SUMMARY Information processing in the nervous system, essential for brain function, is predominantly mediated by chemical neurotransmission. Since Katz and his colleagues first revealed that Ca2+ triggers ultrafast vesicle exocytosis at the presynaptic terminal, studies have focused on the roles of the Ca2+ sensor proteins in this process. Biochemical, structural, and functional studies have found that two members of the synaptotagmin protein family, synaptotagmin-1 and -7 (Syt1 and Syt7), play critical roles in synaptic vesicle exocytosis by acting as primary and secondary Ca2+ sensors at many vertebrate synapses. Deletion of these Ca2+ sensors leads to severe synaptic transmission defects, and mutations in Syt1 and Syt7 have been linked to multiple neurological disorders. Despite the work done on synaptotagmins in rodents, a number of questions remain unresolved regarding how dual Ca2+-sensors coordinate to regulate synaptic transmission. In the last 20 years, the nematode C. elegans has been widely used to study the molecular and cellular mechanisms of synaptic transmission, and exhibit remarkably well-conserved synaptic machinery. Using behavioral and electrophysiological screens of synaptotagmin isoforms in C. elegans, our recent work has shown that the worm NMJs also use a dual Ca2+ sensor system (SNT-1 and SNT-3) to regulate neurotransmitter release. Loss of SNT-1 (the primary Ca2+ sensor) and SNT-3 (the secondary Ca2+ sensor) completely eliminate evoked release, while loss of either alone, has differential effects. The fact that the dual Ca2+ sensor systems of worms and mice share many similarities, allows us to study how synaptotagmins coordinately function at synapses, using the strengths of C. elegans as a genetic model organism and our unique technical expertise. In this project, we propose three aims to address how SNT-1 and SNT-3 work together to regulate neurotransmission. Aim1 focuses on potential interactions between SNT-1/3 with SNARE complexes and the plasma membrane. A number of highly conserved SNT-1and SNT-3 residues known to interface with SNAREs and the plasma membrane in vertebrates, will be mutated to test their effects on SV fusion. Aim 2 will investigate spatial and kinetic aspects of SNT-1 and SNT-3 function in coordinating synaptic release, and determine the interplay between SNT-1, SNT-3 and the key priming factor UNC-13. In Aim 3, we will determine how SNT-1 and SNT-3 regulate synaptic transmission under conditions of high release probability. The findings should significantly expand our understanding of how dual Ca2+ sensor systems operate to meet the demands of synapses under basal as well as elevated-release conditions.