cAMP sensitivity of HCN2 NA/DC mutant channel. (A) Representative whole-cell current traces of mouse HCN2 WT (top) and N520D/A521C (NA/DC) mutant (bottom) recorded in HEK 293T cells transiently expressing the channels with control solution (left) or with 1 µM cAMP (right) in the patch pipette. Arrowheads indicate the current 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 of HCN2 NA/DC in control solution (orange filled circle) and cAMP (orange open circle) obtained from tail currents collected at −40 mV (see arrowheads 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) = −93.8 ± 0.9 mV; HCN2 WT with cAMP (blue open circle) = −86.5 ± 0.7 mV; HCN2 NA/DC in control solution (orange filled circle) = −93 ± 0.4 mV; and HCN2 NA/DC with cAMP (orange open circle) = −87.2 ± 0.7 mV. Data are plotted as dots, and the line represents the mean value ± SEM. The number of cells was ≥5. Statistical analysis performed with one-way ANOVA, followed by post-hoc Tukey test (P values are denoted within the scatterplot graph).