Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play a critical role in regulating neuronal and cardiac rhythmicity, with their function being modulated by cyclic nucleotide binding. Dysfunction of HCN ion channels leads to the genesis of several diseases such as arrhythmia, bradycardia, or epilepsy. This study employs a multidisciplinary approach integrating mutagenesis, ligand binding assays, and molecular dynamics (MD) simulations combined with dynamic pharmacophore studies to investigate the impact of single residue mutations within the cyclic nucleotide-binding domain (CNBD) of HCN4 channels. Utilizing an autodisplay-based ligand binding assay, surface-displayed HCN4 CNBD mutants were evaluated for their interaction with 8-Fluo-cAMP, providing insights into the ligand binding properties. While some known mutational effects could be confirmed (R669, T670), we identified L652 to be crucial for successful ligand binding. Surprisingly, C662, located in the center of the binding pocket, was discovered to play a negligible role in cAMP-binding. Both E660 and R710 were shown to substantially affect 8-Fluo-cAMP-binding, uncovering the direct ligand binding capability of the R710A mutant for the first time. Furthermore, MD simulations coupled with dynamic pharmacophore analysis offered detailed insights into dynamic ligand-protein interactions, elucidating the structural basis of ligand binding and modulation induced by single residue mutations. Here, a novel bypass mechanism of R713 that interacts with cAMP in the absence of R710 was demonstrated. These findings unveil new perspectives on cyclic nucleotide binding in HCN4 channels, providing a foundation for future studies of pathogenic HCN4 ion channel mutations.
© 2024 The Authors. Published by American Chemical Society.