Figure 7.

VAMP2 G₁₀₀/C₁₀₃ influence fusion pore opening. Measurements of quantal release of ATP via P2X₂ receptor mediated currents from INS-1 832/13 cells expressing VAMP2 WT (G₁₀₀/C₁₀₃) or VV (G₁₀₀V/C₁₀₃V) after knock-down of endogenous VAMP2. (a) Currents triggered by ATP release. Top traces show INS-1 832/13 cells transfected with shC (black trace) and infused with media containing 0 or 2 μM free calcium. The 3 bottom traces are from cells co-transfected with plasmid P2X₂-YFP, shC or shV, and VAMP2-pHL (WT^R or VV^R), respectively (black, shC; red, shV + empty vector; blue, shV + VAMP2pHL WT^R; green; shV + VAMP2pHL VV^R). (b i) Frequency of events per second (ANOVA and Tukey shC vs. shV p = 0.004, vs WT^R p = 0.004, vs. VV^R p = 0.001; shC, 12 cells, n = 802 events total; shV, 6 cells, n = 30 events total; WT^R 12 cells, n = 388 events; VV^R, 8 cells, n = 695 events; _°, outliers). (b) Response delay defined as time required for the first event to be detected (ANOVA and Tukey, shC = 4 ± 0.86 s, WT^R = 14, 43 ± 3.21 s, VV^R = 36.90 ± 11.48 s. (c) Schematic illustration of the charge, the 10% to 90% rise time, the rise slope, and the half width of the current generated by P2X₂R. These parameters are measured for the fusion pore kinetics study. (d,e) Mean values of the medians for the charge, the half width (d) and rise time and its slope (e), respectively (*p < 0.05, **p < 0.01, t test). (f) Superimposition of currents of similar amplitude elicited from cells expressing VAMP2pHL WT^R (blue) and VV^R (green).