Project Summary/Abstract In this competitive renewal, we continue our studies of synaptotagmin (syt) isoforms that regulate the release of neurotransmitters and hormones. The founding member of this gene family, syt1, was discovered in 1981 and has been shown to function as the major Ca2+ sensor for synchronous synaptic vesicle (SV) exocytosis in neurons. Surprisingly, the most fundamental property of syt1—it's ability to bind and become activated by Ca2+—remains one of its least understood properties. Syt1 senses Ca2+ via tandem C2 domains, C2A and C2B, and five acidic Ca2+ binding residues have been proposed to coordinate 2-3 Ca2+ ions per domain. However, substitution of these residues in C2A has led to considerable confusion, with conflicting results as to whether these mutations are loss- or gain-of-function or have no effect at all; C2B has been studied in even less detail. In Aim 1 we propose quantitative experiments to compare the roles of each of these putative ligands in binding Ca2+, via isothermal titration calorimetry, and in release, via Ca2+ dose-response measurements of exocytosis using iGluSnFR. In Aim 2, we address the function of syt9, which is perhaps the most misunderstood isoform. Syt9 is in the same clade as syt1 and was proposed to trigger SV release in striatal neurons. Our preliminary data revealed that this is not the case; rather, syt9 is mainly targeted to dense core vesicles in striatal neurons where it regulates the release of substance P, to indirectly control spontaneous SV fusion rates. We will delve into this model by conducting syt9 structure-function studies using both reconstituted fusion assays and syt9 KO neurons, and by conducting pharmacological experiments. We will also expand this work to striatal slices, where substance P has been shown to strongly modulate dopamine release. Importantly, dopaminergic transmission in the striatum plays a key role in addiction and schizophrenia. So, we will conduct experiments to determine whether syt9-regulated substance P release modulates dopamine release in this brain region. Finally, in Aim 3 we return to syt1, to compare its role in activity- dependent SV docking with another isoform, syt7. SV docking dynamics have recently emerged as a key step in numerous aspects of release and short-term plasticity. Using our newly acquired zap-and-freeze instrument, combined with cryo-EM tomography, we will test the idea that syt1 mediates activity-dependent SV docking on short time scales to impact synchronous release, whereas syt7 mediates activity-dependent SV docking on longer time scales to impact asynchronous release, paired-pulse facilitation, and synaptic depression. Collectively, the three Aims proposed here will significantly advance our understanding of the functions of three distinct syt isoforms. In short, we will: 1) address the most elementary questions concerning how the founding member, syt1, senses Ca2+, 2) conduct a rigorous analysis of syt9 function in stria...