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.
Highlights
Cardiac f- and their neuronal relatives h-channels play a key role in the control of heart rate and neuronal excitability
To identify residues potentially involved in drug binding, we first explored the spatial orientation of residues facing this inner cavity by means of an in silico homology 3D model of the pore region (S5–S6 region) of hHCN4
We selected KcsA as a model rather than Kv1.2 for three main reasons: (i) the KcsA structure is known in both the closed and open forms, while only the open structure is available for Kv1.2; (ii) the KcsA structure has been already used successfully as a model template for the HCN2 channel [24]; (iii) the C-terminal part of the TM2/S6 helix in Kv1.2 hosts two almost consecutive proline residues (P405, P407) which confer to the helix a structure divergent from that of KcsA, where these proline residues are missing (Fig. 1B)
Summary
Cardiac f- and their neuronal relatives h-channels play a key role in the control of heart rate and neuronal excitability. These channels have a tetrameric composition, with single subunits belonging to the Hyperpolarization-activated Cyclic Nucleotidegated (HCN) channel family. As well as playing a basic role in cardiac pacemaking, HCN channels have several important functions in neurons [7]. It is important to understand the molecular details of ivabradine block of HCN4 channels, the main isoform expressed in the pacemaker region of the heart. Some of the basic properties of the molecular interaction between ivabradine molecules and native f/HCN channels have been already clarified. A tentative interpretation of this phenomenon predicts that the positively charged quaternary N+ ion of ivabradine antagonizes Na+/K+ permeating ions in their binding sites in the pore [9,10,11], but no evidence has been provided yet to support this hypothesis
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