Cellular and Molecular Life Sciences

, Volume 74, Issue 1, pp 173–181 | Cite as

Ca2+ signals, cell membrane disintegration, and activation of TMEM16F during necroptosis

  • Jiraporn Ousingsawat
  • Inês Cabrita
  • Podchanart Wanitchakool
  • Lalida Sirianant
  • Stefan Krautwald
  • Andreas Linkermann
  • Rainer Schreiber
  • Karl Kunzelmann
Original Article

Abstract

Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca2+. We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT29, 16HBE, H441, and L929. Time-dependent changes in intracellular Ca2+, cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca2+, which was paralleled by the activation of outwardly rectifying Cl currents, which were typical for TMEM16F/ANO6. Ca2+ oscillations were due to Ca2+ release from endoplasmic reticulum, and were independent of extracellular Ca2+. The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death.

Keywords

Cell death Necroptosis Apoptosis TMEM16F Anoctamin 6 Chloride channel 

Notes

Acknowledgments

This work was supported by DFG SFB699-A7/A12, DFG KU756/12-1, Volkswagenstiftung AZ 87 499, and Medical Faculty of Kiel University (F355910 to SK).

Supplementary material

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Supplementary material 1 (PDF 746 kb)
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Supplementary material 5 (PDF 136 kb)
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Supplementary material 6 (PDF 99 kb)

