Calcium and pH dependence dye uptake of N2a cells expressing Panx1 proteins.

<p>N2a cells expressing EYFP or EYFP-tagged mPanx1, drPanx1a or drPanx1b were used for dye uptake assays 48 h post transient transfection. Ethidium bromide (EtBr) fluorescence was compared 5 min after EtBr (20 µM) application. Each bar represents the mean + SEM of 135 analyzed cells. (<b>A</b>) <i>Time </i><i>course </i><i>and</i> (<b>B</b>) <i>statistical </i><i>analyses </i><i>of </i><i>the </i><i>dye </i><i>uptake </i><i>of </i><i>EYFP</i> or <i>Panx1 </i><i>expressing </i><i>N2a </i><i>cells</i>. All values were normalized to the averaged EtBr uptake of drPanx1a expressing cells, which was set to 100%. (p<0.001 = ***) (<b>C</b>, <b>D</b>) <i>Pharmacological reduction of dye uptake in (C) drPanx1a and (D) drPanx1b transfectants</i>. Multiple well-described connexin hemichannel and Panx1 blockers were applied together with EtBr to the external solution. All values were normalized and compared to the control condition of drPanx1a (C) or drPanx1b (D) expressing cells (Ctrl), which was set to 100%. (<b>E</b>) <i>Elevation of dye uptake mediated by Ca²⁺/ionomycin stimulation in EYFP or Panx1 expressing N2a cells</i>. Ionomycin (Iono) was applied together with EtBr. All values were normalized to the averaged EtBr uptake of drPanx1a expressing cells, which was set to 100%. The dye uptake was compared between ionomycin stimulated (+) cells and the respective control condition (-). (<b>F</b>) <i>Influence of the extracellular pH on dye uptake in drPanx1 transfected N2a cells</i>. All values were normalized to the control condition at pH 7.4 of either drPanx1a (blue) or drPanx1b (green) expressing cells, set to 100%. A sigmoidal curve fit was performed. The EYFP control at pH 7.4 is depicted in yellow. (PEG = PEG1500; n = 135 cells per group; p>0.05 = ns; p<0.05 = *; p<0.01 = **; p<0.001 = ***).