Dissecting G Protein Pathways

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DESCRIPTION (provided by applicant): Heterotrimeric G proteins are widely thought to play a critical role in regulating synaptic strength, yet relatively little is known about how these signaling pathways regulate synaptic vesicle exocytosis and recycling. We have undertaken a comprehensive analysis of how two G proteins (Ga0 and Gaq) regulate neurotransmitter release in the nematode C. elegans. We have shown that goa-1 Ga0 and egl-30 Gaq antagonistically regulate acetylcholine (ACh) release at C. elegans neuromuscular junctions (NMJs). We identified agonists that activate each of these pathways. Serotonin, acting via goa-1 Ga0, inhibits ACh release whereas muscarinic agonists, acting via egl-30 Gaq, stimulate ACh release. We showed that these G proteins are likely to directly regulate some aspect of synaptic vesicle recycling, since they control the abundance of a pre-synaptic syntaxin binding protein (UNC-l3) at ACh release sites. And we identified two proteins UNC-13 and protein kinase C (kin13 PKC) that are required for phorbol ester-mediated stimulation of ACh release. We propose three new aims to determine the mechanisms by which these G proteins regulate neurotransmitter release. First, we will determine what aspect of synaptic vesicle dynamics is regulated by goa-1 Ga0, egl-30 Gaq, UNC-13, and kin-13 PKC. Second, we will test the functional importance of the UNC-13:Syntaxin complex. Third, we will determine where and when kin-13 PKC acts to regulate ACh release. And we will test the functional importance of potential kin-13 PKC phosphorylation sites in SNAP-25 and UNC-18. In summary, G protein regulation of synaptic strength has been implicated in addiction, mood disorders, learning, and memory. Given the strong conservation of these pathways across phylogeny, it is likely that our experiments will provide new insights into the mechanisms underlying these fundamental aspects of signal transduction in the brain.