KCNN4 expression in HEK293 cells. (A) Immuno-staining showing expression of WT and mutated KCNN4 at the plasma membrane. WT SK4-HA or SK4 p.Arg352His-HA were transfected in HEK293 cells and stained with an anti-HA antibody (Red). Nuclei were stained with Hoechst (blue). Images are representative of 3 independent experiments. Scale bars, 10 µm. (B) Activation kinetic of WT and p.Arg352His KCNN4 recorded in whole cell configuration. WT KCNN4 or p.Arg352His KCNN4 was expressed in HEK293 cells and then subjected to patch-clamp experiments in whole cell configuration. Currents were recorded immediately after break-in using a 150-ms voltage ramp protocol from −120 to + 80 mV from a holding potential of −60 mV. The current at −20 mV was plotted as a function of time. Values are mean ± SEM of 8 to 12 experiments. (C) Representative current/voltage curves for HEK293 expressing WT or p.Arg352His KCNN4. Inset (upper left) represents reversal potentials just after break-in and at the steady state. Values are represented as a Tukey’s plot (n = 8-12). Statistical analyses were done using the Kruskal-Wallis test followed by a Tukey post hoc test. (D) Tukey’s plots showing current density at −20 mV in WT and mutated SK4 (n = 8-12; ***P < .001). Statistical analysis was performed using a Mann-Whitney test. (E) Representative traces of Ca²⁺-dependent activation of WT and p.Arg352His KCNN4 current recorded in an inside-out macropatch configuration. Currents were elicited by 150-ms voltage ramps from −120 to + 80 mV. Each trace corresponds to a different concentration of Ca²⁺ indicated on the right side of the I/V (in micromolar). (F) Normalized K⁺ current measured at −45 mV in response to [Ca²⁺]_i was plotted as a function of [Ca²⁺]_I for WT (black squares) and p.Arg352His-mutated KCNN4 (red circles). The experimental values (mean ± SEM) were fitted with the Hill equation (Origin Software, Northampton, MA): _, where Y is the relative KCNN4 current at −45 mV (I/I_max) for each [Ca²⁺], Y_max is the maximum current (I_max), K_0.5 is the apparent dissociation constant, and n is the Hill coefficient. Inset (lower right) shows Hill equation parameters K_0.5 and n_h. Values are represented as a Tukey plot (n = 4; *P < . 05). Statistical analysis was performed using a Mann-Whitney test.