Figure 3.
Summary of the effect of GTP analogs and Ca2+ on vesicle mobilization. A, Average traces of FM2-10 destaining in permeabilized synapses after application of 1 μm Ca2+. Dashed line indicates background fluorescence loss (including photobleaching) without application of Ca2+ (typically <10% of the initial fluorescence). Incubation with GTP (n = 6) and GTP-γ-S (n = 6) significantly increased the extent of vesicle mobilization obtained with Ca2+ alone (n = 4) (p < 0.01). Preincubation with GDP-β-S (n = 4) decreased this amount (p < 0.01), whereas addition of ATP (n = 3) did not change it (p > 0.8). B, Bars represent the amount of total recycling pool mobilized after 250 sec of stimulation with 0.1, 1, 10, 100, 300, and 1000 μm Ca2+. Permeabilized synapses were pretreated with Ca2+-free media alone (gray bars; n = 2 for 0.1 μm; n = 4 for 1 μm; n = 7 for 10 μm; n = 7 for 100 μm; n = 4 for 300 μm; n = 2 for 1 mm), with GDP-β-S (open bars; n = 2 for 0.1 μm; n = 4 for 1 μm; n = 2 for 10 μm; n = 4 for 100 μm; n = 4 for 300 μm; n = 2 for 1 mm), or with GTP-γ-S (black bars; n = 2 for 0.1 μm; n = 6 for 1 μm; n = 3 for 10 μm; n = 5 for 100 μm; n = 4 for 300 μm; n = 2 for 1 mm). Differences among the bars are statistically significant for Ca2+ concentrations between 1 and 300 μm (p < 0.05). Except at 300 μm Ca2+, the difference between preincubation with Ca2+-free media alone and GDP-β-S preincubation is significant at p = 0.07. C, The plot of fastest time constants of destaining versus Ca2+ concentration shows a bell-shaped relationship. Preincubation with GTP-γ-S did not alter the rate of destaining seen with preincubation with Ca2+-free media alone.