Figure 2.

Mutation of the C-terminal dileucine-like motif slows, but does not eliminate VGLUT1 endocytosis. A, Time course of changes in fluorescence intensity (ΔF/F₀) in hippocampal neurons transfected with WT VGLUT1-pH (black), FV/AA VGLUT1-pH (red), or FV/GG VGLUT1-pH (blue). The traces, normalized to peak fluorescence, exhibit increases during stimulation at 30 Hz for 1 min (bar) and decreases after stimulation, consistent with exocytosis followed by endocytosis. Endocytosis of FV/GG VGLUT1-pH is less efficient than either WT or FV/AA. B, The mean poststimulus fluorescence decay, fit to a single exponential, is significantly faster for WT (36.9 ± 4.3 s) than FV/AA (59.8 ± 5.0 s; *p < 0.05) or FV/GG (89.4 ± 10.0 s; **p < 0.001, one-way ANOVA, Tukey's post-test). C, The fraction of VGLUT1-pH present on the cell surface at steady-state before stimulation (solid bars; stim/recovery −) and 3 min after stimulation (striped bars; stim/recovery +) was estimated by subtracting the fluorescence upon acid quenching with Tyrode's solution containing MES, pH 5.5, from the fluorescence at rest, normalized to total fluorescence upon alkalinization with NH₄Cl. The steady-state surface level of the FV/GG mutant (6.0 ± 1.0%) is significantly higher than both the FV/AA mutant (3.2 ± 0.7%; p < 0.05, one-way ANOVA, Tukey's post-test) and WT (2.2 ± 0.4%; p < 0.01, one-way ANOVA, Tukey's post-test). Fluorescence after recovery from stimulation of both FV/AA (8.2 ± 1.1%) and FV/GG (18.8 ± 1.4%) mutants are significantly higher than WT (2.3 ± 0.9%; WT vs FV/AA, p < 0.01; WT vs FV/GG, p < 0.001; one-way ANOVA, Tukey's post-test). D, The total amount of transporter released by stimulation at 30 Hz for 1 min was determined in the presence of bafilomycin. The fraction of the total internal pool released is similar between WT (69.6 ± 4.1%), FV/AA (65.0 ± 2.6%), and FV/GG (65.8 ± 2.2%) VGLUT1-pH (p = 0.55, one-way ANOVA). The total internal pool was determined by subtraction of the initial fluorescence from the total fluorescence, as measured by application of 50 mm NH₄Cl. E, Total protein expression of WT (117.7 ± 12.7 a.u.), FV/AA (98.7 ± 7.9 a.u.), and FV/GG (92.1 ± 12.2 a.u.) VGLUT1-pH at boutons are not significantly different (p = 0.26, one-way ANOVA). Data are the mean ± SEM of at least 20 boutons per coverslip from 5–10 coverslips from at least two independent cultures.