Fig. 3: Effect of NB5 in the presence of cAMP.

a Representative whole-cell currents of rbHCN4 wt recorded in control solution (top left, black dot), in presence of 2 µM NB5 in the extracellular solution (top right, blue dot), 30 µM cAMP in the pipette solution (bottom left, purple dot) or both (bottom right, green dot). Traces shown are from −30 mV to −150 mV. Black arrowheads indicate current at −90 mV. Scale bars: 250 pA and 500 ms. b Activation curves obtained from rbHCN4 wt in control solution (black circles) or in the presence of 2 µM NB5 (blue circles), 30 µM cAMP (purple circles) or both (green 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 (c). c Half-activation voltages (V_1/2) of control (black circles), NB5 (blue circles), cAMP (purple circles) and NB5 + cAMP (green circles)-treated cells expressing rbHCN4 wt. Mean V_1/2 ± SEM (in mV) for ctrl = −104.7 ± 1.0, +NB5 = −96.2 ± 1.6, +cAMP = −86.8 ± 2.2, +NB5 +cAMP = −76.2 ± 2.1. d Mean activation (solid circles) and deactivation (empty circles) time constants (τ) of rbHCN4 wt in control solution (black), or in the presence of 2 µM NB5 (blue), 30 µM cAMP (purple) or both (green). 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) shown in (e). Data points are mean ± SEM. Each data point is an average of n ≥ 3 experiments (exact number reported in Source data file). e Voltage clamp protocol (scale bars: 15 mV and 500 ms) used to obtain rbHCN4 deactivation time constants and representative control (ctrl, black dot) current trace (scale bars: 250 pA and 500 ms). 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 4 and 5 along with the details on statistical analysis.