In the nervous system, the primary mode of communication between neurons and their target cells (synaptic transmission), occurs when calcium stimulates the fusion of neurotransmitter-filled vesicles with the plasma membrane. It is a highly regulated process, underlying virtually all neuronal pathways from simple reflex responses to higher order brain function. Our long term objectives are to understand the molecular mechanisms mediating calcium regulated secretion and how short- and long- term changes in synaptic transmission contribute to alterations in synaptic strength in normal and pathological states. To analyze the role of calcium responsive proteins in this process, we have established a novel microinjection system in neuroendocrine PC12 cells, a model system for calcium regulated secretion. Our preliminary microinjection studies into PC12 cells demonstrated that antibodies against the synaptic vesicle protein synaptotagmin (syt) and a recombinant syt fragment, blocked the secretion of catecholamines from PC12 cells, identifying putative functional domains. The inhibitory syt fragment used in these microinjection studies is phosphorylated in vitro by casein kinase II and binds phospholipids in a calcium responsive fashion. On the basis of these observations, we hypothesize that syt functions as a general regulator of calcium regulated secretion, an activity which involves calcium dependent interactions with phospholipids and is dependent on the phosphorylation state of syt. We propose four specific aims to test this hypothesis; l. Define the functional domains of syt l for calcium regulated secretion from PC12 cells. The functional consequences of mutations in the putative calcium binding domain of syt (C2A) on calcium regulated secretion, will be determined by microinjection into PC12 cells. 2. Characterization of mutations in the C2A domain of syt I on calcium dependent phospholipid binding. Effect of the mutations in the C2A domain on syt phospholipid binding in vitro will be examined and the importance of calcium for these interactions determined. 3. Investigate the physiological role of syt I phosphorylation on calcium regulated secretion. To examine the role of syt phosphorylation in vivo, microinjection studies will be performed. The effect of mutant syt fragments and peptides which are poor substrates for casein kinase phosphorylation, on catecholamine release will be determined. 4. Investigate the function of syt I in calcium regulated secretion from SLMVs in neuroendocrine PC12 cells. Using specific cell surface markers for two different populations of secretory vesicles (LDCVs and SLMVs) in PC12 cells, the functional domains of syt regulating the calcium dependent fusion of these two vesicle populations with the plasma membrane will be compared. Discernible differences in syt activity may offer some insight into the molecular mechanisms underlying differential neurotransmitter release, an issue fundamental to higher order brain function.
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