565.07 and G.0A61.13), and the Research Council of the KU Leuven (GOA 2009/07, EF/95/010 and PF-TRPLe) (GO, BN).


  1. 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 284:28571–28578PubMedCrossRefGoogle Scholar
  2. 2.
    Bevers EM, Comfurius P, van Rijn JL, Hemker HC, Zwaal RF (1982) Generation of prothrombin-converting activity and the exposure of phosphatidylserine at the outer surface of platelets. Eur J Biochem 122:429–436PubMedCrossRefGoogle Scholar
  3. 3.
    Bevers EM, Wiedmer T, Comfurius P, Shattil SJ, Weiss HJ, Zwaal RF, Sims PJ (1992) Defective Ca2+-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome. Blood 79:380–388PubMedGoogle Scholar
  4. 4.
    Bevers EM, Williamson PL (2010) Phospholipid scramblase: an update. FEBS Lett 584:2724–2730PubMedCrossRefGoogle Scholar
  5. 5.
    Billig GM, Pál B, Fidzinski P, Jentsch TJ (2011) Ca2+-activated Cl currents are dispensable for olfaction. Nat Neurosci 14:763–769PubMedCrossRefGoogle Scholar
  6. 6.
    Castoldi E, Collins PW, Williamson PL, Bevers EM (2011) Compound heterozygosity for 2 novel TMEM16F mutations in a patient with Scott syndrome. Blood 117:4399–4400PubMedCrossRefGoogle Scholar
  7. 7.
    Chen HJ, Schulman H, Gardner P (1989) A cAMP-regulated chloride channel in lymphocytes that is affected in cystic fibrosis. Science 243:657–660PubMedCrossRefGoogle Scholar
  8. 8.
    Dachary-Prigent J, Pasquet JM, Fressinaud E, Toti F, Freyssinet JM, Nurden AT (1997) Aminophospholipid exposure, microvesiculation and abnormal protein tyrosine phosphorylation in the platelets of a patient with Scott syndrome: a study using physiologic agonists and local anaesthetics. Br J Haematol 99:959–967PubMedCrossRefGoogle Scholar
  9. 9.
    Dekkers DW, Comfurius P, Bevers EM, Zwaal RF (2002) Comparison between Ca2+-induced scrambling of various fluorescently labelled lipid analogues in red blood cells. Biochem J 362:741–747PubMedCrossRefGoogle Scholar
  10. 10.
    Dekkers DW, Comfurius P, Vuist WM, Billheimer JT, Dicker I, Weiss HJ, Zwaal RF, Bevers EM (1998) Impaired Ca2+-induced tyrosine phosphorylation and defective lipid scrambling in erythrocytes from a patient with Scott syndrome: a study using an inhibitor for scramblase that mimics the defect in Scott syndrome. Blood 91:2133–2138PubMedGoogle Scholar
  11. 11.
    Dutta AK, Woo K, Khimji AK, Kresge C, Feranchak AP (2013) Mechanosensitive Cl secretion in biliary epithelium mediated through TMEM16A. Am J Physiol 304:G87–G98Google Scholar
  12. 12.
    Elliott JI, Sardini A, Cooper JC, Alexander DR, Davanture S, Chimini G, Higgins CF (2006) Phosphatidylserine exposure in B lymphocytes: a role for lipid packing. Blood 108:1611–1617PubMedCrossRefGoogle Scholar
  13. 13.
    Ferrera L, Caputo A, Ubby I, Bussani E, Zegarra-Moran O, Ravazzolo R, Pagani F, Galietta LJ (2009) Regulation of TMEM16A chloride channel properties by alternative splicing. J Biol Chem 284:33360–33368PubMedCrossRefGoogle Scholar
  14. 14.
    Frizzell RA, Rechkemmer GR, Shoemaker RL (1986) Altered regulation of airway epithelial cell chloride channels in cystic fibrosis. Science 233:558–560PubMedCrossRefGoogle Scholar
  15. 15.
    Gabriel SE, Clarke LL, Boucher RC, Stutts MJ (1993) CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship. Nature 363:263–268PubMedCrossRefGoogle Scholar
  16. 16.
    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 141:585–600PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Guggino WB, Stanton BA (2006) New insights into cystic fibrosis: molecular switches that regulate CFTR. Nat Rev Mol Cell Biol 7:426–436PubMedCrossRefGoogle Scholar
  18. 18.
    Hartzell HC, Putzier I, Arreola J (2005) Calcium-activated chloride channels. Annu Rev Physiol 67:719–758PubMedCrossRefGoogle Scholar
  19. 19.
