The NA/DC double mutation prevents TRIP8b regulation of HCN1 gating in cortical neurons derived from hiPSCs. (A) Representative whole-cell current traces of human HCN1 WT (top) and N478D/A479C (NA/DC) mutant (bottom) recorded in cortical neurons derived from hiPSCs transiently expressing the channels alone (left) or with TRIP8b (1a-4; right) and analyzed as described in Materials and methods. (B) Mean current–voltage relationship of HCN1 WT (black filled circle), HCN1 WT + TRIP8b (1a-4; black open circle), HCN1 NA/DC (red filled circle), and HCN1 NA/DC + TRIP8b (1a-4; red open circle). The values of the current recorded at −80, −90, −110, −110, and −120 mV of both HCN1 WT and mutant coexpressed with TRIP8b are significantly higher of the current of HCN1 WT and mutant alone. All values and exact P values are reported in Table 4. Mean current density (pA/pF) measured at −120 mV of HCN1 WT = −27.2 ± 7.7 pA/pF; HCN1 WT + TRIP8b (1a-4) = −71.5 ± 19.5 pA/pF; HCN1 NA/DC = −22.5 ± 12.7 pA/pF; and HCN1 NA/DC + TRIP8b (1a-4) = −62.4 ± 19 pA/pF. In mock-transfected neurons, the current density at −120 mV was −12.5 ± 2 pA/pF, not reported in the graph for the sake of clarity. (C) Normalized mean activation curves of HCN1 WT + TRIP8b (1a-4; black open circle) and HCN1 NA/DC + TRIP8b (1a-4; red open circle) channels obtained as described in Materials and methods. Data are presented as means ± SEM. The number of cells was ≥3. Statistical analysis was performed with one-way ANOVA, followed by post-hoc Fisher test.