PROJECT ABSTRACT/SUMMARY All known cognitive, affective, and related behavioral processes rely on circuits formed by neuronal ensembles. High-fidelity communication between neurons requires the regulated release of neurotransmitters, which are usually contained in membrane-enclosed vesicles at presynaptic terminals. In most neurons, Ca2+ influx from voltage-gated channels acts upon presynaptic proteins to trigger fusion of these vesicles with the plasma membrane. The principal Ca2+ sensors for fast neurotransmitter release are members of the Synaptotagmin (Syt) families, principally Syt-1. De novo missense mutations in Syt-1 have been found in human patients with profound global developmental delays, underscoring the essential role this protein plays in brain function. A pair of closely-related proteins, Doc2α and Doc2β (collectively “Doc2”), have similar structural features but trigger release on a slower timescale as compared to Syt-1. Both Syt-1 and Doc2 contain tandem C2 domains that interact with membranes in a Ca2+-dependent fashion. But despite intensive study, it remains unclear how Syt-1 and Doc2 act upon presynaptic membranes and other proteins to trigger fusion. Candidate mechanisms include (1) the action of Syt-1/Doc2 on presynaptic membranes, and (2) direct interactions with soluble N- ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, which catalyze membrane fusion. This proposal seeks to address major unanswered questions about the Syt-1/Doc2—membrane and Syt- 1/Doc2—SNARE interactions that enable fast, Ca2+-triggered membrane fusion. Using a set of biophysical approaches, these experiments will define how SNAREs and physiologic phospholipids cooperate to shape the Syt-1/Doc2—membrane interface before, during, and after membrane fusion. Syt-1 mutations from human patients, two of which have not yet been described in the literature, will be studied using a combination of biophysical approaches and high-speed imaging of glutamate release in live neurons. By defining critical structure-function relationships in Syt-1, these results will establish a biophysical and physiologic basis for how Syt-1 mutations cause disease in human patients. Together, the proposed experiments stand to significantly deepen our mechanistic understanding of neurotransmission in health and disease.