Figure 4.

Sub-cellular Localization and Electrophysiology Analysis of the Dominant-Negative Effect of Human K_V6.4-Met419

(A) HEK293 and HeLa cells (separate experiments) were transfected with K_V2.1 and wild-type K_V6.4, K_V6.4-Met419, or equimolar concentrations of K_V6.4/K_V6.4-Met419. Cell membranes were stained with Na⁺/K⁺ ATPase (red channel) and HA-tagged K_V6.4 (green channel). HA-tagged K_V6.4 localized to the cell membrane, showing significant co-localization with Na⁺/K⁺ ATPase. K_V6.4-Met419 and K_V6.4/K_V6.4-Met419 co-expression showed cytoplasmic retention of K_V6.4 and no evidence of co-localization with Na⁺/K⁺ ATPase. The graphs display the profiles of signals for the membrane and K_V6.4.HA along the plane of the white line in the merged image. Note that the red and green signal co-localize in the K_V6.4 experiment and are distinct in the K_V6.4-Met419 and heterozygote experiment. Scale bars indicate 20 μm.

(B) Quantification of Pearson’s co-localization co-efficient between K_V6.4.HA and Na⁺/K⁺ ATPase under each experimental condition. For each condition, at least 17 cells were counted from three independent experiments.

(C) V_0.5 act from inactivation protocols shown in Figures 1D–1G. Co-expression of K_V6.4 and K_V6.4-Met419 with K_V2.1 failed to evoke a shift in the voltage dependence of inactivation.

Bars indicate mean values, error bars indicate SEM, n = 9–15, ^∗∗∗p < 0.001. The statistics in (B) and (C) represent one-way ANOVA with Bonferroni’s multiple comparisons test.