Dopaminergic neurotransmission plays an important role in the regulation of cognitive, behavioral, and motor functions. Abnormalities in the dopamine (DA) system have been implicated in neuropsychiatric disorders including drug addiction, schizophrenia, attention deficit hyperactivity disorder, and autism spectrum disorder (ASD). The DA transporter (DAT) is a presynaptic protein that regulates DA neurotransmission by mediating the re-uptake of synaptically released DA. The DAT is a major molecular target of the psychostimulant amphetamine (AMPH), which causes an elevation in extracellular DA by inducing a reversal in DAT function.
Since DA neurotransmission is heavily dictated by DAT function, understanding the regulators of DAT transport may be instrumental in understanding DA-related neuropsychiatric disorders. In this dissertation, I report multiple avenues of research that explore the regulatory events associated with DAT-mediated reverse transport of DA. First, I present that the phospholipid PIP2 directly binds, through electrostatic interactions, to positively charged DAT N-terminal residues. This interaction is required for robust AMPH-induced, DA efflux, yet does not affect the process of DA uptake. I also describe an ASD-associated de novo mutation in the DAT gene. This de novo mutation causes profound abnormalities in DAT function, including a persistent reverse transport of DA. Exposing the mutant DAT to Zn2+ partially reverses the functional deficits observed in the transporter. Lastly, I functionally characterize the effect of two separate gene variations associated with ASD, one in the DAT-interacting protein syntaxin 1 (STX1) and one in the DAT itself. I observe that the STX1 variant is hypo-phosphorylated at a key regulatory residue, resulting in a reduction in the capacity of the DAT to reverse transport DA. In parallel, I observe that the hDAT variant has reduced interactions with STX1, which also results in a reduction in the capacity of the DAT to transport DA in reverse. Collectively, these data outline multiple molecular and structural regulators of the reversal of DAT function and may contribute to a more complete understanding of the etiology of DA-related neuropsychiatric disorders.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-12052014-153138 |
| Date | 08 December 2014 |
| Creators | Hamilton, Peter James |
| Contributors | Laurence Zwiebel, Kevin Currie, Hassane Mchaourab, David Jacobson, Aurelio Galli |
| 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-12052014-153138/ |
| Rights | unrestricted, 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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