Fig. 4: Effect of NB5 on hHCN1/rbHCN4 heterotetramers.

a Representative whole-cell currents of hHCN1 wt/rbHCN4 wt (co-transfected) recorded in control solution (left, black dot) or with 2 µM NB5 in the extracellular solution (right, blue dot). Traces shown are from −30 mV to −120 mV. Black arrowheads indicate current at −75 mV. Scale bars: 250 pA and 500 ms. b I/V relationships of hHCN1 wt/rbHCN4 wt in control solution (black) or with 2 µM NB5 (blue). Data are mean ± SEM. Current density values (in pA/pF) recorded at −120 mV, HCN1/4 = −147.7 ± 19.4, +NB5 = −148.8 ± 22.9 are not statistically different (^§p = 0.4, two-sided Student’s t test). c Activation curves obtained from hHCN1 wt/rbHCN4 wt currents in control solution (black circles) or with 2 µM NB5 (blue circles). Data fit to the Boltzmann equation are plotted as solid lines. Data points are mean ± SEM. d Half-activation voltages (V_1/2) of control (black circles) and NB5-treated (blue circles) cells expressing hHCN1 wt/rbHCN4 wt. Mean V_1/2 ± SEM for HCN1/4 = −84.8 ± 1.6 mV, +NB5 = −72.2 ± 2.0 mV. Shift in V_1/2 (ΔV_1/2) ± SEM = 12.7 ± 2.5 mV. V_1/2 of cells expressing hHCN1 (V_1/2 = −73.0 ± 0.9 mV) or rbHCN4 (V_1/2 = −104.0 ± 0.9 mV) in control solution shown as empty black circles. e Mean activation (solid) and deactivation (empty) time constants (τ) of hHCN1 wt/rbHCN4 wt in control solution (black circles) or with 2 µM NB5 (blue 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). V_1/2, ΔV_1/2, inverse slope factors (k), kinetics (τ_act, τ_deact) and number of cells (n) are reported in Supplementary Tables 3 and 5 along with the details on statistical analysis.