FIGURE 4.

Mutations in the trigger site greatly decrease the ability of synaptobrevin to support neurotransmitter release. Chromaffin cells of synaptobrevin 2/cellubrevin double null mice were isolated, and mutant or wild-type (WT) synaptobrevin was overexpressed. Catecholamine release was measured electrochemically and by membrane capacitance increase, which is proportional to changes in surface area. A, overexpression of synaptobrevin carrying the point mutations I45A and M46A or M46A shows a drastically reduced secretory response compared with wild-type synaptobrevin. Top panel, means ± S.E. of intracellular Ca²⁺ concentration after UV-induced Ca²⁺ uncaging at time = 0.5 s. Middle panel, mean capacitance increase. Bottom panel, mean amperometric current (thick traces, left ordinate) and amperometric charge (thin traces, right ordinate). Note that because the double mutant showed only very little response, it was not analyzed in more detail. B, a detailed view of normalized capacitance traces of wild-type synaptobrevin and the M46A mutant (A) shows that the kinetics of the remaining release is unaffected. C, burst and sustained release is reduced in the M46A mutant (wild-type rescue: n = 11; M46A: n = 17; double knock-out (DKO): n = 15. *, p < 0.05; **, p < 0.01; ***, p < 0.001 in nonparametric Mann-Whitney U test). The basal cell size is smaller in the double knock-out cells, probably because of impaired exocytosis before the stimulus.