The NA/DC double mutation prevents TRIP8b regulation of HCN1 gating in cortical neurons from neonatal rats. (A) Representative whole-cell current traces of human HCN1 WT (top) and N478D/A479C (NA/DC) mutant (bottom) recorded in primary cultures of cortical neurons from neonatal rats transiently expressing the channels alone (left) or with TRIP8b (1a-4; right) and analyzed as described in Materials and methods. (B) Top: Mean current–voltage relationship of HCN1 WT (black filled circle) and HCN1 WT + TRIP8b (1a-4; black open circle). Bottom: Mean current–voltage relationship of HCN1 NA/DC (red filled circle) and HCN1 NA/DC + TRIP8b (1a-4; red open circle). Empty triangles in both panels represent the current recorded in the mock-transfected neurons. Mean current density (pA/pF) measured at −120 mV of HCN1 WT = −57.5 ± 16.3 pA/pF; HCN1 WT + TRIP8b (1a-4) = −132.4 ± 28 pA/pF; HCN1 NA/DC = −36.3 ± 10.5 pA/pF; and HCN1 NA/DC + TRIP8b (1a-4) = −79 ± 11.5 pA/pF. 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 3. (C) Mean normalized activation curves 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) channels obtained as described in Materials and methods. (D) Mean half-activation potential (V_1/2) of HCN1 WT (black filled circle) = −81.9 ± 1.4 mV; HCN1 WT + TRIP8b (1a-4; black open circle) = −88.6 ± 1.1 mV; HCN1 NA/DC (red filled circle) = −82.4 ± 1 mV; and HCN1 NA/DC + TRIP8b (1a-4; red open circle) = −82.2 ± 1.2 mV. P values are denoted within the scatterplot graph. The number of cells was ≥5. Statistical analysis performed with one-way ANOVA, followed by post-hoc Fisher test.