The computational and cognitive abilities of the brain arise from the sheer number and variety of its internal communication links. The structural and functional unit of these communication links is the synapse. Modulation of synaptic strength is the basic property of all neuronal circuits and is believed to be a fundamental mechanism underlying the plasticity of the CNS. In recent years fundamental progress in the isolation and characterization of a variety of nerve terminal-enriched proteins has been made. The present study will examine the biochemical machinery that orchestrates synaptic vesicle docking and fusion. We will directly compare the effects on neurotransmitter release of the post-translational modification (protein phosphorylation) of different components of the release machinery. The first part of these studies will determine which of the selected proteins are substrates for a set of protein kinases implicated in nerve terminal function. The nerve terminal proteins VAMP, synaptotagmin, synaptophysin and SNAP-25 will be characterized as physiological substrates for Ca2+/ calmodulin-dependent protein kinase II, protein kinase C, cAMP-dependent protein kinase and casein kinase II using in vitro and in vivo assays. In each case, the sites of phosphorylation will be identified. We will then use direct injections of these proteins into the presynaptic element as a way to dissect their specific roles in the regulation of synaptic vesicles trafficking and neurotransmitter release. Using electron microscopy techniques we will compare ultrastructural changes in the nerve terminal resulting from these injections. Effects of phosphorylation of each of these proteins on specific protein-protein interactions that have been shown as physiologically relevant, will be assessed using quantitative in vitro binding assays. Phosphorylation state-specific antibodies will also be used for ultrastructural studies to localize these phosphorylation events within the synapse. Finally, fluorescently-labeled phosphorylation state-specific antibodies will be employed in injection experiments into living axons to monitor phosphorylation driven regulation in a functioning synapse. These studies will reveal the physiological role for phosphorylation of selected nerve terminal proteins in neurotransmitter release and release modulation.
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