Functional analysis of the Gardos channel variant p.Arg352His. (A) Current-voltage curves of WT and mutated KCNN4 in oocyte membranes with quantification. I/V curves correspond to the maximal current recorded for WT or p.Arg352His KCNN4-expressing oocytes (around 135 seconds) in gluconate medium with 1 µM A23187. Data are means of ramps recorded on 15 (WT) or 22 (p.Arg352His) oocytes. NI are control (noninjected) oocytes (n = 4). (Inset) Western blot detection of WT and mutated KCNN4 indicated by the arrow (around 50 kDa). (B) Activation kinetic. For oocytes expressing WT KCNN4 or p.Arg352His KCNN4, the current measured in gluconate medium at 0 mV was plotted as a function of time (left). The maximal intensity of the current being different between WT and mutated KCNN4, a ratio between I at different times and the I_max was calculated for each condition and plotted as a function of time. Data are means of 15 (WT) or 22 (p.Arg352His) oocytes coming from 3 different batches. The arrow indicates the opening of calcium ionophore perfusion. The addition of A23187 did not stimulate any current in noninjected oocytes; for clarity, the NI trace was not plotted on this graph. The bar graph (right) quantifies the remaining current at 220 seconds in oocytes expressing WT or p.Arg352His KCNN4. The current at 220 seconds was divided by I_max (at about 135 seconds) for each recording. Data are means ± standard error of the mean (SEM) of 15 (WT) or 22 (p.Arg352His) oocytes. Statistical analysis was done using the Mann-Whitney test; the 2 bars are different with a risk of 0.2% (bidirectional). (C) TRAM34 inhibition: once the maximal current was reached in oocytes expressing p.Arg352His mutant, 10 µM TRAM34 was added. This induced a rapid current decrease. The mean value of maximal currents at 50 mV was calculated (white bar) and compared with the mean value of minimal currents at 50 mV after TRAM34 addition (gray bar). Data are means of 4 oocytes ± SEM.