Structural determinants of ivabradine block of the open pore of HCN4

Area of Science:

• Structural biology
• Molecular pharmacology
• Cardiovascular research

Background:

• Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (HCN1-4) regulate cardiac and neuronal excitability.
• Dysfunction of HCN channels is linked to conditions like sinoatrial block (HCN4), epilepsy (HCN1), and chronic pain (HCN2).
• There is a need for subtype-specific treatments targeting HCN channels.

Purpose of the Study:

• To determine the high-resolution cryo-electron microscopy (cryo-EM) structure of the HCN4 channel in complex with ivabradine.
• To elucidate the molecular mechanism of ivabradine binding and block within the HCN4 channel.
• To provide a structural basis for the rational design of novel HCN channel modulators.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to solve the structure of HCN4-ivabradine complex at 3 Å resolution.
• Molecular dynamics (MD) simulations to investigate the ion-blocking mechanism.
• Analysis of key amino acid residues involved in drug binding and channel function.

Main Results:

• The cryo-EM structure reveals ivabradine bound within the open pore of the HCN4 channel.
• Specific residues (Y507, I511, F510) in the inner cavity and S6 segment are identified as critical for ivabradine binding.
• A unique cysteine residue (C479) in the selectivity filter (SF) was found to accelerate the block kinetics.
• MD simulations suggest ivabradine blocks permeating ions via electrostatic repulsion within the SF.

Conclusions:

• The structure provides unprecedented atomic detail on how ivabradine, the only marketed HCN channel drug, interacts with HCN4.
• The identified binding site and residues offer a foundation for designing more selective HCN channel inhibitors.
• Understanding the precise mechanism of block can guide the development of targeted therapies for HCN channelopathies.