The neuron-specific electroneutral potassium (K+) and chloride (Cl-) cotransporter, KCC2, is a key regulator of neuronal Cl-. KCC2 has been shown to play a critical role in controlling neuronal excitability, yet little is known about its regulation. Protein-protein interactions are generally well known to provide insight into membrane transporter regulation. Therefore, I have chosen to identify novel KCC2 protein-protein interactions, with the goal of studying how these interactions affect KCC2 activity.
I performed a yeast-two hybrid screen of a mouse brain cDNA library and identified several novel binding partners of the carboxyl terminus of KCC2 (KCC2-CT). One of these identified binding partners is Protein Associated with Myc, or PAM. Binding between KCC2 and the RCC1 domain of PAM (RCC1/PAM) was demonstrated using yeast two-hybrid, GST-pull-down assay, and coimmunoprecipitation. In order to study the functional role of PAM binding to KCC2, I identified the binding site of RCC1/PAM on the KCC2-CT, and within this site created a point mutant which disrupts RCC1/PAM binding. This point mutation was then transferred into full-length KCC2 and compared with wild-type KCC2 in 86Rb+/K+ uptake experiments to assess differences in transport activity. These flux experiments, along with experiments to measure changes in RNA and protein levels, lead to the conclusion that PAM binding to KCC2 does not primarily affect K-Cl transport by altering cell-surface expression of the cotransporter. Rather, PAM binding to the carboxyl terminus of KCC2 likely participates in the net dephosphorylation of KCC2, which in turn leads to activation of KCC2-mediated ion transport.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03312008-182826 |
| Date | 28 April 2008 |
| Creators | Garbarini, Nicole Jodela |
| Contributors | Randy Blakely, Eric Delpire, Louis DeFelice, Anne Kenworthy, Bih-Hwa Shieh |
| 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-03312008-182826/ |
| 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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