Panel A shows representative confocal microscopy images of HEK293 cells expressing a membrane marker in red, Kv1.3-YFP in green, and KCNE4-CFP variants in blue. Separate columns display membrane staining, Kv1.3 localization, KCNE4 localization, merged fluorescence images, and corresponding fluorescence intensity plot profiles measured along white arrowed lines. Rows compare Kv1.3 alone, and Kv1.3 coexpressed with KCNE4 variants plus 145D, plus 145E, and plus 145A. Panel B shows a bar plot with bar graphs quantifying the Manders overlap coefficient (MOC) for Kv1.3 localization at the plasma membrane in cells expressing Kv1.3 alone or together with KCNE4 variants. Panel C shows a bar plot with bar graphs comparing the plasma membrane localization of KCNE4 variants in the presence of Kv1.3 using MOC values. Panel D shows a bar plot with bar graphs comparing the colocalization between Kv1.3 and KCNE4 variants using MOC measurements.
Both KCNE4 (145D and 145E) variants similarly impair the plasma membrane targeting of Kv1.3. Representative confocal microscopy images of a triple-colocalization experiment of Kv1.3, KCNE4, and DsRed membrane marker. (A) Representative images of HEK293 cells transfected with a membrane marker (red), Kv1.3-YFP (green), and KCNE4-CFP variants (blue). In the right panels, the plot profiles correspond to the sections indicated with white arrows in the merged panels. Scale bar: 10 µm. (B) MOC of Kv1.3 at the plasma membrane (Mb) in the absence (Kv1.3 alone, 45 cells) or in the presence of KCNE4 variants (+145D, 39 cells; +145E, 51 cells; or +145A, 27 cells). P: <0.0001. (C) MOC for KCNE4 present at the plasma membrane (Mb) in the presence of Kv1.3. P: 0.764. (D) MOC for Kv1.3 colocalization with KCNE4. P: 0.093. Individual values and mean ± SE were plotted. Statistical analysis: Kruskal–Wallis with Dunn’s multiple comparisons test. n.s., not significant. ***P < 0.001. Black circles, Kv1.3 alone; blue squares, +145D; orange triangles, +145E; and green diamonds, +145A.