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Title page for ETD etd-03252016-105615

Type of Document Dissertation
Author Zurawski, Zack P.
Author's Email Address zack.p.zurawski@vanderbilt.edu
URN etd-03252016-105615
Title The molecular requirements of the G- protein betagamma-SNARE interaction.
Degree PhD
Department Pharmacology
Advisory Committee
Advisor Name Title
Brian Wadzinski, Ph.D Committee Chair
Craig Lindsley, Ph.D Committee Member
Heidi Hamm, Ph.D Committee Member
Kevin Currie Committee Member
Qi Zhang, Ph.D Committee Member
  • synaptic transmission
  • exocytosis
  • SNAREs
  • neuroscience
  • G betagamma
  • G proteins
Date of Defense 2016-01-11
Availability unrestricted
Gi/o-coupled G-protein coupled receptors (GPCRs) can exert an inhibitory effect on vesicle release through several G-protein driven mechanisms, more than one of which may be concurrently present in individual presynaptic terminals. G protein betagamma subunits inhibit exocytosis via directly binding to the synaptosomal-associated protein of 25 kDa (SNAP25), competing with the fusogenic calcium sensor synaptotagmin 1 (Syt1) in a calcium-dependent manner for binding sites on SNAP25. Here, we generate several SNAP25 C-terminal mutants that are deficient in G protein betagamma binding while retaining normal vesicle release. The SNAP25-8A mutant features 8 G protein betagamma-binding residues mutated to Ala, and the SNAP25delta3 mutant, in which residue G204 is replaced by a stop codon, both feature a partial reduction in G protein beta1gamma2 binding in vitro. SNAP25-8A exhibits a reduction in the ability of the lamprey serotonin receptor to reduce excitatory postsynaptic current (EPSC) amplitudes, an effect previously shown to be mediated through the G protein betagamma-SNARE interaction. Syt1 binding to these mutants is largely intact. We conclude that the extreme C-terminus of SNAP25 is a critical region for the G protein betagamma-SNARE interaction. To further investigate the physiological relevance of the G protein betagamma-SNARE interaction, we have developed small molecule modulators of the G protein betagamma-SNARE interaction with micromolar potency. A transgenic mouse has been made containing the SNAP25delta3 mutation using the CRISPR-Cas9 reaction. Characterization of the phenotype of this animal is ongoing. In summary, we have identified key residues for the G protein betagamma-SNARE interaction and generated new experimental tools to investigate the importance of this interaction in tissues and disease states where its relevance is not currently known.
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