Neurotransmitter release is regulated at many steps, which in part confers upon synapses their plasticity, adaptability, and individuality. Two key steps, Ca2+ 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 Ca2+ entry and increases release by promoting a downstream step of Ca2+-triggered fusion. Together with Hsc70, CSP might direct protein interactions among Ca 2v channels, G proteins, syntaxin, and synaptotagmin. To better understand CSP's action, we need to know: (1) whether CSP indeed promotes Gbeta/gamma inhibition of Ca2+ 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 Ca2+ entry, Ca2+-triggered fusion, short-term plasticity of release, and Ca2+ 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 Gbeta/gamma inhibition of Ca2+ 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS038274-04A1
Application #
6580118
Study Section
Special Emphasis Panel (ZRG1-MDCN-1 (01))
Program Officer
Talley, Edmund M
Project Start
1998-12-03
Project End
2005-11-30
Budget Start
2002-12-15
Budget End
2003-11-30
Support Year
4
Fiscal Year
2003
Total Cost
$311,278
Indirect Cost
Name
University of Arizona
Department
Type
Organized Research Units
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721
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Bronk, Peter; Nie, Zhiping; Klose, Markus K et al. (2005) The multiple functions of cysteine-string protein analyzed at Drosophila nerve terminals. J Neurosci 25:2204-14
Song, Wei; Ranjan, Ravi; Dawson-Scully, Ken et al. (2002) Presynaptic regulation of neurotransmission in Drosophila by the g protein-coupled receptor methuselah. Neuron 36:105-19
Bronk, P; Wenniger, J J; Dawson-Scully, K et al. (2001) Drosophila Hsc70-4 is critical for neurotransmitter exocytosis in vivo. Neuron 30:475-88
Zinsmaier, K E; Bronk, P (2001) Molecular chaperones and the regulation of neurotransmitter exocytosis. Biochem Pharmacol 62:1-11