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First published online 26 April 2005
doi: 10.1242/jcs.02348

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Targeting of voltage-gated potassium channel isoforms to distinct cell surface microdomains

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

View larger version (101K): [in a new window]   Fig. 1. Expression of YFP-tagged Kv channels in HEK cells. Confocal images of YFP-Kv2.1HA (A), YFP-Kv1.4myc (B) and YFP-Kv1.3 (C). Surface channels were detected by incubation of live cells with either anti-HA (to detect Kv2.1HA) or anti-myc (to detect Kv1.4myc) antibodies before fixation and Alexa 594-labeled secondary antibody addition as described in Materials and Methods. Colour coding is as follows: red, immunofluorescence surface labeling of the extracellular HA or myc epitopes; green, YFP fluorescence. YFP-Kv1.3 was not epitope tagged, therefore only YFP fluorescence is shown. Optical sections correspond to the relative middle of each cell. Bars, 10 µm.

View larger version (92K): [in a new window]   Fig. 2. CFP-Kv2.1HA and YFP-Kv1.4myc do not colocalize when co-expressed. Confocal image of a live HEK cell expressing both CFP-Kv2.1HA and YFP-Kv1.4myc. (A) The CFP fluorescence, (B) the YFP and (C) the overlay of the CFP and YFP signals. CFP is pseudocolored green and YFP is pseudocolored red. (D) Plot of CFP and YFP pixel intensity verses cell surface length within the boxed region shown in C. Note that while both channels are expressed in the same regions, the intensity profiles indicate that any true colocalization is unlikely to occur.

View larger version (94K): [in a new window]   Fig. 3. YFP-Kv2.1HA and YFP-Kv1.4myc exhibit a differential polarization in HEK cells. Confocal images of YFP fluorescence in live cells were taken of the bottom surface of expressing cells (top panels in both A and B). Three-dimensional confocal stacks were used to generate X-Z images of each cell, which were two-dimensional deconvolved using Zeiss LSM510 v3.2 software (bottom panel, A and B). Kv2.1 clearly has a preference for the basal surface, whereas Kv1.4 is evenly distributed. Bars, 10 µm.

View larger version (55K): [in a new window]   Fig. 4. YFP-Kv2.1HA exhibits limited mobility after photobleach. (A) Images from a FRAP time series in a YFP-Kv2.1HA-expressing HEK cell. The region highlighted by the white box was bleached and recovery in that region was followed by scanning every 15 seconds for 20 minutes (see Materials and Methods). The inset shows enlargement of the bleach region and shows that recovery of YFP fluorescence does not occur via lateral diffusion of YFP-Kv2.1 from adjacent nonbleached membrane. (B) Average recovery after photobleach for YFP-Kv2.1HA. Fluorescence at each time point after bleach (bleach=time 0) was normalized to the prebleach intensity and averaged for all cells (n=25). The solid line through the data is a two-exponential fit to the average data (see Materials and Methods). Fit parameters: 1=36.1 seconds, A1=0.13; 2=308 seconds, A2=0.24, Mf=0.41±0.04. Bar, 10 µm.

View larger version (72K): [in a new window]   Fig. 5. Use of photoactivatable-GFP to monitor Kv2.1 mobility. HEK cells were transfected with PA-GFP-Kv2.1HA and mRFP to mark transfected cells (mRFP not shown). Before activation, PA-GFP-Kv2.1HA-expressing cells displayed no GFP fluorescence (Preactivation). Photoactivation was via illumination of an ROI (red box) using a 405 nm laser for 500 milliseconds at 10-15% attenuation. This protocol resulted in bright green fluorescence with minimal photobleach. After activation, GFP was excited by 488 nm laser light (emission bandpass set at 505-530 nm) and monitored by scanning every 5 seconds for 250 scans (41 minutes). At 640 seconds post-photoactivation, green fluorescence is apparent on the cell membrane opposite to the activation ROI (red arrow). Note that even 2500 seconds following activation, most of the fluorescence is still restricted to the photoactivation ROI, consistent with the low recovery observed with FRAP. Bar, 10 µm.

