Fig. 5: Effect of NB5 on native If current.
From: Extracellular activation of HCN4 by a subtype-specific nanobody
a Voltage-clamp protocol (scale bars: 20 mV and 500 ms) and representative whole-cell If currents obtained from rabbit SAN cells in control solution (black dot) or pre-incubated with 2 µM NB5 in the extracellular solution (blue dot). Scale bars: 100 pA and 500 ms. b Related mean activation curves with data points shown as mean ± SEM. Data fit to the Boltzmann equation (solid lines) yielded half-activation voltages (V1/2) of −86.7 ± 2.3 mV and −75.5 ± 2.2 mV for control (black circles) and NB5-treated (blue circles) cells, respectively. The shift in V1/2 (ΔV1/2) ± SEM = 11.2 ± 3.2 mV. c, d Representative recordings and action potential rate (bpm) of single rabbit SAN cells spontaneous activity before (top, black dot) and after (bottom, blue dot) superfusion of 2 µM NB5. Scale bars: 10 mV and 0.25 s. Data points are individual cells. Mean AP rate ± SEM for ctrl = 52.3 ± 8.7 bpm, +NB5 = 68.7 ± 12.7 bpm (two-sided Student’s paired t-test *p = 0.049). e Representative whole-cell If currents recorded from hiPSC-CMs in control solution (black dot) or pre-incubated with 2 µM NB5 (blue dot). Scale bars: 100 pA and 500 ms. f Normalized tail current amplitude plotted against voltage for hiPSC-CMs recorded in control solution (black circles) or with NB5 (blue circles). Concatenated fits of data to the Boltzmann equation (solid lines) yielded half-activation voltages (V1/2) of −87.7 mV and −78.4 mV for control (black circles) and NB5-treated (blue circles) cells, respectively. g, h Representative recordings and action potential rate (bpm) of hiPSC-CMs spontaneous activity in control solution (top, black dot) or pre-incubated with 2 µM NB5 (bottom, blue dot). Scale bars: 10 mV and 0.25 s. Data points are individual cells. Mean AP rate ± SEM for ctrl = 36.6 ± 5.5 bpm, +NB5 = 65.4 ± 7.9 bpm (two-sided unpaired Student’s t test **p = 0.008). V1/2, ΔV1/2, inverse slope factors (k), number of cells (n) and statistical analysis for all experiments are reported in Supplementary Table 3.
