Contribution of HCN1 variant to sinus bradycardia: A case report

FIGURE 1. 12‐lead ECG recordings. (A) Index patient (resting heart rate was 31 bpm). (B) Patient's son (resting heart rate was 67 bpm). (C) Volunteer (resting heart rate was 50 bpm)

FIGURE 2. HCN4 and HCN1 gene mutations viewed in IVG. (A) HCN4 synonymous mutations. (B) HCN1 N‐del mutation. (C) HCN1 C‐terminal missense mutation. P, patient, PS, patient's son

FIGURE 3. Western blots of HCN1 mutants in HEK293. In cell membrane preparation (left), immunoblots are shown for WT, P851A, and N‐del channel proteins (A) and for N‐del + wtHCN1, N‐del + P851A, and wtHCN1wt channel proteins (B). (C) Average surface channel protein expression of WT, N‐del, N‐del + P851A, P851A, and N‐del + wtHCN1, normalized to β‐actin. In total cell lysate (right), immunoblots are shown for WT, P851A, and N‐del channel proteins (D) and for N‐del + wtHCN1, N‐del + P851A, and wtHCN1wt channel proteins (E). (H) Average total channel protein expression of WT, N‐del, N‐del + P851A, P851A, and N‐del + wtHCN1, normalized to β‐actin

FIGURE 4. Flow cytometry and confocal imaging studies of HCN1 mutant channel surface expression. Representative flow experiments for surface staining of wtHCN1, N‐del + wtHCN1, and N‐del + P851A. (B) Averaged percentage of channel surface staining of wtHCN1, N‐del + wtHCN1, and N‐del + P851A, normalized to wild‐type HCN1. An HA‐APC antibody was used to recognize the HA‐tag in the channel constructs. (C) Confocal images of wtHCN1, (D) N‐del + P851A, (E) N‐del + wtHCN1. (F) Average ratio of GFP area (representing number of channels) to the total cell area. * indicates statistical significance (P < .05)

FIGURE 5. Biophysical properties of HCN1 mutant channels. (A) Wild‐type channel currents were elicited by 1‐s hyperpolarizing pulses from −40 to −110 mV in 10 mV increments. (B) N‐del channel currents were elicited by 1.5s hyperpolarizing pulses from −60 to −110 mV in 10 mV increments. (C) P851A channel currents were elicited by 1s hyperpolarizing pulses from −80 to −130 mV in 10 mV increments. (D) N‐del + P851A channel currents were elicited by 3.5s hyperpolarizing pulses from −90 to −140 mV in 10 mV increments. (E) N‐del + wtHCN1 channel currents were elicited by 1s hyperpolarizing pulses from −50 to −100 mV in 10 mV increments. The holding potential was −20 mV for all cells studied

FIGURE 6. Average HCN1 mutant channel threshold activation (A) and current density (B)

FIGURE 7. ZD7288 blockade of HCN1 mutant channel activation. The channel current is shown in black and ZD7288 (10 μM) in gray line. ZD7288 blocked wild‐type HCN1 channel current at −90 mV (A), N‐del channel current at −110 mV (B), P851A channel current at −130 mV (C). N‐del + P851A channel current at −120 mV (D), and N‐del + wtHCN1 at −100 mV (E). The holding potential was −20mV for all cells studied

FIGURE 8. Effect of HCN1 variants on sinoatrial pacemaker activity. (A) Control action potential (upper) and I(f) current (lower). (B) Effects of a decreased I(f) channel conductance by 33% on action potential (upper) and I(f) current (lower). Vertical dotted line: I(f) current amplitude