Figure 6

The VV mutation in the TMD of VAMP2 alters exocytosis and fusion pore kinetics. Exocytosis and release kinetics were determined by near-field TIRF microscopy in PC12 and in INS-1 832/13 clonal β-cells expressing VAMP2 WT or the VAMP2 VV mutant after knock-down of endogenous VAMP2. (a) PC12 cells were transiently co-transfected with plasmids encoding VAMP2-pHluorin (WT^R or VV^R) and shRNA directed against VAMP2. Fusion events were recorded by TIRF microscopy and sum of fusion events per 10 s is given for cells stimulated by 90 mM [K⁺]_o (WT, n = 6 cells, 237 events; VV n = 4 cells, 139 events total) *2p < 0.05 (Student’s t-test). (b) Same as in (a) but measurements performed on INS-1 832/13. Sum of fusion event per 20 s in transfected cells were stimulated by 35 mM [K⁺]_o for 30 s (WT n = 9 cells, 1114 events total; VV n = 9 cells, 476 events total. *2p < 0.05; **2p < 0.01 (Student’s t-test). (c) Representative films of fusion events in INS-1 832/13 cells. The events are temporally synchronized to the frame of their maximum fluorescence. (d) Mean changes in normalized fluorescence immediately before and after exocytosis in INS-1 832/13 cells expressing either VAMP2pHL WT^R (open circles) or VV^R (closed circles). For display the fluorescent mean events were synchronized to their maximum fluorescence. Note narrower peak for VV as compared to WT. *2p < 0.05; **2p < 0.01 VAMP2 WT vs. VV (Student’s t-test). INSERT: Events synchronized for first event with a mean >10% of maximal fluorescence and curves fitted for first order exponential growth. Note shorter time constant τ for VV (closed circles) as compared to WT (open circles). The time constants τ_up for the fluorescence increases averaged 189 ± 28 ms for WT^R and 80 ± 7 ms for VV^R (p < 0.05; F-test). Similarly, the decay of fluorescence following the peak was slower for WT^R (time constant τ_down: 558 ± 57 ms) than for VV^R (τ_down: 315 ± 17 ms) (p < 0.05; F-test).