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| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
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MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 580 |
Targeting
of voltage-gated potassium channel isoforms to distinct cell surface microdomains |
Kristen
M. S. O’Connell and Michael M. Tamkun |
Kv
channels, Membrane trafficking, Lipid rafts |
For live cell imaging, HEK cells
were grown in 60 mm culture dishes and, 24 hours post-transfection, were passed
at a 1:4 dilution onto collagen coated glass bottom 35 mm dishes
(MatTek, Ashland, MA).
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Confocal
microscopy |
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HEK-293 |
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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, PAGFP- 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. |
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