Ca2+ signals, cell membrane disintegration, and activation of TMEM16F during necroptosis
- 936 Downloads
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.
KeywordsCell death Necroptosis Apoptosis TMEM16F Anoctamin 6 Chloride channel
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).
- 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
- 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
- 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
- 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
- 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