Fig. 6: Effect of NB5 on HCN4 pathogenic variant K531N.

a Representative whole-cell currents of rbHCN4 wt (left, black dot) and wt/K531N (center, black empty dot) recorded in control solution or in the presence of 0.2 µM NB5 in the extracellular solution (wt/K531N, right, empty light blue dot). Traces shown are from −30 mV to −150 mV. Black arrowheads indicate current at −105 mV. Scale bars: 250 pA and 500 ms. b Activation curves obtained from rbHCN4 wt (black solid circles) and wt/K531N (black empty circles) currents in control solution or with 0.2 µM NB5 (wt/K531N, light blue empty circles). Data fit to the Boltzmann equation are plotted as solid lines. Data points are mean ± SEM. Individual V_1/2 values are shown in (d). c Mean activation and deactivation time constants (τ) of rbHCN4 wt (black solid circles) and wt/K531N (black empty circles) in control solution or with 0.2 µM NB5 (wt/K531N, light blue empty circles). Time constants were calculated, at the indicated voltages, by fitting a single exponential function to current traces of a (activation) and to current traces obtained with a deactivating protocol (deactivation, see Methods). Data points are mean ± SEM. Each data point is an average of n ≥ 3 experiments (exact number reported in Source data file). d V_1/2 of control wt (black solid circles), control wt/K531N (black empty circles) and wt/K531N cells treated with 0.2 µM NB5 (light blue empty circles) or 2 µM NB5 (dark blue empty circles). Mean V_1/2 ± SEM for wt = −104.8 ± 0.8 mV, wt/K531N = −112.7 ± 1.0 mV, wt/K531N + 0.2 µM NB5 = −101.9 ± 1.1 mV, wt/K531N + 2 µM NB5 = −94.7 ± 0.7 mV. Statistical analysis performed with One-way ANOVA test compared to wt (***p < 0.001). V_1/2, ΔV_1/2, inverse slope factors (k), kinetics (τ) and number of cells (n) for each experiment shown are reported in Supplementary Tables 3 and 5 along with the details on statistical analysis.