Both constitutive fusion and synaptic vesicle fusion are mediated by SNARE proteins. However, they differ in one important aspect – synaptic vesicle fusion is regulated by calcium. Recent studies suggest that calcium sensitivity is conferred by two proteins: complexin and synaptotagmin. Current models suggest that complexin acts as a brake on vesicle fusion that synaptotagmin can relieve upon calcium influx. Loss of complexin (
cpx-1) from C. elegans results in a >90% reduction in evoked release, an increase in mini frequency and a 50% reduction in the number of synaptic vesicles at the synapse. These results suggest that synaptic vesicles are rapidly released and depleted at the synapse in
cpx-1 mutants, supporting a role for
cpx-1 as a brake on fusion. Loss of synaptotagmin-1 (
snt-1) in C. elegans results in a 50% reduction in evoked neurotransmitter release and a >90% reduction in mini frequency.
snt-1 mutants also exhibit a dramatic reduction in the number of synaptic vesicles at the synapse.
cpx-1;
snt-1 double mutants are additive for these phenotypes. However, the analysis of
snt-1 mutants is complicated by its role in synaptic vesicle endocytosis as well as in synaptic vesicle exocytosis. To circumvent this issue we need to rescue
snt-1 for endocytosis, but leave exocytosis defective. We used mosSCI to insert a single copy of the
snt-1 gene into
snt-1 null mutants. These constructs rescue the
snt-1 null phenotypes and provide a reagent to introduce
snt-1 point mutations. To separate out SNT-1's role in synaptic vesicle endocytosis and synaptic vesicle exocytosis, we have blocked SNT-1's ability to bind calcium by mutating the calcium coordinating aspartate residues in either the C2A (C2A*), C2B (C2B*) or both C2A and B (C2A*B*) domains of
snt-1. All of the mutants are expressed and trafficked to the synapse and partially rescue the locomotion phenotype of
snt-1 null mutants. Both the C2B* and C2A*B* mutants exhibit resistance to aldicarb suggesting they cause reduced neurotransmitter release. Mutations in the C2A* domain exhibit hypersensitivity to aldicarb, suggesting that this mutation, like
cpx-1 mutants, exhibits excessive neurotransmitter release. We are in the process of analyzing these
snt-1 mutants on their own and in a
cpx-1 null mutant background.