Fig. 3: The ion channel CNGA3 is a target of H2S.
From: Hydrogen sulfide as a potent predator-derived kairomone mediating fear-related responses in mice
a Transfection of HEK 293 cells with a plasmid encoding for GFP to validate the transfection and a plasmid coding for CNGA3. After 24–48 h, transfected cells are used for calcium imaging experiments (designed by BioRender). Microscopy images of the transfected cells with a DIC contrast (Hv) showing cells in green (GFP reporter plasmid) and after Fura-2 loading. b Immunostainings of HEK cells transfected with GFP and CNGA3. White squares: zoomed areas. Green, GFP. Red, CNGA3. Blue, Dapi staining. Scale bars: 20 μm. c CNGA3 expressed in HEK cells are activated by 8-bromo-cGMP (1000 µM). Representative examples of the intracellular calcium changes observed in one CNGA3-negative cell (top; CNGA3−) and in one CNGA3-positive cell (bottom; CNGA3+). The Fura-2AM ratio is given in arbitrary units and time in minutes. Bar indicates perfusion time. d Representative examples of intracellular calcium changes observed in one CNGA3-negative cell (top; CNGA3−) and in one CNGA3-positive cell (bottom; CNGA3+) in the presence of ATP (100 μM; to control the viability of the cells) and in the presence of different concentrations of H2S (gradient). The Fura-2AM ratio is given in arbitrary units (a.u) and time in minutes. Bars indicate perfusion times. e Graphs representing the percentage of responding cells (left) and the amplitude of the responses (right) to different H2S concentrations. Number of cells: 50–150 from 1 to 3 different experiments. White bars, CNGA3-negative cells (CNGA3−); grey bars, CNGA3-positive cells (CNGA3+). f Graphs representing the percentage of responding CNGA3-positive cells (left) and the amplitude of the responses observed (right) to H2S (25 µM), and to the other known GG ligands, SBT, 2-PT and TMT (100 µM) (number of cells: 50). g Fluorimetric biochemical assessment of CNGA3 S-sulfhydration by H2S. Schematization of DTNB effect on the free thiol (SH−) groups performed on a lysate of HEK cells expressing CNGA3 (on the left; BioRender). Measurement of relative absorbance (arbitary units; a.u) of TNB over time in minutes at 412 nm (on the right). Three different conditions were tested : DTNB alone (0.3 mM; black line); DTNB + IAA (0.5 mM; magenta line) and DTNB + H2S (25 µM; orange line).