View larger version (41K): [in a new window]   Fig. 6. YFP-Kv1.4myc and YFP-Kv1.3 are freely mobile in the plasma membrane. (A) Images from FRAP times series in a YFP-Kv1.4myc-expressing HEK cell. The region highlighted in the white box was bleached and recovery in that region followed by scanning every 15 seconds for a total of 80 scans (see Materials and Methods). (B) Average recovery after photobleach for YFP-Kv1.4myc. Fluorescence at each time point was normalized to prebleach intensity and averaged for all cells (n=16). The solid line through the data is a single exponential fit to the average data (see Materials and Methods). Fit parameters: =133.0 seconds, A=0.52; Mf=0.78±0.07. (C) Average recovery following photobleach for YFP-Kv1.3 (n=31). Curve and fit was generated as for YFP-Kv1.4 with the solid line representing a single exponential fit. Fit parameters: =117.6 seconds, A=0.56; Mf=0.78±0.04. Bar, 10 µm.

View larger version (100K): [in a new window]   Fig. 7. Use of photoactivatable-GFP to observe diffusion of Kv1.4. Photoactivation and imaging was performed as described in Fig. 5. No fluorescence is apparent before photoactivation of PA-GFP-Kv1.4myc; following illumination by 405 nm light, there is bright green fluorescence in the ROI. Unlike PA-GFP-Kv2.1, 1 minute after activation, there is GFP signal apparent throughout the cell membrane, consistent with rapid diffusion of Kv1.4 throughout the plasmalemma. Approximately 13 minutes after photoactivation, PA-GFP-Kv1.4 is evenly distributed over the entire cell membrane. Bar, 10 µm.

View larger version (32K): [in a new window]   Fig. 8. YFP-Kv2.1 and YFP-Kv1.4 sediment differently on a sucrose density gradient. Whole-cell homogenates from HEK cells expressing either YFP-Kv2.1HA (top) or YFP-Kv1.4myc (bottom) were sedimented through a 5-20% sucrose density gradient (see Materials and Methods) to estimate channel-containing complex size. Kv2.1 and Kv1.4-containing gradient fractions were identified via western blot analysis using anti-GFP antibody. Thyroglobulin (660 kDa) was used as a size standard, giving a symmetrical peak in fraction 4. P represents the pellet material collected from the bottom of the tube.

View larger version (74K): [in a new window]   Fig. 9. Depletion of membrane cholesterol alters Kv2.1 cluster size. Cells were cholesterol depleted by incubation with 2% 2-hydroxypropyl-ß-cyclodextrin for 1.5 hours at 37°C, then imaged within 2 hours (see Materials and Methods). Cluster size was determined by confocal imaging of the basal surface of expressing cells and measurement of six randomly selected puncta from each cell. The average cluster size for control cells (A) was 1.6±0.2 µm2 (n=102) and 4.4±0.6 µm2 (n=132)* (*=P<0.001) following cholesterol depletion (B). Note that only discrete aggregates were measured as opposed to the large plaques in B, therefore the noted size increase is probably an underestimate. Bars, 5 µm.

View larger version (18K): [in a new window]   Fig. 10. Effect of cholesterol depletion on FRAP kinetics for Kv2.1, Kv1.4 and Kv1.3. HEK cells were cholesterol depleted by incubation with 2% 2-hydroxypropyl-ß-cyclodextrin (CD) and FRAP measured as described in Materials and Methods. Average recovery after photobleach for YFP-Kv2.1HA (A), YFP-Kv1.4myc (B) and YFP-Kv1.3 (C). The dashed line in each plot represents the FRAP under control conditions for each channel, and solid lines are exponential fits to the average data following cholesterol depletion. Fit parameters – Kv2.1 (n=8): 1=51.0 seconds, A1=0.12; 2=2889.1 seconds, A2=0.54; Mf=0.32±0.03. Kv1.4 (n=9): =195.7 seconds, A=0.39; Mf=0.58±0.07. Kv1.3 (n=18): 1=39.2 seconds, A1=0.34; 2=535.7 seconds, A2=0.42; Mf=0.89±0.06.

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