Fig. 1.

Gap19 inhibits [Ca²⁺]_i-triggered ATP release in C6-Cx43 cells. a Topology of Cx43 and location of Gap19 in the L2 domain, part of the CL. The underlined sequence is a putative membrane translocation motif and the greyed zone is crucial for CT–CL interactions. b Confocal micrographs of C6-Cx43 cells, counter-stained for F-actin (red fluorescence), illustrating cellular uptake of fluorescein-labeled (green) Gap19 (FITC-Gap19), Gap19I^130A (FITC-Gap19^130A) and fluorescein only (Fluo). Scale bar is 20 μm. The bar chart below reports fluorescence intensities (A.U., arbitrary units) measured in the cells. Uptake of fluorescein-labeled peptides was significantly stronger as compared to fluorescein only (n = 4). Stars indicate significance compared to Fluo. c Inducing [Ca²⁺]_i changes with the Ca²⁺ ionophore A23187 (inset shows experimental approach) triggered significant ATP release in C6-Cx43 but not in C6 cells stably transfected with the empty vector (C6-pLTR) (n = 12). Gap19 (200 μM, 30 min) strongly inhibited [Ca²⁺]_i-triggered ATP release. d Gap19-inhibition of triggered ATP release was concentration-dependent (n = 6). e Junctional conductance (G_j) measurements in Cx43 expressing Novikoff cell pairs at different time points in the absence or presence of Gap19 (400 μM) in the recording pipette solution. G_j was normalized to the corresponding values at the beginning of the experiment. Gap19 had no effect on G_j (n = 4–16). f Representative images of a FRAP experiment in C6-Cx43 cells preloaded with CFDA. Images were acquired before photobleaching (pre-bleach), just after photobleaching the cell marked with the dotted line (bleach) and 5 min later to assess fluorescence recovery in the bleached cell. g Quantification of the fluorescence recovery 5 min after photobleaching: 30 min Gap19 (200 μM) had no influence while 24–48 h incubations promoted dye transfer (n = 5). Inset above illustrates that 24 h incubation with Gap19 inhibited ATP release equally strong as 30 min incubation (n = 12). h Exposure of C6-Panx1 cells to 143 mM [K⁺]_e-triggered ATP release that was blocked by carbenoxolone (Cbx, 10 μM, 30 min) or ¹⁰Panx1 (200 μM, 30 min), and absent in C6-WT cells. Gap19 or Scr¹⁰Panx1 had no effect on high [K⁺]_e-triggered ATP release (n = 12). i Gap19 (200 μM, 30 min) did not inhibit [Ca²⁺]_i-triggered ATP release (brought about by 2 μM A23187 applied during 5 min) in HeLa-Cx40 cells (n = 6). Stars indicate statistical significance compared to the neighboring grey baseline bar (except in b); number signs mark comparisons to the black control bar; one symbol p < 0.05, two symbols p < 0.01, three symbols p < 0.001