title: Ca2+signals, cell membrane disintegration, and activation of TMEM16F during necroptosis.
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.
authors.affiliation: Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany.
authors.forename: Jiraporn
authors.initials: J
authors.lastname: Ousingsawat
authors.affiliation: Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany.
authors.forename: Inês
authors.initials: I
authors.lastname: Cabrita
authors.affiliation: Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany.
authors.forename: Podchanart
authors.initials: P
authors.lastname: Wanitchakool
authors.affiliation: Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany.
authors.forename: Lalida
authors.initials: L
authors.lastname: Sirianant
authors.affiliation: Division of Nephrology and Hypertension, Christian-Albrechts-University, Kiel, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Krautwald
authors.affiliation: Division of Nephrology and Hypertension, Christian-Albrechts-University, Kiel, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Linkermann
authors.affiliation: Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Schreiber
authors.affiliation: Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany. karl.kunzelmann@ur.de.
authors.forename: Karl
authors.initials: K
authors.lastname: Kunzelmann
mesh_terms: D000818:Animals; D000075368:Anoctamins; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D009336:Necrosis / Q000378:metabolism* / Q000473:pathology; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: C576687:ANO6 protein, mouse; D000075368:Anoctamins; D049294:Phospholipid Transfer Proteins; D014409:Tumor Necrosis Factor-alpha; D002118:Calcium
keywords: Anoctamin 6; Apoptosis; Cell death; Chloride channel; Necroptosis; TMEM16F