    Heemskerk JW, Mattheij NJ, Cosemans JM (2013) Platelet-based coagulation: different populations, different functions. J Thromb Haemost 11:2–16PubMedCrossRefGoogle Scholar
  20. 20.
    Jacobsen KS, Zeeberg K, Poulsen KA, Hoffmann EK, Schwab A (2013) The role of TMEM16A (ANO1) and TMEM16F (ANO6) in cell migration. Pflügers Arch. (in press).Google Scholar
  21. 21.
    Jung J, Nam JH, Park HW, Oh U, Yoon JH, Lee MG (2012) Dynamic modulation of ANO1/TMEM16A HCO3 permeability by Ca2+/calmodulin. Proc Natl Acad Sci U S A 110(1):360–365PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Keuren JF, Wielders SJ, Ulrichts H, Hackeng T, Heemskerk JW, Deckmyn H, Bevers EM, Lindhout T (2005) Synergistic effect of thrombin on collagen-induced platelet procoagulant activity is mediated through protease-activated receptor-1. Arterioscler Thromb Vasc Biol 25:1499–1505PubMedCrossRefGoogle Scholar
  23. 23.
    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:e611PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    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 46:233–241PubMedCrossRefGoogle Scholar
  25. 25.
    Kunzelmann K, Pavenstädt H, Greger R (1989) Properties and regulation of chloride channels in cystic fibrosis and normal airway epithelial cells. Pflügers Arch 415:172–182PubMedCrossRefGoogle Scholar
  26. 26.
    Kunzelmann K, Schreiber R, Kmit A, Jantarajit W, Martins JR, Faria D, Kongsuphol P, Ousingsawat J, Tian Y (2012) Expression and function of epithelial anoctamins. Exp Physiol 97:184–192PubMedGoogle Scholar
  27. 27.
    Kunzelmann K, Tian Y, Martins JR, Faria D, Kongsuphol P, Ousingsawat J, Thevenod F, Roussa E, Rock JR, Schreiber R (2011) Anoctamins. Pflügers Arch 462:195–208PubMedCrossRefGoogle Scholar
  28. 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 44:1897–1900PubMedCrossRefGoogle Scholar
  29. 29.
    Kunzelmann K, Tilmann M, Hansen CP, Greger R (1991) Inhibition of epithelial chloride channels by cytosol. Pflügers Arch 418:479–490PubMedCrossRefGoogle Scholar
  30. 30.
    Lhermusier T, Chap H, Payrastre B (2011) Platelet membrane phospholipid asymmetry: from the characterization of a scramblase activity to the identification of an essential protein mutated in Scott syndrome. J Thromb Haemost 9:1883–1891PubMedCrossRefGoogle Scholar
  31. 31.
    Li M, McCann JD, Liedtke CM, Nairn AC, Greengard P, Welsh MJ (1988) Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium. Nature 331:358–360PubMedCrossRefGoogle Scholar
  32. 32.
    Magenheimer BS, St John PL, Isom KS, Abrahamson DR, De Lisle RC, Wallace DP, Maser RL, Grantham JJ, Calvet JP (2006) Early embryonic renal tubules of wild-type and polycystic kidney disease kidneys respond to cAMP stimulation with cystic fibrosis transmembrane conductance regulator/Na+, K+,2Cl- co-transporter-dependent cystic dilation. J Am Soc Nephrol 17:3424–3437PubMedCrossRefGoogle Scholar
  33. 33.
    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 108:18168–18172PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Mattheij NJ, Braun A, van Kruchten R, Cosemans JM, van der Meijden PE, Baaten C, Ehlen HW, Schreiber R, Vortkamp A, Collins PW, Bevers EM, Ousingsawat J, Kunzelmann K, Nieswandt B, Heemskerk JW (2013) The Scott syndrome protein anoctamin 6 (TMEM16F) regulates multiple cell death responses including membrane phospholipid scrambling in platelets. J. Thromb. Haemost. (Abstract) (in press)Google Scholar
  35. 35.
    Mattheij NJ, Gilio K, van Kruchten R, Jobe SM, Wieschhaus AJ, Chishti AH, Collins P, Heemskerk JW, Cosemans JM (2013) Dual mechanism of integrin αIIbβ3 closure in procoagulant platelets. J Biol Chem 288:13325–13336PubMedCrossRefGoogle Scholar
  36. 36.
