A gain-of-function HCN4 mutant in the HCN domain is responsible for inappropriate sinus tachycardia in a Spanish family

Fig. 1.. A family with inappropriate sinus tachycardia (IST). (A) Pedigree of the IST proband’s family. The arrow indicates the proband (III.2); circles and squares represent females and males, respectively. (B) Twelve‐lead ECGs of II.1, III.2, and IV.9 family members (paper speed: 25 mm/s).

Fig. 2.. Macroscopic currents generated by WT, p.V240M, and WT+p.V240M HCN4 channels. (A and B) IHCN4 traces (A) and density–voltage relationships (B) generated in CHO cells transiently expressing WT, p.V240M, and WT+p.V240M HCN4 channels by applying the protocol depicted at the top. The Inset in B shows data at potentials between −90 and −50 mV in an expanded scale. (C) Fully activated IHCN4 density–voltage relationships generated by WT, p.V240M, and WT+p.V240M channels. Continuous lines represent a linear regression fit to the data. (D and E) Time constants of IHCN4 activation (D) and deactivation (E) measured at different membrane potentials. In (B–E), each point represents the mean ± SEM of ≥5 experiments/cells from ≥3 dishes. *P < 0.01 vs. HCN4 WT. #P < 0.05 vs. HCN4 WT and P < 0.05 vs. p.V240M. ANOVA followed by Tukey's test and multilevel mixed‐effects model.

Fig. 3.. Effects of p.V240M on the voltage dependence of IHCN4 activation. (A) Typical IHCN4 generated by WT+p.V240M channels when applying the protocol shown at the top, which consists of a test hyperpolarizing pulse of variable duration (from 1.5 s at −160 mV to 12 s at −20 mV) followed by a 1.5-s pulse to 0 mV from a holding potential of −40 mV. The first (−160 mV) and last (−20 mV) pulses of the protocol are shown in red and blue, respectively. (B) Non-steady-state activation curves of WT, p.V240M, and WT+p.V240M channels expressed in CHO cells constructed by plotting the normalized tail amplitudes as a function of the membrane potential of the test pulse. *P < 0.05 vs. HCN4 WT. #P < 0.05 vs. HCN4 WT and p.V240M. (C) Example of superimposed tail currents elicited by the 1.5-s pulse to 0 mV generated by WT and p.V240M channels. (D–F) Non-steady-state activation curves of WT (D), p.V240M (E), and WT+p.V240M (F) channels expressed in HEK293 cells in the presence or absence of 10 μM cAMP. *P < 0.05 vs. cells nondialyzed with cAMP. In (B) and (D–F), tail current densities were normalized and plotted against the membrane potential of the test pulse. Continuous lines represent the Boltzmann…

Fig. 4.. Membrane expression of WT and p.V240M HCN4 channels. Representative western blot images (A) and densitometric analyses (B) of biotinylation assays showing the total (input) or surface (membrane) expression of HCN4 in CHO cells expressing WT or p.V240M channels. The cytosolic protein ezrin was used as a negative control. In (B), each dot represents one experiment and each bar the mean ± SEM of five experiments.

Fig. 5.. Single-channel recordings generated by WT and p.V240M HCN4 channels. Single-channel recordings obtained by applying 3-s pulses from −35 to −90 mV in cells expressing or not (A) WT (B) or p.V240M (C) HCN4 channels in the absence and presence (D–F) of ivabradine 5 μM. Continuous and dotted lines represent the current amplitude generated by closed (C) and open (O) channels, respectively.

Fig. 6.. Gating properties of WT and p.V240M HCN4 channels. Mean fo (A), Po (B), and open time (C) for iHCN4 recorded in cells expressing WT or p.V240M HCN4 channels by applying 3-s pulses from −35 to −90 mV. (D) iHCN4 amplitude generated by WT and p.V240M channels after applying pulses to −90 mV. (E) iHCN4-voltage relationships generated by WT and p.V240M channels. In (A–D), each dot represents one experiment/cell and each bar the mean ± SEM of n experiments/cells as indicated in the figure. In (E), each point represents the mean ± SEM of 5 experiments/cells. *P < 0.05 vs. WT. Student’s t test and multilevel mixed‐effects model.

Fig. 7.. Mathematical model of human sinoatrial node cell action potential. (A and B) APs (A) and If (B) obtained when running the model under basal conditions (WT; Left), or after introducing the experimentally measured IHCN4 changes induced by p.V240M alone (center) or together with WT (Right). The dotted lines represent the zero membrane potential (A) and current (B) levels.

Fig. 8.. Molecular modeling of WT and p.V240M HCN4 channels. (A) Ribbon representation of the cryo-EM structure of human HCN4 (PDB: 6GYN). Only two diagonal subunits are shown for clarity. The transmembrane segments (TM) are gray, the HCND is orange, the C-linker is red, the CNBD is blue, and the distal C helix is green. (B) Close view of superimposed WT and V240M models (color coded as in A). Sticks representation shows amino acids side-chains from WT (cyan) and V240M (purple). V240 and M240 are shown as spheres. (C) Interaction of HCND with the VSD showing the hydrophobic interactions of F229 with I404 and M407 from TM S4 (VSD) and Y258 from the loop connecting HCND to TM S1, and polar contacts of K228 and Y409 in WT (red dashed line) or K228 and N227 in V240M (yellow dashed line). Spheres represent the van der Waals surface occupied by the side chains.