Figure 5.
The NA/DC double mutation in HCN1 prevents TRIP8b regulation of channel function. (A) Representative whole-cell current traces of human HCN1 WT (top) and N478D/A479C (NA/DC) mutant (bottom) recorded in HEK 293T cells transiently expressing the channels alone (left) or with TRIP8b (1a-4; right) and analyzed as described in Materials and methods. Arrows indicate the current selected for analysis in B. (B) Mean 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 from tail currents collected at −40 mV (see arrows in A). The currents were normalized for the cell capacitance and plotted (changed in sign) as a function of test voltages. Solid and dashed lines indicate the half-activation potential (V1/2) value, while arrowheads indicate the current density value at saturation, plotted respectively in C and D. (C) Mean half-activation potential (V1/2) of HCN1 WT (black filled circle) = −72.7 ± 0.4 mV; HCN1 WT + TRIP8b (1a-4; black open circle) = −78.2 ± 0.5 mV; HCN1 NA/DC (red filled circle) = −72.6 ± 0.6 mV; and HCN1 NA/DC + TRIP8b (1a-4; red open circle) = −73.2 ± 0.4 mV. P values are denoted within the scatter plot graph. (D) Mean maximal tail current density of HCN1 WT (black filled bar) = 10.1 ± 2.7 pA/pF; HCN1 WT + TRIP8b (1a-4; black open bar) = 23 ± 8 pA/pF; HCN1 NA/DC (red filled bar) = 9.6 ± 4 pA/pF; and HCN1 NA/DC + TRIP8b (1a-4; red open bar) = 23.8 ± 4.5 pA/pF. P values are denoted within the column bar graph. (E) Mean activation and deactivation time constants (before and after the x-axis break, respectively) 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). Time constant of activation and deactivation were calculated, as described in Materials and methods. All values and exact P values are reported in Table 2. Data are plotted as a dot and the line represents the mean value ± SEM. n = 8 cells. Statistical analysis performed with one-way ANOVA, followed by post-hoc Tukey test.

The NA/DC double mutation in HCN1 prevents TRIP8b regulation of channel function. (A) Representative whole-cell current traces of human HCN1 WT (top) and N478D/A479C (NA/DC) mutant (bottom) recorded in HEK 293T cells transiently expressing the channels alone (left) or with TRIP8b (1a-4; right) and analyzed as described in Materials and methods. Arrows indicate the current selected for analysis in B. (B) Mean 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 from tail currents collected at −40 mV (see arrows in A). The currents were normalized for the cell capacitance and plotted (changed in sign) as a function of test voltages. Solid and dashed lines indicate the half-activation potential (V1/2) value, while arrowheads indicate the current density value at saturation, plotted respectively in C and D. (C) Mean half-activation potential (V1/2) of HCN1 WT (black filled circle) = −72.7 ± 0.4 mV; HCN1 WT + TRIP8b (1a-4; black open circle) = −78.2 ± 0.5 mV; HCN1 NA/DC (red filled circle) = −72.6 ± 0.6 mV; and HCN1 NA/DC + TRIP8b (1a-4; red open circle) = −73.2 ± 0.4 mV. P values are denoted within the scatter plot graph. (D) Mean maximal tail current density of HCN1 WT (black filled bar) = 10.1 ± 2.7 pA/pF; HCN1 WT + TRIP8b (1a-4; black open bar) = 23 ± 8 pA/pF; HCN1 NA/DC (red filled bar) = 9.6 ± 4 pA/pF; and HCN1 NA/DC + TRIP8b (1a-4; red open bar) = 23.8 ± 4.5 pA/pF. P values are denoted within the column bar graph. (E) Mean activation and deactivation time constants (before and after the x-axis break, respectively) 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). Time constant of activation and deactivation were calculated, as described in Materials and methods. All values and exact P values are reported in Table 2. Data are plotted as a dot and the line represents the mean value ± SEM. n = 8 cells. Statistical analysis performed with one-way ANOVA, followed by post-hoc Tukey test.

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