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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.007351
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 9 May 2007
The Kv2.1 delayed-rectifier channel trafficks to 1-3 µm2 clusters on the surface of neurons and transfected HEK cells. Single quantum dot (Qdot) tracking and FRAP approaches were used to quantify the diffusion of GFP-labeled Kv2.1 channels on the cell surface and address the mechanisms underlying the formation of these unique membrane structures. Mean square displacement analysis of single Kv2.1 channel tracks inside or outside the surface clusters yielded mean diffusion coefficients of 0.03±0.02 µm2/second and 0.06±0.05 µm2/second, respectively. Kv2.1 channels outside the clusters effectively ignore the cluster boundary, readily diffusing through these microdomains. However, in 5% of the tracks analyzed, single, non-clustered channels were observed to cross into a cluster and become corralled within the cluster perimeter. Alexa Fluor 594-labelled phalloidin staining and mCherry-Kv2.1 co-expression with GFP-actin indicated that the Kv2.1 surface clusters form where the cortical actin cytoskeleton is reduced. Kv2.1 channels lacking the C-terminus do not form clusters, freely diffusing over the cell surface with a mean diffusion coefficient of 0.07±0.04 µm2/second. These data support a model whereby the Kv2.1 clusters are formed by sub-membrane cytoskeletal structures that limit the lateral diffusion of only the sub-population of Kv2.1 channels carrying the appropriate modifications on the Kv2.1 C-terminus.
Key words: Cytoskeletal fence, Fluorescence, Live cell imaging, Localization, Potassium channel, Quantum dot tracking
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