Evolutionarily conserved and divergent mechanisms of dual Ca²⁺ sensors in synaptic vesicle exocytosis

Neurotransmitter release at the Caenorhabditis elegans neuromuscular junction is governed by a dual Ca²⁺ sensor system composed of SNT-1 and SNT-3, which function analogously to the Ca²⁺ sensor systems found in certain mammalian neurons, such as synaptotagmin-1 and -7 (Syt1/Syt7) in the hippocampus. In this study, we investigated how SNT-1 and SNT-3 mediate fast and slow neurotransmitter release through their potential interactions with the SNARE complex and their polybasic motifs. AlphaFold 3 models of SNT-1-SNARE and SNT-3-SNARE complexes predicted a C2B-SNARE arrangement consistent with the canonical Syt1-SNARE primary interface [Zhou et al., Nature 525, 62-67 (2015)] and precisely identified conserved binding residues within the C2B domains, as well as in SNAP-25 and Syntaxin, highlighting the evolutionary conservation of this interaction. Electrophysiological analyses using targeted mutagenesis demonstrated that both SNT-1 and SNT-3 require C2B-SNARE interactions and polybasic motifs within their C2 domains to drive evoked fast and slow neurotransmitter release. Notably, SNT-1 and SNT-3 exhibited differential dependence on distinct regions of the C2B-SNARE interface and their respective polybasic motifs, suggesting that Ca²⁺-triggered fast and slow release operate via distinct mechanistic strategies. Furthermore, we found that SNT-1 mediates spontaneous neurotransmitter release through multiple pathways, involving not only the primary C2B-SNARE interface but also additional putative SNARE-binding interactions. Together, our findings uncover both conserved and divergent mechanisms for synaptic exocytosis regulated by the dual Ca²⁺ sensors in C. elegans.