Figure 4. TRIP8b_nano abolishes cAMP effect on HCN channel gating.

(A-C) Representative whole-cell HCN4, HCN2 and HCN1 currents recorded, at the indicated voltages, with control solution or with cAMP (1 μM for HCN4 and 5 μM for HCN2) or with cAMP + 10 µM TRIP8b_nano in the patch pipette (for HCN1, 10 µM TRIP8b_nano only was added). (D-F) Mean activation curves measured from HCN4, HCN2 and HCN1 in control solution (open circles), cAMP (filled circles), cAMP +TRIP8b_nano, or TRIP8b_nano only in the case of HCN1 (filled triangles). Solid and dashed lines indicate Boltzmann fitting to the data (see Materials and methods). (G) Half activation potential (V_1/2) values of HCN4 (blue), HCN2 (black) HCN1 (red) in control solution (open circle), cAMP (filled circle) and cAMP +TRIP8b_nano, or TRIP8b_nano only in the case of HCN1 (filled triangle). HCN4, control = −102.8 ± 0.3 mV; HCN4 +1 µM cAMP = −89.2 ± 0.6 mV; HCN4 +1 µM cAMP +10 µM TRIP8b_nano = −102.1 ± 0.6 mV, HCN4 +1 µM cAMP in the patch pipette +10 µM TRIP8b_nano in the bath solution = −91.7 ± 0.3 mV; HCN2, control = −93.7 ± 0.3 mV; HCN2 +5 µM cAMP = −83.5 ± 0.3 mV; HCN2 +5 µM cAMP +10 µM TRIP8b_nano = −94.5 ± 0.6 mV; HCN1, control = −72.8 ± 0.2 mV; HCN1 +10 µM TRIP8b_nano = −82 ± 0.5 mV. Data are presented as mean ± SEM. Number of cells (N) ≥ 11. There is no significant difference between the controls and the addition of TRIP8b_nano with (HCN4, HCN2) or without (HCN1) cAMP in the pipette. No significant difference was observed following the addition of TRIP8b_nano in the bath. Statistical analysis performed with two-way ANOVA, followed by post-hoc Tukey test (p<0.001).

Figure 4—figure supplement 1. Comparison of half activation potentials (V_1/2) of HCN1 WT and HCN1 R₅₄₉E mutant (Chen et al., 2001).

(A) Currents were measured at the indicated voltages (- 50,–60, −70,–120 mV), from HEK 293 T cells with control solution or with 15 μM cAMP in the patch pipette. (B) Mean activation curves obtained from tail currents were fitted to a Boltzmann equation (see Materials and methods). (C) Half activation potential (V_1/2) determined from Boltzmann fitting. HCN1 WT (black filled circle)=−72.7 ± 0.2 mV; HCN1 WT + cAMP (black open circle)=−73.1 ± 0.3 mV; HCN1 R₅₄₉E (red filled circle)=−80.4 ± 0.2 mV; HCN1 R₅₄₉E + cAMP (red open circle) = −81 ± 0.3 mV. Note that in human HCN1, used here, R₅₄₉E corresponds to mouse HCN1 R₅₃₈E used in Chen et al. (2001). Data are presented as mean ± SEM. Number of experiments (N) ≥ 8. Wt values are significantly different from mutant. Statistical analysis performed with two-way ANOVA, followed by post-hoc Tukey test (p<0.001).

Figure 4—figure supplement 2. Voltage-dependency of activation time constant (τ_on) of HCN4 channels in control solution (open circles), 1 µM cAMP (filled circles), 1 µM cAMP + 10 µM TRIP8b_nano (filled triangles).

Data are presented as mean ± SEM. Number of cells (N) = 5.