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, Doc2? and Doc2? (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.
Studying the fundamentals of how neurons communicate can provide valuable information as we seek to better understand and treat brain illnesses. This grant application proposes experiments that will deepen our mechanistic understanding of neurotransmission and define how genetic mutations in regulators of neurotransmitter release cause disease in human patients. The knowledge gained from these experiments will better position researchers and clinicians to tackle the increasingly heavy burden of brain illnesses on children and adults in the U.S. and abroad.
