Dissection of the Roles of Synaptotagmins in Calcium Dependent Neurotransmitter Release

Neuronal communication is mediated by neurotransmitter release, which is triggered in response to a calcium influx into the presynaptic cell, caused by the arrival of an action potential. Synaptotagmins (Syts) mediate this calcium-dependent regulation of neurotransmitter release by mechanisms that still remain elusive. Syts have two calcium-binding C2 domains (C2A and C2B) which are crucial for their function. Clarifying how Syt C2 domains interact with the neuronal membrane fusion machinery and the membranes is essential for gaining insights into the mechanisms of calcium-triggered neurotransmitter release. To understand the role of Syts in neurotransmitter release, we explored the origins of functional differences between the C2 domains of two Syt isoforms, Syt1 and Syt7, which mediate synchronous and asynchronous release respectively. Calcium binding to the C2A domain is critical for Syt7 functions whereas Syt1 function depends more on calcium binding ability of its C2B domain. By solving the structures of Syt7 C2A and C2AB fragments and by analyzing their intrinsic calcium binding properties by ITC, we showed that these properties do not give rise to the functional differentiation between Syt1 and Syt7. By characterizing the calcium-dependent phospholipid binding of C2 domains by FRET, we demonstrated that C2A and C2B dominate membrane binding in Syt7 and Syt1, respectively. This suggests that membrane affinity of C2 domains might dictate their functional importance for Syt function. In addition to membrane binding, Syt1 C2B domain is also involved in interactions with the SNARE complex, which is the central component of neuronal membrane fusion machinery. Clarifying the molecular details of these interactions is crucial for understanding how Syt1 cooperates with SNARE complex to trigger calcium-dependent membrane fusion. Previous structural studies have shown three distinct SNARE complex-binding interfaces on Syt1 C2B domain, two of which overlap with membrane binding regions. By using NMR, we showed that only two of these interfaces exist in solution. By using FRET to characterize interactions on membranes, we showed that Syt1-SNARE complex binding primarily occurs via one of the remaining interfaces (primary interface), and this interaction is almost abolished in the presence of calcium. Together, these results suggest a model where, a release of Syt1-SNARE complex interactions by calcium triggers membrane fusion.