Functional impact of selected de novo HCN1 variants using whole-cell patch-clamp. (A) Representative traces of whole-cell currents recorded in CHO cells transfected with constructs for wild-type (WT), M153I, M243R, M305L, G391D, S399P or R590Q human HCN1 channels. Currents were elicited by test pulses ranging from −20 mV to −130 mV in 10 mV increments from a holding potential of −20 mV. (B) Plot of mean current density as a function of test voltage for wild-type, M153I, M243R, K261E, M305L, G391D, S399P and R590Q human HCN1 channels (two-way ANOVA, *P < 0.05). (C) Mean tail current activation curves for wild-type, M153I, M243R, and R590Q HCN1 channels. (D) M153I mutant channels have faster activation time constants than wild-type HCN1 channel (two-way ANOVA, *P < 0.05). The M153I mutant channels also display significantly increased deactivation time constants compared with those of wild-type HCN1 (one-way ANOVA, **P < 0.01 for voltage −60 mV). (E) Plot of the per cent of cells expressing the I_h current for all variants. (F) Mean tail current activation curves for wild-type, M153I and wild-type/M153I HCN1 channels. (G) Plot of the per cent of cells expressing the I_h current for all variants tested. Data are presented as means ± SEM with the numbers of experiments for each condition indicated in parentheses. Values for half activation potential (V_1/2) and inverse slope factor (k) are reported in Supplementary Table 4.