Figure 5: Graded increase in current and ATP/dye permeation via concatemeric PANX1 channels with decreasing numbers of intact C termini.
From: A quantized mechanism for activation of pannexin channels
(a) Whole-cell current density was obtained from HEK293T cells expressing various GFP-tagged hexameric PANX1 concatemers, with (blue) or without (black) TEVp co-expression. Numbers of recorded cells are provided in the parenthesis. Inset: averaged CBX-sensitive I–V relationships, normalized to current density at +80 mV, from cells co-expressing concatemers plus TEVp. Note that 6(6CT) is not depicted because that construct generated no CBX-sensitive current, even with TEVp co-expression. (b) Cell-surface biotinylation from HEK293T cells expressing GFP-tagged hexameric PANX1 concatemers. Representative Western blots (top) and grouped data (bottom, n=4) show a similar ratio of cell-surface expression for all GFP-tagged PANX1 concatemers (normalized to total expression). Loading controls for streptavidin pull-down samples and total cell lysate samples were Na+/K+ ATPase and α-tubulin. (See Supplementary Fig. 6 for uncropped images). (c) Averaged Trovan-sensitive ATP release from HEK293T cells expressing GFP-tagged hexameric PANX1 concatemers with (blue) or without (black) TEVp co-expression (n=4 experiments; 4 h collection). (d) TO-PRO-3 uptake in Jurkat T cells expressing GFP-tagged hexameric PANX1 concatemers. Representative flow cytometry data shows GFP fluorescence (left) or TO-PRO-3 uptake (right) in cells with or without TEVp co-expression. Bar graph shows grouped data from four independent experiments; mean fluorescence intensity (MFI) of TO-PRO-3 uptake was normalized to the corresponding MFI for GFP within an experiment, and those values were normalized to the value for 6(0CT)+TEVp across independent experiments. Predicted whole-cell current (a), ATP release (c) and TO-PRO-3 uptake (d) based on single-channel properties are overlaid (grey data points). All data are presented as mean±s.e.m.; *P<0.05 using two-way analysis of variance followed by Fisher’s LSD test; NS, no statistical significance.
