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Figure 4


Fig. 4. FRAP analysis of Kv2.1 mobility outside the cell surface cluster. (A) Imaging of a HEK cell expressing GFP-Kv2.1 before and after photobleach within the indicated region of interest (yellow circle). Prebleach and postbleach images and the fluorescence recovery at 28.8 seconds postbleach are shown. The perimeters of the clusters observed in the prebleach image are indicated in red. Note that the fluorescence intensity within the cluster perimeter recovers to the prebleach background level over this time interval as illustrated in the lower right panel. (B) Time course of fluorescence recovery within the bleach ROI, illustrating a FRAP time constant of 9.0 seconds in this representative experiment. Using this value, we estimated that non-clustered Kv2.1 has a diffusion coefficient of 0.09 µm2/second. The mean diffusion coefficient calculated for non-clustered channels using this approach was 0.08±0.03 µm2/second, n=10. Cells were imaged every 1.1 seconds.





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