Role of CSP/Hsc70 in Regulated Neurotransmitter Release

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EXCEED THE SPACE PROVIDED. Neurotransmitter release is regulated at many steps, which in part confers upon synapses their plasticity, adaptability, and individuality. Two key steps, Ca 2+ entry and vesicle fusion, appear to be regulated by the synaptic vesicle-associated cysteine-string protein (CSP) - but in "opposing" ways. Previous work supports the hypothesis that CSP reduces release by inhibiting presynaptic Ca z+entry and increases release by promoting a downstream step of CaZ+-triggered fusion. Together with Hsc70, CSP might direct protein interactions among Ca 2¿ channels, G proteins, syntaxin, and synaptotagmin. To better understand CSP's action, we need to know: (1) whether CSP indeed promotes G_7 inhibition of Ca z+ channels at nerve terminals, (2) which of the other known CSP interactions mediates which function of CSP, and (3) which functions are regulated by PKA-phosphorylation at nerve terminals. To resolve these issues, I propose to test the above hypotheses by exploiting the genetic model system Drosophila to examine the effects of systematically targeted CSP mutations on neurotransmission at neuromuscular junctions, accomplishing a complete in vivo structure/function analysis. Specifically, Aim 1 will (a) correlate protein interactions of the J-, L-, C-, and Ct-domain with CSP's synaptic roles, including Ca 2¿ entry, Ca2+-triggered fusion, short-ter m plasticity of release, and Ca 2+ homeostasis. Aim 1 will also (b) resolve the significance of PKA-mediated phosphorylation of CSP at nerve terminals. Aim 2 will determine (a) whether CSP is critical for G_' inhibition of Ca > entry and/or (b) vesicular fusion. From this systematic analysis a substantial framework will emerge for understanding the apparently opposing actions of CSP. The proposed work will also expand our understanding of important regulatory mechanisms of synaptic transmission and their relation to the functional plasticity of the nervous system and human health. PERFORMANCE SITE ========================================Section End===========================================