Mechanism of Synaptotagmin Action in Neurotransmitter Release

Neurotransmitter release occurs by fusion of the synaptic vesicle membrane with the plasma membrane. Formation of a highly stable complex, known as the SNARE (soluble NSF-attachment protein receptors) complex, brings the two membranes close in space. SNARE complex formation is required but probably not sufficient for fusion to occur. An increase in the local Ca2+ concentration at the synaptic terminal rapidly triggers neurotransmitter release. The mechanism of Ca2+ action is still unknown. Synaptotagmin 1, a brain-specific vesicular transmembrane protein, is the Ca2+ sensor in neurons. It has two cytoplasmic C2 domains (C2A and C2B) that bind Ca2+. Both C2 domains interact with negatively charged phospholipids in a Ca2+ dependent manner. The interaction of synaptotagmin 1 with the SNARE complex is also reported. We investigated whether the interaction of synaptotagmin 1 with membranes or with the SNARE complex is critical for membrane fusion. A new method to detect protein-protein interactions by 1D NMR spectroscopy was developed. Either the 13C signal of the SNARE complex or synaptotagmin 1 was monitored to perform competition experiments between SNAREs and lipid vesicles for binding to synaptotagmin 1. In the presence of both lipids and the SNARE complex, synaptotagmin 1 binds to lipids but cannot bind to the SNARE complex. This result suggests that Ca2+-dependent membrane binding is the primary activity of synaptotagmin 1. We investigated the mechanism of Ca2+-dependent phospholipid binding to synaptotagmin 1 C2 domains. A combination of crosslinking and FRET experiments showed that synaptotagmin 1 does not oligomerize upon Ca2+-dependent binding to phospholipid vesicles. Intriguingly, it binds to two membranes simultaneously and brings them into close proximity as visualized by cryo-EM experiments. We showed that the isolated C2B domain is sufficient to induce close membrane proximity. Mutational analysis suggested that the abundance of basic residues around the C2B surface, which generates a highly positive electrostatic potential together with the bound Ca2+ ions, is essential for this activity. We suggest that the ability of the C2B domain to bring membranes into close proximity can explain why the C2B domain has a more critical function in vivo than the C2A domain.nn