UVM Theses and Dissertations
Format:
Print
Author:
Markey, Jonathan Charles
Dept./Program:
Pharmacology
Year:
2006
Degree:
MS
Abstract:
The delayed rectifier voltage-gated potassium channel Kv1.2 is involved in a variety of neurological and cardiovascular processes. Kv1.2 has been shown to be regulated through tyrosine kinase dependent mechanisms that may involve actincytoskeleton dependent endocytosis of the channel. Kv1.2 has also been shown to directly interact with the actin binding protein cortactin, thus providing a link to tyrosine kinases and the actin cytoskeleton. Development of a FRET based system to study Kv1.2-cortactin interactions in live cells is described. Utilization of YFP-tagged Kv1.2 is required.
Rigorous tests were performed to ensure that FRET occurs between YFP-Kv1.2 and cortactin-CFP in fixed HEK293 cells, including donor and acceptor photobleaching, spectral-bleed through correction, and micro-FRET analysis. Micro-FRET analysis in living cells suggests that YFP-Kv1.2 and cortactin-CFP interaction occurs at the cell's surface and within intracellular vesicles. When quantified, data suggests that this interaction is significantly stronger within cellular protrusions and membrane ruffles than at the other areas of the cell's surface. In contrast, the data suggest that this interaction is significantly weaker within intracellular vesicles than at the cell's surface.
Rigorous tests were performed to ensure that FRET occurs between YFP-Kv1.2 and cortactin-CFP in fixed HEK293 cells, including donor and acceptor photobleaching, spectral-bleed through correction, and micro-FRET analysis. Micro-FRET analysis in living cells suggests that YFP-Kv1.2 and cortactin-CFP interaction occurs at the cell's surface and within intracellular vesicles. When quantified, data suggests that this interaction is significantly stronger within cellular protrusions and membrane ruffles than at the other areas of the cell's surface. In contrast, the data suggest that this interaction is significantly weaker within intracellular vesicles than at the cell's surface.