References

  1. 1.
    Kunzelmann K (2016) Ion channels in regulated cell death. Cell Mol Life Sci 73(11–12):2387–2403CrossRefPubMedGoogle Scholar
  2. 2.
    Lang F, Hoffmann EK (2012) Role of ion transport in control of apoptotic cell death. Compr Physiol 2:2037–2061PubMedGoogle Scholar
  3. 3.
    Planells-Cases R, Lutter D, Guyader C, Gerhards NM, Ullrich F, Elger DA, Kucukosmanoglu A, Xu G, Voss FK, Reincke SM, Stauber T, Blomen VA, Vis DJ, Wessels LF, Brummelkamp TR, Borst P, Rottenberg S, Jentsch TJ (2015) Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs. EMBO J 34:2993–3008CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Voss FK, Ullrich F, Munch J, Lazarow K, Lutter D, Mah N, Andrade-Navarro MA, von Kries JP, Stauber T, Jentsch TJ (2014) Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC. Science 344:634–638CrossRefPubMedGoogle Scholar
  5. 5.
    Qiu Z, Dubin AE, Mathur J, Tu B, Reddy K, Miraglia LJ, Reinhardt J, Orth AP, Patapoutian A (2014) SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel. Cell 157:447–458CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Linkermann A, Green DR (2014) Necroptosis. N Engl J Med 370:455–465CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Chen X, Li W, Ren J, Huang D, He WT, Song Y, Yang C, Li W, Zheng X, Chen P, Han J (2014) Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell Res 24:105–121CrossRefPubMedGoogle Scholar
  8. 8.
    Ousingsawat J, Wanitchakool P, Kmit A, Romao AM, Jantarajit W, Schreiber S, Kunzelmann K (2015) Anoctamin 6 mediates effects essential for innate immunity downstream of P2X7-receptors in macrophages. Nat Commun 6:6245CrossRefPubMedGoogle Scholar
  9. 9.
    Tian Y, Schreiber R, Kunzelmann K (2012) Anoctamins are a family of Ca2+ activated Cl channels. J Cell Sci 125:4991–4998CrossRefPubMedGoogle Scholar
  10. 10.
    Yu K, Whitlock JM, Lee K, Ortlund EA, Yuan CY, Hartzell HC (2015) Identification of a lipid scrambling domain in ANO6/TMEM16F. Elife. doi: 10.7554/eLife.06901 Google Scholar
  11. 11.
    Tait SW, Oberst A, Quarato G, Milasta S, Haller M, Wang R, Karvela M, Ichim G, Yatim N, Albert ML, Kidd G, Wakefield R, Frase S, Krautwald S, Linkermann A, Green DR (2013) Widespread mitochondrial depletion via mitophagy does not compromise necroptosis. Cell Rep 5:878–885CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Depeursinge C, Colomb T, Emery Y, Kuhn J, Charriere F, Rappaz B, Marquet P (2007) Digital holographic microscopy applied to life sciences. Conf Proc IEEE Eng Med Biol Soc 2007:6244–6247PubMedGoogle Scholar
  13. 13.
    Marquet P, Depeursinge C, Magistretti PJ (2013) Exploring neural cell dynamics with digital holographic microscopy. Annu Rev Biomed Eng 15:407–431CrossRefPubMedGoogle Scholar
  14. 14.
    Molder A, Sebesta M, Gustafsson M, Gisselson L, Wingren AG, Alm K (2008) Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography. J Microsc 232:240–247CrossRefPubMedGoogle Scholar
  15. 15.
    Okada Y (2006) Cell volume-sensitive chloride channels: phenotypic properties and molecular identity. Contrib Nephrol 152:9–24CrossRefPubMedGoogle Scholar
  16. 16.
    Cai Z, Jitkaew S, Zhao J, Chiang HC, Choksi S, Liu J, Ward Y, Wu LG, Liu ZG (2014) Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol 16:55–65CrossRefPubMedGoogle Scholar
  17. 17.
    Wanitchakool P, Ousingsawat J, Sirianant L, MacAulay N, Schreiber R, Kunzelmann K (2016) Cl channels in apoptosis. Eur Biophys J. doi: 10.1007/s00249-016-1140-3
  18. 18.
    Kunzelmann K, Tian Y, Martins JR, Faria D, Kongsuphol P, Ousingsawat J, Thevenod F, Roussa E, Rock JR, Schreiber R (2011) Anoctamins. Pflugers Arch 462:195–208CrossRefPubMedGoogle Scholar
  19. 19.
    Namkung W, Thiagarajah JR, Phuan PW, Verkman AS (2010) Inhibition of Ca2+ -activated Cl channels by gallotannins as a possible molecular basis for health benefits of red wine and green tea. FASEB J 24:4178–4186CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Ko EA, Jin BJ, Namkung W, Ma T, Thiagarajah JR, 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):1120–1129CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Suzuki J, Umeda M, Sims PJ, Nagata S (2010) Calcium-dependent phospholipid scrambling by TMEM16F. Nature 468:834–838CrossRefPubMedGoogle Scholar
  22. 22.
    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 141:585–600CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    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:C748–C759CrossRefPubMedGoogle Scholar
  24. 24.
    Sirianant L, Ousingsawat J, Wanitchakool P, Schreiber R, Kunzelmann K (2015) Cellular volume regulation by anoctamin 6:Ca2+, phospholipase A2, osmosensing. Pflügers Arch 468:335–349CrossRefPubMedGoogle Scholar
  25. 25.
    Kunzelmann K, Nilius B, Owsianik G, Schreiber R, Ousingsawat J, Sirianant L, Wanitchakool P, Bevers EM, Heemskerk JW (2014) Molecular functions of anoctamin 6 (TMEM16F): a chloride channel, cation channel or phospholipid scramblase? Pflügers Arch 466:407–414CrossRefPubMedGoogle Scholar
  26. 26.
    Mattheij NJ, Braun A, van Kruchten R, Castoldi E, Pircher J, Baaten CC, Wulling M, Kuijpers MJ, Kohler R, Poole AW, Schreiber R, Vortkamp A, Collins PW, Nieswandt B, Kunzelmann K, Cosemans JM, Heemskerk JW (2015) Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage. FASEB J 30:727–737CrossRefPubMedGoogle Scholar
  27. 27.
    Liu G, Liu G, Chen H, Borst O, Gawaz M, Vortkamp A, Schreiber R, Kunzelmann K, Lang F (2015) Involvement of Ca2+ activated Cl channel Ano6 in platelet activation and apoptosis. Cell Physiol Biochem 37:1934–1944CrossRefPubMedGoogle Scholar
  28. 28.
    Hammer C, Wanitchakool P, Sirianant L, Papiol S, Monnheimer M, Faria D, Ousingsawat J, Schramek N, Schmitt C, Margos G, Michel A, Kraiczy P, Pawlita M, Schreiber R, Schulz TF, Fingerle V, Tumani H, Ehrenreich H, Kunzelmann K (2015) A coding variant of ANO10, affecting volume regulation of macrophages, is associated with Borrelia seropositivity. Mol Med 21:26–37CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Karch J, Kanisicak O, Brody MJ, Sargent MA, Michael DM, Molkentin JD (2015) Necroptosis interfaces with MOMP and the MPTP in mediating cell death. PLoS One 10:e0130520CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Henriquez M, Armisen R, Stutzin A, Quest AF (2008) Cell death by necrosis, a regulated way to go. Curr Mol Med 8:187–206CrossRefPubMedGoogle Scholar
  31. 31.
    Segawa K, Nagata S (2015) An apoptotic ‘eat me’ signal: phosphatidylserine exposure. Trends Cell Biol 25:639–650CrossRefPubMedGoogle Scholar
  32. 32.
    Hayslett JP, Gögelein H, Kunzelmann K, Greger R (1987) Characteristics of apical chloride channels in human colon cells (HT29). Pflügers Arch 410:487–494CrossRefPubMedGoogle Scholar
  33. 33.
    Kunzelmann K, Koslowsky T, Gruenert DC, Greger R (1994) CAMP-dependent activation of ion conductances in bronchial epithelial cells. Pflügers Arch 428:590–596CrossRefPubMedGoogle Scholar
  34. 34.
    Faria D, Lentze N, Almaca J, Luz S, Alessio L, Tian Y, Martins JP, Cruz P, Schreiber R, Rezwan M, Farinha CM, Auerbach D, Amaral MD, Kunzelmann K (2012) Regulation of ENaC biogenesis by the stress response protein SERP1. Pflugers Arch 463:819–827CrossRefPubMedGoogle Scholar
  35. 35.
    Linkermann A, Brasen JH, De Zen F, Weinlich R, Schwendener RA, Green DR, Kunzendorf U, Krautwald S (2012) Dichotomy between RIP1- and RIP3-mediated necroptosis in tumor necrosis factor-alpha-induced shock. Mol Med 18:577–586 (Cambridge, Mass) CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Schreiber R, Uliyakina I, Kongsuphol P, Warth R, Mirza M, Martins JR, Kunzelmann K (2010) Expression and Function of Epithelial Anoctamins. J Biol Chem 285:7838–7845CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing 2016

Authors and Affiliations

  • Jiraporn Ousingsawat
    • 1
  • Inês Cabrita
    • 1
  • Podchanart Wanitchakool
    • 1
  • Lalida Sirianant
    • 1
  • Stefan Krautwald
    • 2
  • Andreas Linkermann
    • 2
  • Rainer Schreiber
    • 1
  • Karl Kunzelmann
    • 1
  1. 1.Institut für PhysiologieUniversität RegensburgRegensburgGermany
  2. 2.Division of Nephrology and HypertensionChristian-Albrechts-UniversityKielGermany

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