cAMP sensitivity of HCN2 R632E channel. (A) Representative whole-cell current traces of mouse HCN2 WT (top) and R632E mutant (bottom) recorded in HEK 293T cells transiently expressing the channels with control solution (left) or with 5 µM cAMP (right) in the patch pipette. Arrows indicate the currents selected for analysis in B. (B) Normalized mean activation curves of HCN2 WT in control solution (blue filled circle) and cAMP (blue open circle) and HCN2 R632E in control solution (green filled circle) and cAMP (green open circle) obtained from tail currents collected at −40 mV (see arrows in A). Lines show data fit to a Boltzmann function (see Materials and methods), from which we derived the half-activation potentials (V_1/2) plotted in C. (C) Mean half-activation potential (V_1/2) of HCN2 WT in control solution (blue filled circle) = −96.3 ± 0.4 mV; HCN2 WT with cAMP (blue open circle) = −83.7 ± 0.3 mV; HCN2 R632E in control solution (green filled circle) = −96.4 ± 0.3 mV; HCN2 R632E with cAMP (green open circle) = −86.7 ± 0.3 mV. Data are plotted as dots and the line represents the mean value ± SEM. The number of cells was ≥10. Statistical analysis performed with one-way ANOVA, followed by post-hoc Tukey test (P values are denoted within the scatterplot graph).