Mechanism of Synaptotagmin Action in Neurotransmitter Release

dc.contributor.advisorRizo-Rey, Joséen
dc.creatorArac-Ozkan, Demeten
dc.description.abstractNeurotransmitter 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.nnen
dc.format.digitalOriginborn digitalen
dc.subjectSNARE Proteinsen
dc.subjectSynaptotagmin Ien
dc.titleMechanism of Synaptotagmin Action in Neurotransmitter Releaseen
dc.type.materialTexten School of Biomedical Sciencesen Biophysicsen Southwestern Medical Centeren of Philosophyen


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