Fig. 5. Detection of Kv1.4 within the Kv2.1 cell surface cluster. FRAP analysis of YFP-Kv1.4-myc mobility. HEK cells expressing both CFP-Kv2.1-HA and YFP-Kv1.4-myc were imaged for each fluorophore. (A) Localization of CFP-Kv2.1-HA. (B) Homogeneous distribution of YFP-Kv1.4-myc. Note that Kv1.4 is present at the same cell surface density on both sides of the Kv2.1 cluster perimeter (outlined in white). (C) Photobleach of YFP-Kv1.4-myc contained within the red ROI. This field of view is identical to that presented in A and B. (D) YFP fluorescence recovery at 146 seconds, illustrating that the mobile YFP-Kv1.4-myc channels have diffused into the CFP-Kv2.1-HA clusters outlined in white. (E) Time course of fluorescence recovery within the bleach ROI, illustrating a FRAP time constant of 42.4 seconds in this representative experiment. Using this value, we calculated that the mobile YFP-Kv1.4-myc channels in this cell have a diffusion coefficient of 0.2 µm²/second. The mean diffusion coefficient for YFP-Kv1.4-myc was 0.28±0.13 µm²/second, n=11. Cells were imaged every 1.6 seconds.