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First published online 26 April 2005
doi: 10.1242/jcs.02348
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Research Article |
1 Department of Biomedical Sciences, Colorado State University, Ft Collins, CO 80523, USA
2 Department of Biochemistry and Molecular Biology, Colorado State University, Ft Collins, CO 80523, USA
* Author for correspondence (e-mail: tamkunmm{at}lamar.colostate.edu)
Accepted 23 February 2005
Voltage-gated potassium (Kv) channels regulate action potential duration in nerve and muscle; therefore changes in the number and location of surface channels can profoundly influence electrical excitability. To investigate trafficking of Kv2.1, 1.4 and 1.3 within the plasma membrane, we combined the expression of fluorescent protein-tagged Kv channels with live cell confocal imaging. Kv2.1 exhibited a clustered distribution in HEK cells similar to that seen in hippocampal neurons, whereas Kv1.4 and Kv1.3 were evenly distributed over the plasma membrane. Using FRAP, surface Kv2.1 displayed limited mobility; approximately 40% of the fluorescence recovered within 20 minutes of photobleach (Mf=0.41±0.04). Recovery occurred not by diffusion from adjacent membrane but probably by transport of nascent channel from within the cell. By contrast, the Kv1 family members Kv1.4 and Kv1.3 were highly mobile, both showing approximately 80% recovery (Kv 1.4 Mf=0.78±0.07; Kv1.3 Mf=0.78±0.04; without correction for photobleach); unlike Kv2.1, recovery was consistent with diffusion of channel from membrane adjacent to the bleach region. Studies using PA-GFP-tagged channels were consistent with the FRAP results. Following photoactivation of a small region of plasma membrane PA-GFP-Kv2.1 remained restricted to the photoactivation ROI, while PA-GFP-Kv1.4 rapidly diffused throughout the cell surface. Additionally, PA-GFP-Kv2.1 moved into regions of the cell membrane not adjacent to the original photoactivation ROI. Sucrose density gradient analysis indicated that half of Kv2.1 is part of a large, macromolecular complex while Kv1.4 sediments as predicted for the tetrameric channel complex. Disruption of membrane cholesterol by cyclodextrin minimally altered Kv2.1 mobility (Mf=0.32±0.03), but significantly increased surface cluster size by at least fourfold. By comparison, the mobility of Kv1.4 decreased following cholesterol depletion with no change in surface distribution. The mobility of Kv1.3 was slightly increased following cyclodextrin treatment. These results indicate that (1) Kv2.1, Kv1.4 and Kv1.3 exist in distinct compartments that exhibit different trafficking properties, (2) membrane cholesterol levels differentially modulate the trafficking and localization of Kv channels and (3) Kv2.1 expressed in HEK cells exhibits a surface distribution similar to that seen in native cells.
Key words: Kv channels, Membrane trafficking, Lipid rafts
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