title: Identification of the molecular site of ivabradine binding to HCN4 channels.
abstract: Ivabradine is a specific heart rate-reducing agent approved as a treatment of chronic stable angina. Its mode of action involves a selective and specific block of HCN channels, the molecular components of sinoatrial "funny" (f)-channels. Different studies suggest that the binding site of ivabradine is located in the inner vestibule of HCN channels, but the molecular details of ivabradine binding are unknown. We thus sought to investigate by mutagenesis and in silico analysis which residues of the HCN4 channel, the HCN isoform expressed in the sinoatrial node, are involved in the binding of ivabradine. Using homology modeling, we verified the presence of an inner cavity below the channel pore and identified residues lining the cavity; these residues were replaced with alanine (or valine) either alone or in combination, and WT and mutant channels were expressed in HEK293 cells. Comparison of the block efficiency of mutant vs WT channels, measured by patch-clamp, revealed that residues Y506, F509 and I510 are involved in ivabradine binding. For each mutant channel, docking simulations correctly explain the reduced block efficiency in terms of proportionally reduced affinity for ivabradine binding. In summary our study shows that ivabradine occupies a cavity below the channel pore, and identifies specific residues facing this cavity that interact and stabilize the ivabradine molecule. This study provides an interpretation of known properties of f/HCN4 channel block by ivabradine such as the "open channel block", the current-dependence of block and the property of "trapping" of drug molecules in the closed configuration.
authors: The PaceLab, Department of Life Sciences, Università degli Studi di Milano, Milano, Italy.
authors: Annalisa
authors: A
authors: Bucchi
authors: Mirko
authors: M
authors: Baruscotti
authors: Marco
authors: M
authors: Nardini
authors: Andrea
authors: A
authors: Barbuti
authors: Stefano
authors: S
authors: Micheloni
authors: Martino
authors: M
authors: Bolognesi
authors: Dario
authors: D
authors: DiFrancesco
mesh_terms: D000595:Amino Acid Sequence; D001552:Benzazepines / Q000494:pharmacology*; D001665:Binding Sites; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D000077550:Ivabradine; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D015221:Potassium Channels; D016415:Sequence Alignment; D012849:Sinoatrial Node / Q000378:metabolism
chemical_list: D001552:Benzazepines; D054815:Cyclic Nucleotide-Gated Cation Channels; C119587:HCN4 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D009124:Muscle Proteins; D015221:Potassium Channels; D000077550:Ivabradine