Structural mechanism of human HCN1 hyperpolarization-activated channel inhibition by ivabradine

Figures

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  Figure 1. Functional and cryo-EM characterization of HCN1 inhibition by ivabradine.A, the inhibitory effect of ivabradine on WT hHCN1 overexpression, measured by whole-cell patch-clamp electrophysiological recordings. B, concentration-response curve for ivabradine inhibition. Data are shown as n = 6 independent experiments. C, cryo-EM density maps of ivabradine-bound HCN1 is shown in side view. The purple, green, light, and dark pink colors represent each HCN1 subunit, red color shows cryo-EM densities for ivabradine. D, single subunit ribbon diagram of ivabradine-bound HCN1 structure is shown in side view. Red surface shows cryo-EM densities for ivabradine. HCN, hyperpolarization-activated cyclic nucleotide-gated; hHCN1, human HCN1.

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  Figure 2. Structure of the HCN1 channel in complex with ivabradine.A, overview of the ivabradine-binding site in HCN1. Ivabradine is shown as yellow sticks. B, detailed view of the binding site, illustrating the interactions between ivabradine and HCN1. The side chains of crucial residues are shown in sticks. C, the detailed binding site density map at 3.5σ is shown. D, the density is shown in blue mesh for ivabradine, which is depicted in sticks. E, cartoon representation of the interaction between ivabradine and HCN1. Green solid lines represent π-π stacking interaction. F, the inhibitory effects of ivabradine on various HCN1 mutants (D140A, F143A, F285A, W281A, and Y289A), measured by electrophysiological recordings. HCN, hyperpolarization-activated cyclic nucleotide-gated.

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  Figure 3. MD simulations.A, representative MD structure (blue cartoon and brownish sticks), aligned to the cryo-EM structure (gray cartoon and yellow sticks). B, RMSD of ivabradine with HCN1 complex over simulation time. C, RMSF values of HCN1 complex residues interacting with ivabradine. Residues that interact with the ligand are marked with green-colored vertical bars. D, the protein-ligand contacts within the HCN1-ivabradine complex. HCN, hyperpolarization-activated cyclic nucleotide-gated; MD, molecular dynamics; RMSF, root mean square fluctuation.

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  Figure 4. Structure comparison between apo hHCN1, hyperpolarized hHCN1 and ivabradine-bound hHCN1.A and B, the overall conformational difference in TMD between apo hHCN1 (magenta, PDB: 5U6O), hyperpolarized hHCN1 (cyan, PDB: 6UQF) and ivabradine-bound hHCN1 (light blue). C and D, comparison of ivabradine binding site between apo hHCN1 (magenta, PDB: 5U6O), hyperpolarized hHCN1 (cyan, PDB: 6UQF) and ivabradine-bound hHCN1 (light blue). HCN, hyperpolarization-activated cyclic nucleotide-gated; hHCN1, human HCN1; PDB, Protein Data Bank; TMD, transmembrane domain.

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  Figure 5. Structure-based virtual screening for HCN1.A, schematic representation of the virtual screening strategy. B, representative current and structures of haloperidol and nebivolol. C, concentration-response curve for haloperidol inhibition. Data are shown as n = 3 independent experiments. HCN, hyperpolarization-activated cyclic nucleotide-gated.

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  Figure 6. The mechanism model of HCN1 inhibition by ivabradine. The inhibitor ivabradine acts like a wedge, binds to the pocket formed by the S4, S1, and HCN domain, thus preventing the conformational change of the S4 helix and maintaining the HCN1 channel in a closed conformation. HCN, hyperpolarization-activated cyclic nucleotide-gated.