Disease-causing mutations in Synaptotagmin can act via dominant-negative, gain-of-function or haploinsufficient mechanisms.

Members of the Synaptotagmin (SYT) family of synaptic vesicle (SV) proteins contain two Ca2+ binding C2 domains (C2A and C2B) that regulate the timing and probability of SV fusion. Recently, dominant mutations in SYT1, present at most CNS synapses, and SYT2, abundant in the PNS, have been found to cause human neurological diseases. Most pathogenic alleles localize to the C2B Ca2+ binding pocket, although mutations outside of this region have been identified. Here we report the effects of a large array of disease-associated human SYT point mutations on synaptic transmission using the Drosophila model to define their mechanism of action. Mutations in the C2B Ca2+ binding pocket were the most severe and acted in a dominant-negative manner to decrease evoked release. Indeed, mutations in the C2B Ca2+ binding pocket were a privileged site for dominant-negative effects on SV fusion. Molecular dynamics simulations indicate these mutations alter the topology of the C2B Ca2+ binding pocket and disrupt Ca2+-triggered membrane insertion. Several mutations outside of this region acted through a gain-of-function mechanism to enhance neurotransmitter release, while others caused the protein to undergo degradation. Consistent with this latter subset acting via haploinsufficiency, heterozygotes lacking one copy of SYT1 displayed a ~40% decrease in evoked release. These data indicate C2B Ca2+ binding pocket mutations act dominantly to poison the fusion machinery and block release sites, while mutations outside of this region cause haploinsufficiency or gain-of-function effects with milder effects on synaptic output and behavioral phenotypes.