Vesicular neurotransmitter transporters (VNTs) are a small family of proteins responsible for packaging neurotransmitter into secretory vesicles. Their presence and function are required for regulated secretion from neuronal and neuroendocrine cells. During both the biogenesis and the activity-dependent recycling of secretory vesicles, VNTs undergo trafficking that can determine the quality, quantity, and location of packaged neurotransmitter. Thus understanding the signals and mechanisms of VNT trafficking is essential to understanding the regulation of neurotransmission.
Here, the synaptic vesicle specific trafficking of Vesicular Acetylcholine Transporter (VAChT) is investigated. A dileucine containing targeting motif, with dual properties for internalization and synaptic vesicle targeting, is identified in the C-terminus of VAChT. Chimeras between this motif and an unrelated plasma membrane protein localize to synaptic-vesicle-like vesicles in a neuroendocrine cell line. The specificity and generalization of this motif is assessed. Next, sorting nexin 5 (SNX5), implicated in the regulation of membrane traffic, is identified as a novel regulator of VAChT targeting to synaptic vesicles. Disruption of SNX5 function leads to a decrease in VAChT-directed synaptic vesicle targeting and a concomitant increase in targeting to large dense core vesicles. This shift between secretory granules suggests an important mechanism of VNT regulation with the potential to shape properties of neurotransmission.
In order to understand the physiologic importance of VNT regulation, vesicular transport and its influence on activity-dependent release must be assessed in living neurons. However, this has not been possible. Therefore, a live cell assay was established to measure vesicular transport and its contributions to release in brain slice. Using a pH sensitive, fluorescent serotonin analog visualized by two-photon microscopy, activity dependent somatic release and vesicular monoamine transporter (VMAT) activity were measured in the dorsal raphe nucleus. Interestingly, while a portion of monoamine packaged at rest was held in reserve, monoamine packaged during stimulation was released efficiently. The work presented in this thesis provides a greater understanding of VNT trafficking and activity-dependent function. Furthermore, it provides the foundation for the comprehensive study of the active role of VNTs in shaping the properties of neurotransmission.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-02262009-143623 |
| Date | 11 March 2009 |
| Creators | Colgan, Lesley Anne |
| Contributors | Edwin Levitan, Gonzalo Torres, Thomas Martin, John Horn, Susan Amara, Yongjian Liu, Adrian Michael |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-02262009-143623/ |
| 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 University of Pittsburgh 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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