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

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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.

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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).

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Authors and Affiliations

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

    Jiraporn Ousingsawat, Inês Cabrita, Podchanart Wanitchakool, Lalida Sirianant, Rainer Schreiber & Karl Kunzelmann

  2. Division of Nephrology and Hypertension, Christian-Albrechts-University, Kiel, Germany

    Stefan Krautwald & Andreas Linkermann

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  1. Jiraporn Ousingsawat
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  2. Inês Cabrita
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  3. Podchanart Wanitchakool
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Correspondence to Karl Kunzelmann.

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Ousingsawat, J., Cabrita, I., Wanitchakool, P. et al. Ca2+ signals, cell membrane disintegration, and activation of TMEM16F during necroptosis. Cell. Mol. Life Sci. 74, 173–181 (2017). https://doi.org/10.1007/s00018-016-2338-3

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  • DOI: https://doi.org/10.1007/s00018-016-2338-3

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