Functional Characterization of Synaptic Proteins in Calcium Triggered Exocytosis




Chang, Wen-Pin

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Release of neurotransmitter involves fusion of the membrane of synaptic vesicle with the presynaptic plasma membrane, a process that is tightly regulated by calcium. One of the central goals is to understand the molecular machinery underline the fundamental fusion mechanism used at all synapses, thus it is important to characterize the physiological function of unknown synaptic proteins and to identify new members that might have functions in synaptic vesicle fusion. In this thesis, I first characterize the function of synaptic vesicle protein 2 (SV2), which is one of the first synaptic vesicle proteins identified. SV2 is essential for survival in mice; its deletion impairs neurotransmitter release, although the exact point at which step of release is affected remains unclear. Using electrophysiological approaches, our data demonstrate that SV2 acts downstream of the priming, but upstream of the Ca2+-triggering of vesicle fusion. By using rescue experiments, we also demonstrate that mutations of charged residues within the transmembrane regions or of the intravesicular glycosylation sequences of SV2 block its function, probably by impairing the folding and trafficking of SV2. In contrast, deletion of the conserved N-terminal putative synaptotagmin-binding sequence of SV2 did not abolish SV2 function, nor did mutation of another conserved cytoplasmic sequence. These observations suggest that SV2 functions in a maturation step of primed vesicles that converts the vesicles into a Ca2+- and synaptotagmin-responsive state. Second, SNAREs and Sec1/Munc18 (SM) proteins are critical for intracellular membrane fusion. The neuronal SM protein Munc18-1 binds to SNARE complexes and syntaxin-1. The interaction to SNARE complex likely represents the general mode of SMARE/SM protein coupling, but the understanding of its physiological relevance to vesicle fusion and precise point of its function during the process is hindered by the duality of Munc18-1/SNARE binding modes. Here we designed three mutations that preserve Munc18-1/syntaxin-1 binding but differentially impairs the Munc18-1/SNARE complex binding. By utilizing rescue experiments, we showed that the impairment correlates with disruption of vesicle priming and evoked release, and suggest that Munc18-1/SNARE complex assemblies generally govern membrane traffic. Third, we reported the primary structure and biochemical properties of a family of evolutionarily conserved mammalian proteins, E-Syts, which contain multiple C2 domains, a common Ca2+ binding module, and a transmembrane region. Our findings suggest that E-Syts function as Ca2+-regulated intrinsic membrane proteins and expand the repertoire of multiple C2 domains proteins to a fourth class beyond synaptotagmins, ferlins, and MTCPs (multiple C2 domain and transmembrane region proteins).

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