    Munnix IC, Harmsma M, Giddings JC, Collins PW, Feijge MA, Comfurius P, Heemskerk JW, Bevers EM (2003) Store-mediated calcium entry in the regulation of phosphatidylserine exposure in blood cells from Scott patients. Thromb Haemost 89:687–695PubMedGoogle Scholar
  37. 37.
    Nilius B, Droogmans G (2003) Amazing chloride channels: an overview. Acta Physiol Scand 177:119–147PubMedCrossRefGoogle Scholar
  38. 38.
    Nilius B, Honore E (2012) Sensing pressure with ion channels. Trends Neurosci 35:477–486PubMedCrossRefGoogle Scholar
  39. 39.
    Owsianik G, Prenen J, Hermans C, Eggermont J, and Nilius B (2010) Functional characterization of TMEM16 anion channels. FASEB J. (Abstract) 608.12.Google Scholar
  40. 40.
    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 109:10376–10381PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Ponissery SS, Stephan AB, Talaga AK, Zhao H, Reisert J (2013) Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel anoctamin 2. J Gen Physiol 141(6):691CrossRefGoogle Scholar
  42. 42.
    Schoenwaelder SM, Yuan Y, Josefsson EC, White MJ, Yao Y, Mason KD, O’Reilly LA, Henley KJ, Ono A, Hsiao S, Willcox A, Roberts AW, Huang DC, Salem HH, Kile BT, Jackson SP (2009) Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function. Blood 114:663–666PubMedCrossRefGoogle Scholar
  43. 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 285:7838–7845PubMedCrossRefGoogle Scholar
  44. 44.
    Segawa K, Suzuki J, Nagata S (2011) Constitutive exposure of phosphatidylserine on viable cells. Proc Natl Acad Sci U S A 108:19246–19251PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Shimizu T, Iehara 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(8):C748–C759PubMedCrossRefGoogle Scholar
  46. 46.
    Sims PJ, Wiedmer T, Esmon CT, Weiss HJ, Shattil SJ (1989) Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity. J Biol Chem 264:17049–17057PubMedGoogle Scholar
  47. 47.
    Sinke AP, Deen PM (2011) The physiological implication of novel proteins in systemic osmoregulation. FASEB J 25:3279–3289PubMedCrossRefGoogle Scholar
  48. 48.
    Stephan AB, Shum EY, Hirsh S, Cygnar KD, Reisert J, Zhao H (2009) ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification. Proc Natl Acad Sci U S A 106:11776–11781PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Stout JG, Basse F, Luhm RA, Weiss HJ, Wiedmer T, Sims PJ (1997) Scott syndrome erythrocytes contain a membrane protein capable of mediating Ca2+-dependent transbilayer migration of membrane phospholipids. J Clin Invest 99:2232–2238PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Suzuki J, Fujii T, Imao T, Ishihara K, Kuba H, Nagata S (2013) Calcium-dependent phospholipid scramblase activity of TMEM16 family members. J Biol Chem 288(19):13305–13316PubMedCrossRefGoogle Scholar
  51. 51.
    Suzuki J, Umeda M, Sims PJ, Nagata S (2010) Calcium-dependent phospholipid scrambling by TMEM16F. Nature 468:834–838PubMedCrossRefGoogle Scholar
  52. 52.
    Szabo I, Lepple-Wienhues A, Kaba KN, Zoratti M, Gulbins E, Lang F (1998) Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes. Proc Natl Acad Sci U S A 95:6169–6174PubMedCentralPubMedCrossRefGoogle Scholar
  53. 53.
    Tian Y, Kongsuphol P, Hug MJ, Ousingsawat J, Witzgall R, Schreiber R, Kunzelmann K (2011) Calmodulin-dependent activation of the epithelial calcium-dependent chloride channel TMEM16A. FASEB J 25:1058–1068PubMedCrossRefGoogle Scholar
  54. 54.
    Tian Y, Schreiber R, Kunzelmann K (2012) Anoctamins are a family of Ca2+ activated Cl channels. J Cell Sci 125:4991–4998PubMedCrossRefGoogle Scholar
  55. 55.
    Tien J, Lee HY, Minor DL Jr, Jan YN, Jan LY (2013) Identification of a dimerization domain in the TMEM16A calcium-activated chloride channel (CaCC). Proc Natl Acad Sci U S A 110(16):6352–6357PubMedCentralPubMedCrossRefGoogle Scholar
  56. 56.
    Toti F, Satta N, Fressinaud E, Meyer D, Freyssinet JM (1996) Scott syndrome, characterized by impaired transmembrane migration of procoagulant phosphatidylserine and hemorrhagic complications, is an inherited disorder. Blood 87:1409–1415PubMedGoogle Scholar
  57. 57.
