Human embryonic kidney cell culture, transient transfection, and patch-clamp electrophysiology

Human embryonic kidney (HEK)293 cells were cultured as previously described (18). HEK293 cell cultures (70–80% confluence) were cotransfected with 250 ng K_V2.1 and 1 µg WT or mutant hKCNE5 with Lipofectamine 2000 reagent (Thermo Fisher Scientific). Green fluorescent protein cDNA (0.5 µg) was cotransfected as a transfection marker. Transfected cells were dissociated with trypsin and used for electrophysiological analysis 16–24 h after transfection. Patch-clamp recordings from HEK293 cells were performed as previously described (18). Series resistance was compensated (80%) for, and cells with a voltage error exceeding 5 mV after compensation were excluded from analysis. The Boltzmann equation, y = 1/{1+exp[−(V−V_1/2)/k]}, where V is the voltage applied, V_1/2 is the voltage at which 50% of the channels are (in)activated, and k is the slope factor, was used to fit the voltage-dependence of activation and inactivation. Single and double exponential functions were used to fit the activation and deactivation kinetics, respectively. Student’s t test or Mann-Whitney rank sum test was used for statistical analysis, and values of P < 0.05 were considered statistically significant.