Figure 1
From: Anoctamin-4 is a bona fide Ca2+-dependent non-selective cation channel
Heterologous expression of Ano4 elicits Ca2+-dependent cation conductance that is sensitive to fenamates. (A) Confocal image of HEK293 cells expressing Ano4-c-Myc. Staining with an antibody against c-Myc (red). Scale bar represents 10 µm. (B) Raw currents through HEK293 cells heterologously expressing Ano4 before and after the application of ionomycin (1 µM) as indicated by the bar. Small box illustrating the stimulation protocol applied to the cells: 10 voltage steps between −140 mV and +60 mV of a 50 ms duration applied every 2.5 s. (C) Current density-voltage plot of HEK293 cells expressing Ano4 before (filled circles) and after the application of ionomycin (open circles). (D) Reversal potential of the cells in C before (black bar) and after the application of ionomycin (grey bar). (E) Raw currents through HEK293 cells expressing Ano4 in an ionomycin-containing Ringer solution before and after the application of the fenamate niflumic acid (NFA, 100 µm). Application is indicated by the bar. (F) Current density-voltage plot before (filled circles) and after the application of NFA (open circles). (G) Spline curve of change of current density (ΔpA/pF) of Ano4 transfected HEK293 cells at different [Ca2+]i. (H) Confocal images of HEK293 cells transfected with Ano4-GFP (upper panel) or Ano4-1-1150del-GFP (lower panel). Staining against pan-Cadherin (yellow) and Ano4 (red). Nuclei were stained with DAPI. Scale bar represents 10 µm. (I) Bar chart representing the Pearson Correlation Coefficient between pan-Cadherin and Ano4 pixels (transfection and immunostaining according to (H)). (J) Ionomycin-mediated increase in current density in cells transfected with GFP (black bar), Ano4-GFP (grey) and Ano4-1-1150del-GFP (white bar). (K–M) Assessment of scramblase activity by FACS sorting of annexin A5-labeled HEK293 cells transfected with GFP alone (K), with Ano4 plus GFP under control conditions (L) or after the application of ionomycin (1 µM). (M) X-axis: Fluorescence intensity of Anx A5-6S-IDCC (log); Y-axis: Fluorescence intensity of GFP (log). The right upper square represents the transfected, Annexin A5-positive cell fraction. Values are given as mean ± SEM. ***p < 0.001.
