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Probing the bradycardic drug binding receptor of HCN-encoded pacemaker channels

Yau-Chi Chan1, Kai Wang, Ka-Wing Au

  • 1Division of Cardiology, Department of Medicine, Queen Mary Hospital, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong.

Pflugers Archiv : European Journal of Physiology
|September 17, 2009
PubMed

Insights

This study investigates the drug receptor site on HCN1 channels, crucial for heart pacing. Alanine scanning revealed specific residues in the inner pore vestibule are key for ZD7288 binding, informing antiarrhythmic drug design.

Area of Science:

  • Cardiovascular Physiology
  • Molecular Pharmacology
  • Ion Channel Biophysics

Background:

  • The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel family, particularly HCN1-4, is vital for cardiac pacing.
  • Bradycardic agents targeting HCN channels, like ZD7288, are used clinically, but their precise binding site remains undefined.

Purpose of the Study:

  • To elucidate the molecular structure of the ZD7288 drug receptor within HCN1 channels.
  • To identify specific amino acid residues in the HCN1 channel pore that interact with ZD7288.

Main Methods:

  • Systematic alanine scanning mutagenesis was performed on residues within the selectivity filter, P-S6 linker, and S6 pore vestibule of HCN1 channels.
  • Heterologous expression of mutant HCN1 channels in HEK293 cells followed by patch-clamp electrophysiology to record ionic currents.
  • Dose-response curves were generated to determine the half-blocking concentration (IC50) of ZD7288 for wild-type and mutant channels.
  • Thermodynamic cycle analysis was employed to investigate energetic interactions between S6 residues.

Main Results:

  • Mutations I348A, G349A, Y350A, G351A (P-loop), P355A, and V356A (P-S6 linker) abolished measurable currents.
  • Wild-type HCN1 channels had an IC50 for ZD7288 of 25.8 µM.
  • Mutations C347A, S357A, F378A, and V379A significantly increased the IC50 for ZD7288, indicating reduced drug sensitivity.
  • Mutation M377A in the S6 region showed hypersensitivity to ZD7288 (IC50 = 5.1 µM), suggesting specific site interactions.
  • Thermodynamic analysis revealed significant coupling energies between M377-F378 and F378-V379 residues.

Conclusions:

  • Specific residues within the inner pore vestibule (S6 region) of HCN1 channels are critical for ZD7288 binding.
  • The findings support a refined model of ZD7288 block within the HCN1 channel pore.
  • This detailed understanding can guide the development of improved antiarrhythmic drugs and bioartificial pacemakers.

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