External Ca2+ Dependency of Synaptic Transmission in Drosophila synaptotagmin I Mutants

To resolve some of differences in reports on the function of Synaptotagmin I (Syt I), we re-examined synaptic transmission at the neuromuscular junction of Drosophila embryos that have mutations in the Syt I gene ( syt I). Two major questions addressed were which Ca2+ binding domain, C2A or C2B, sense Ca2+ and is Syt I a negative regulator of spontaneous vesicle fusion. Synaptic currents were induced by nerve stimulation or by high K+ treatment in external solutions containing various Ca2+ concentrations. In a null allele, syt IAD4, synchronous synaptic currents were rarely observed but not abolished. The quantal content was about 1/60 of control but increased linearly with [Ca2+]e with a slope of 0.95 ( N) in the double logarithmic plot, in contrast to 3.01 in control. The slope of 1.06 in an allele, syt IAD1, which lacks the second Ca2+ binding domain, C2B, was not different from in syt IAD4. In another allele, syt IAD3, in which one amino acid in C2B is mutated, synchronous synaptic transmission was also impaired and N was 1.54, which is significantly smaller than in control. In high K+ saline, the [Ca2+]e dependency of vesicle release in syt IAD4 was lower than in controls, whereas that in syt IAD3 was even lower than in syt IAD4, suggesting that syt IAD3 is inhibiting vesicle fusion. These findings led us to conclude that C2B, not C2A, senses Ca2+, and Syt I is a negative regulator of vesicle fusion.