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The role of the dynein motor system on the trafficking of cardiac potassium ion channels

University of British Columbia

In this thesis, we establish a role for the retrograde dynein motor in mediating the trafficking of cardiac potassium channels. Using dynamitin overexpression to disrupt the dynein motor complex, we demonstrate that dynein disruption doubles Kv1 .5 currents and increases surface Kv1.5 protein levels in HEK293 cells. Similar results were seen when these cells where treated with the endocytosis inhibitor, dynamin inhibitory peptide. Confocal images of HEK293 cells and rat atrial myocytes reveal a redistribution of Kv1.5 staining to the plasma membrane when dynein is disrupted, either by dynamitin overexpression or in the presence of the microtubule depolymerizing agent, nocodazole (35 μM). Sustained currents from rat atrial myocytes were increased by 2-fold upon nocodazole pretreatment. These increases in sustained currents were inhibited by the Kv1.5-specific blocker DMM (100 nM). We also demonstrate that Kv1.5 colocalizes with the early endosomal marker, EEAI, suggesting that Kv1.5 enters the early endosomal pathway. Taken together, these experiments suggest that Kv1.5 surface expression is regulated by the dynein-mediated endocytosis. In addition, pretreatment of nocodazole at 37°C reduced Ito currents by one-half. Furthermore, dynamitin overexpression in HEK293 cells reduced Kv4.2 and Kv4.3 channel currents and reduced Kv4.2 staining at the cell surface in confocal images. Dynein motility is coordinated by the anterograde motor, kinesin, and vice versa (Hamm-Alvarez et al., 1993; Valetti et al., 1999; Deacon et al., 2003). Given this and results from a previous report that suggest that the forward trafficking of Kv4.2 is mediated by kinesin, we suggest that dynein disruption impairs the anterograde trafficking of Kv4.2 and Kv4.3 channels (Chu et al., 2006). Lastly, we found no effect on lk1-like currents in nocodazole pretreated rat atrial myocytes at 37°C. Similarly, dynamitin overexpression and nocodazole did not alter the current density of the Ik1-forming channel. Kir2.1, in Itk- cells. This insensitivity of Kir2.1 may suggest an equal dependence for both the dynein and kinesin motor system in regulating the trafficking of Kir2.1 to the cell surface. On the whole, this thesis suggests variable dependencies of the dynein motor in the trafficking of various cardiac potassium channels, and as such, may have important implications in our understanding of the mechanisms that determine potassium channel trafficking in the heart.

Text

2010-01-09

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http://hdl.handle.net/2429/17977

Physiology

University of British Columbia

UBCV

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