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. 2017 Jan;74(1):173-181.
doi: 10.1007/s00018-016-2338-3. Epub 2016 Aug 17.

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

Jiraporn Ousingsawat  1 , Inês Cabrita  1 , Podchanart Wanitchakool  1 , Lalida Sirianant  1 , Stefan Krautwald  2 , Andreas Linkermann  2 , Rainer Schreiber  1 , Karl Kunzelmann  3
Affiliations

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

Jiraporn Ousingsawat et al. Cell Mol Life Sci. 2017 Jan.

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: Anoctamin 6; Apoptosis; Cell death; Chloride channel; Necroptosis; TMEM16F.

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Figures

Fig. 1
Fig. 1
Induction of necroptosis in NIH3T3 cells. Necroptosis in NIH3T3 cells was induced by the necroptotic cocktail TSZ containing TNF (10 ng/ml), smac mimetic birinapant (5 µM), and the pan-caspase inhibitor Z-VAD (25 µM). a, b Flow cytometry indicating time-dependent membrane permeabilization (7-AAD positivity) and phosphatidylserine exposure (annexin V positivity). c Inhibition of necroptosis (AnnV/7-AAD positivity) by necrostatin 1 (Nec-1; 100 µM). Mean ± SEM (number of cells). #Significant increase in AnnV/7-AAD positivity (p = 0.002, 2 h; 0.004, 3 h; 0.00001, 4 h) and inhibition by Nec-1 (p = 0.00001) (ANOVA)
Fig. 2
Fig. 2
Morphology of necroptotic cells. a 3D-analysis of non-stained NIH3T3 cells using holographic images (HoloMonitorTM, I&L Biosystems; Germany). Induction of necroptosis in NIH3T3 cells by necroptotic cocktail TSZ was paralleled by a time-dependent change of cell morphology. TSZ induced rounding up of the cells and increased cell height, leading to an initial cell swelling followed by an overall morphological cell shrinkage. Vertical scale bar indicates cell height ranging from 0 to 15.71 µm. b Holographic analysis of cell volume confirms initial cell swelling followed by subsequent cell shrinkage. c DIC images of NIH 3T3 cells treated with TSZ. d Analyzed volumes from individual cells indicating initial cell swelling followed by cell shrinkage. e Analysis of TSZ-induced cell volume changes by flow cytometry. Initial cell swelling is followed by cell shrinkage. f Measurement of cell membrane capacitance in patch clamp experiments suggesting an increase in cell membrane surface area during necroptosis. Bar indicates 10 µm. Mean ± SEM (number of cells). *Significant change in cell volume by TSZ (b, p = 0.00001–0.0001; d, p = 0.01–0.03) or capacitance (f, p = 001) (paired t test). #Significant change in cell volume by TSZ (e, p = 0.0001–0.001) (ANOVA)
Fig. 3
Fig. 3
Ca2+ signals in necroptotic cells. Analysis of intracellular Ca2+ signals during induction of necroptosis. a Ca2+ oscillations induced by TSZ. b Rise in intracellular Ca2+ levels by TSZ. c Rise in intracellular Ca2+ levels by TSZ was not affected by the (1) inhibitor of IP3 receptors, xestospongin C (1 µM), (2) the inhibitor of ORAI channels, YM58483 (5 µM), or the inhibitor of TRPC channels, SK&F96365 (20 µM), but was blocked by dantrolene (10 µM), an inhibitor of ryanodine receptors. Red bars indicate the presence of TSZ. Mean ± SEM (number of cells). #Significant increase of intracellular Ca2+ levels by TSZ (b, p = 0.0037, 2 h; p = 0.001, 3 h; p = 0.0015, 4 h), and inhibition of Ca2+ increase by dantrolene (c, p = 0.04) (ANOVA)
Fig. 4
Fig. 4
Whole cell currents in necroptotic cells. a Time-dependent whole cell currents activated by TZS, shown at clamp voltages ranging from −100 to +100 mV in steps of 20 mV. b Corresponding current voltage relationships indicate the activation of outwardly rectifying whole cell Cl− currents that are inhibited by replacement of extracellular Cl− with gluconate (5Cl−). c Inhibition of TSZ-activated currents by the ANO6 blocker tannic acid (TA, 5–20 µM). d Inhibition of TSZ-activated whole cell currents upon siRNA knockdown of ANO6. Mean ± SEM (number of cells). #Significant increase of whole cell currents by TSZ (b–d, p = 0.0015–0.04) (unpaired t test). *Significant inhibition of whole cell currents by 5Cl− or TA (b, p = 0.001–0.028; c, p = 0.018) (paired t test). $Significant inhibition of TSZ-induced whole cell currents by siRNA-ANO6 (p = 0.039; unpaired t test)
Fig. 5
Fig. 5
Minor role of TMEM16F in necroptotic cell death. a, b RT-PCR analysis of the expression of TMEM16 proteins in NIH3T3 cells indicates dominant expression of TMEM16F (ANO6) and TMEM16K (ANO10). c Confirmation of successful protein knockdown by siRNA (72 h). d Marginal effects of knockdown of TMEM16F (ANO6) and TMEM16 K (ANO10) on necroptotic cell death. e No effects of inhibitors of TMEM16 proteins on necroptotic cell death. CaCCinhAO1 (10 µM), T16inhAO1 (10 µM), tannic acid (TA, 10 µM), and NS3728 (10 µM). f Percentage of necroptotic cells (Annexin V/7-ADD positivity) induced by TSZ and effects of various compounds on cell death. Removal of extracellular Ca2+ (Ca2+ free), xestospongin C, dantrolene, YM58483, SK&F69365, and the TRPM7-inhibitor NS8593 (10 µM) had no or only marginal effects on cell death. Mean ± SEM (number of cells). #Significant inhibition of necroptosis (d, p = 0.003–0.021; f, p = 0.017) (ANOVA)
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