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.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03252016-105615 |
| Date | 08 April 2016 |
| Creators | Zurawski, Zack P. |
| Contributors | Heidi Hamm, Ph.D, Qi Zhang, Ph.D, Craig Lindsley, Ph.D, Kevin Currie, Brian Wadzinski, Ph.D |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-03252016-105615/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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