Fig. 1: Functional effect of NB5 on rbHCN4.

a Voltage-clamp protocol (scale bars: 15 mV and 500 ms) and representative whole-cell currents of wt rbHCN4 expressed in HEK293T cells and recorded without (left, black dot) or with (center, blue dot) 20 µM NB5 in the extracellular solution. Black arrowheads indicate current at −105 mV. The right panel shows superposition of traces recorded from a cell without (black trace) and a cell with NB5 (blue trace) at −150 mV and tail currents collected at −40 mV. Scale bars: 250 pA and 500 ms. b Mean I/V relationships of steady state current density (I_ss, pA/pF) obtained from cells with (blue) or without (black) 20 µM NB5 in the extracellular solution. Data are mean ± SEM. c Activation curves obtained from cells in (a). 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). d Half-activation voltages (V_1/2) of individual cells plotted in (c). Mean V_1/2 ± SEM for ctrl = −104.8 ± 0.8 mV, +NB5 = −95.7 ± 0.9 mV. The shift in half activation voltage (ΔV_1/2) ± SEM = 9.1 ± 1.2 mV. e ΔV_1/2 (mV), measured as in d, in response to a range of NB5 concentrations (µM). Data fit to the Hill equation (solid line) yielded a half maximal effective concentration (K_1/2) value of 40 nM and a Hill coefficient (n_H) value of 0.65. Data are mean ± SEM. Each data point is an average of n ≥ 3 cells (exact number reported in Source data file). V_1/2, ΔV_1/2, inverse slope factors (k) and number of cells (n) for each experiment shown are reported in Supplementary Table 1 along with the details on statistical analysis.