    Van Kruchten R, Mattheij NJ, Saunders C, Feijge MA, Swieringa F, Wolfs JL, Collins PW, Heemskerk JW, Bevers EM (2013) Both TMEM16F-dependent and TMEM16F-independent pathways contribute to phosphatidylserine exposure in platelet apoptosis and platelet activation. Blood 121(10):1850–1857PubMedCrossRefGoogle Scholar
  58. 58.
    Varga-Szabo D, Braun A, Nieswandt B (2011) STIM and Orai in platelet function. Cell Calcium 50:270–278PubMedCrossRefGoogle Scholar
  59. 59.
    Versteeg HH, Heemskerk JW, Levi M, Reitsma PH (2013) New fundamentals in hemostasis. Physiol Rev 93:327–358PubMedCrossRefGoogle Scholar
  60. 60.
    Viitanen T, Sukumaran P, Lof C, Tornquist K (2013) 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):814–823PubMedCrossRefGoogle Scholar
  61. 61.
    Voets T, Droogmans G, Raskin G, Eggermont J, Nilius B (1999) Reduced intracellular ionic strength as the initial trigger for activation of endothelial volume-regulated anion channels. Proc Natl Acad Sci U S A 96:5298–5303PubMedCentralPubMedCrossRefGoogle Scholar
  62. 62.
    Voets T, Manolopoulos V, Eggermont J, Ellory C, Droogmans G, Nilius B (1998) Regulation of a swelling-activated chloride current in bovine endothelium by protein tyrosine phosphorylation and G proteins. J Physiol 506:341–352PubMedCrossRefGoogle Scholar
  63. 63.
    Wielders SJ, Broers J, ten Cate H, Collins PW, Bevers EM, Lindhout T (2009) Absence of platelet-dependent fibrin formation in a patient with Scott syndrome. Thromb Haemost 102:76–82PubMedGoogle Scholar
  64. 64.
    Williamson P, Christie A, Kohlin T, Schlegel RA, Comfurius P, Harmsma M, Zwaal RF, Bevers EM (2001) Phospholipid scramblase activation pathways in lymphocytes. Biochemistry 40:8065–8072PubMedCrossRefGoogle Scholar
  65. 65.
    Wolfs JL, Comfurius P, Bekers O, Zwaal RF, Balasubramanian K, Schroit AJ, Lindhout T, Bevers EM (2009) Direct inhibition of phospholipid scrambling activity in erythrocytes by potassium ions. Cell Mol Life Sci 66:314–323PubMedCrossRefGoogle Scholar
  66. 66.
    Xiao Q, Yu K, Perez-Cornejo P, Cui Y, Arreola J, Hartzell HC (2011) Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop. Proc Natl Acad Sci U S A 108:8891–8896PubMedCentralPubMedCrossRefGoogle Scholar
  67. 67.
    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 Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation. Cell 151:111–122PubMedCentralPubMedCrossRefGoogle Scholar
  68. 68.
    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 455:1210–1215PubMedCrossRefGoogle Scholar
  69. 69.
    Yu K, Duran C, Qu Z, Cui YY, Hartzell HC (2012) Explaining calcium-dependent gating of anoctamin-1 chloride channels requires a revised topology. Circ Res 110:990–999PubMedCentralPubMedCrossRefGoogle Scholar
  70. 70.
    Zhou Q, Zhao J, Stout JG, Luhm RA, Wiedmer T, Sims PJ (1997) Molecular cloning of human plasma membrane phospholipid scramblase. A protein mediating transbilayer movement of plasma membrane phospholipids. J Biol Chem 272:18240–18244PubMedCrossRefGoogle Scholar
  71. 71.
    Zwaal RF, Schroit AJ (1997) Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 89:1121–1132PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Karl Kunzelmann
    • 1
    Email author
  • Bernd Nilius
    • 2
  • Grzegorz Owsianik
    • 2
  • Rainer Schreiber
    • 1
  • Jiraporn Ousingsawat
    • 1
  • Lalida Sirianant
    • 1
  • Podchanart Wanitchakool
    • 1
  • Edouard M. Bevers
    • 3
  • Johan W. M. Heemskerk
    • 3
  1. 1.Institut für PhysiologieUniversität RegensburgRegensburgGermany
  2. 2.Laboratory of Ion Channel Research, Department of Cellular and Molecular MedicineKU LeuvenLeuvenBelgium
  3. 3.Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM)University of MaastrichtMaastrichtThe Netherlands

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