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. 2017 Nov 24;292(47):19381-19391.
doi: 10.1074/jbc.M117.803049. Epub 2017 Oct 5.

Activation of the phospholipid scramblase TMEM16F by nanosecond pulsed electric fields (nsPEF) facilitates its diverse cytophysiological effects

Claudia Muratori  1 , Andrei G Pakhomov  2 , Elena Gianulis  2 , Jade Meads  2 , Maura Casciola  2 , Peter A Mollica  3 , Olga N Pakhomova  2
Affiliations

Activation of the phospholipid scramblase TMEM16F by nanosecond pulsed electric fields (nsPEF) facilitates its diverse cytophysiological effects

Claudia Muratori et al. J Biol Chem. .

Abstract

Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca2+-dependent phospholipid scramblase and Ca2+-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, Tmem16f expression knockdown and TMEM16F-specific inhibition decreased nsPEF-induced PS exposure by 49 and 42%, respectively. Moreover, the Tmem16f silencing significantly decreased Ca2+-dependent chloride channel currents activated in response to the nanoporation. Tmem16f expression also affected nsPEF-induced cell blebbing, with only 20% of the silenced cells developing blebs compared with 53% of the control cells. This inhibition of cellular blebbing correlated with a 25% decrease in cytosolic free Ca2+ transient at 30 s after nanoporation. Finally, in TMEM16F-overexpressing cells, a train of 120 pulses (300 ns, 20 Hz, 6 kV/cm) decreased cell survival to 34% compared with 51% in control cells (*, p < 0.01). Taken together, these results indicate that TMEM16F activation by nanoporation mediates and enhances the diverse cellular effects of nsPEF.

Keywords: Anoctamin 6; TMEM16F; cell death; chloride channel; electroporation; nanosecond pulsed electric fields (nsPEF); phosphatidylserine; plasma membrane.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
nsPEF-induced phosphatidylserine externalization (A), its dependence on Ca2+ (B), and on TMEM16F activity (C–E). HEK 293 were treated with a single 300-ns 25.5 kV/cm pulse, and PS externalization was monitored by time lapse confocal microscopy using either Cy5-labeled Annexin V (A, C, and E) or lactadherin-FITC (B). The nsPEF treatments were done at 87 s into the experiment (black arrows), after acquiring 3 pre-exposure images as a baseline. Panel A shows for a group of cells treated with nsPEF, DIC and Annexin V fluorescence images taken at the indicated time points. Panel B shows the lack of PS externalization in the absence of extracellular Ca2+. Cells were treated with nsPEF in either 2 or 0 mm CaCl2 bath solutions. DIC and lactadherin-FITC fluorescence images were taken at the indicated time points. In panel C, the scramblase activity of TMEM16F was blocked by incubation of cells with 25 μm CaCCinh-AO-1 for 5 min. In control samples, the inhibitor vehicle DMSO was diluted the same way. Sham-exposed cells were not exposed to nsPEF but subjected to all the same manipulations. In panel D cells were transfected with two siRNA targeting Tmem16f transcripts (siRNA Tmem16f-I and -II) or scrambled siRNA and specific silencing was verified by real-time quantitative PCR. Tmem16f mRNA levels were normalized to b-act mRNA and are shown as relative expression. Panel E shows that silencing Tmem16f with two different siRNA sequences, Tmem16f-I (left) and -II (right), reduced the Annexin V emission curve in response to nsPEF. Mean ± S.E., 35–46 (B), 35–40 (D), and 25–35 (E) cells per each group from 3 to 5 independent experiments.
Figure 2.
Figure 2.
Silencing Tmem16f expression inhibits membrane blebbing (A and B) and reduced calcium transient (C) induced by nsPEF. HEK 293 cells silenced for Tmem16f expression were treated with one 300-ns 25.5 kV/cm pulse in the presence of 2 mm extracellular Ca2+. Panel A shows the DIC images taken at the indicated time points of two representative groups of control (black) and Tmem16f-silenced (red) cells. At 360 s post-nsPEF, the yellow arrows indicate the pseudopod-like blebs. Quantification of the effects seen in A is shown in B. Blebs were measured at 360 s after nsPEF. Cells were considered bleb-positive when the length of the bleb exceeded the cell radius. In cells considered positive for blebbing, the maximum bleb length was calculated by measuring the longest bleb in each cell. Mean ± S.E., 100–120 cells per each group from 3 independent experiments. *, p < 0.01. Panel C compares Ca2+ transients evoked by nsPEF in cells silenced for Tmem16f expression and control cells. The nsPEF treatment was done at 27 s into the experiment, after acquiring 3 pre-exposure images as a baseline, changes in cytosolic free calcium concentration were measured using Fluo-4. Each trace is the average from 50 cells.
Figure 3.
Figure 3.
Nanopore current dependence on extracellular Cl− (A) and intracellular free Ca2+ (B). HEK 293 cells were bathed with 2 mm extracellular CaCl2 and, 2 min after the establishment of the whole cell configuration, subjected to one 300-ns pulse at 4.2 kV/cm. Panel A shows the effect on Cl− replaced by Gluconate− on membrane currents in nsPEF-exposed cells. Currents were measured at 30 s prior to nsPEF and then after at 60 s. Mean ± S.E., 15 cells per each group from 2 independent experiments. Inset shows the position of the PEF-delivering electrodes relative to the exposed cell and the recording pipette. Calibration bar: 20 μm. In panel B, the cytoplasm of the patched cells was dialyzed using pipette solutions containing either 0.5 or 5 mm EGTA, or 5 mm BAPTA. Currents were measured at the indicated time points relative to the moment of nsPEF delivery. Mean ± S.E., 10 cells per each group from 3 independent experiments. *, p < 0.05; **, p < 0.001 for the difference between +30 and +150 s.
Figure 4.
Figure 4.
Silencing Tmem16f expression suppresses currents at positive membrane potentials. I-V curves for control and Tmem16f-silenced cells measured at 30 and 270 s after nsPEF (one 300-ns, 4.2 kV/cm). Values were corrected for their parallel sham exposure measurements. Mean ± S.E., 14–16 cells per each group from 4 independent experiments. *, p < 0.05 for the difference between control and Tmem16f-silenced samples.
Figure 5.
Figure 5.
The effect of pulse number (A) and TMEM16F overexpression on HEK 293 cell death (B and C). A, cells were treated with increasing numbers of 300-ns pulses (20 Hz at 6 kV/cm). Exposures were performed in 1-mm gap electroporation cuvettes and cell survival was assessed in 24 h. Results are expressed in % to sham-exposed parallel control at 24 h. Mean ± S.E. for 9 independent experiments. B, expression level of TMEM16F-3xFLAG analyzed by Western blot and FACS analyses at 48 h after transfection. Extracts of cells were immunoblotted with an anti-FLAG and, as a control, with β-actin antibody. TMEM16F is seen as a broad band around 120 kDa. To measure the % of TMEM16F-positive cells, control and overexpressing cells were permeabilized, stained with an anti-FLAG antibody, and analyzed by FACS. Cells in the gated area were considered positive for TMEM16F expression. C, overexpression of TMEM16F increases the cytotoxicity of exposure to 120 pulses (300-ns width, 6 kV/cm, 20 Hz). The survival was measured at 24 h after nsPEF exposure and expressed in % to sham-exposed parallel control. Mean ± S.E. for 6 independent experiments. *, p < 0.001.
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