Combination of Autodisplay and Dynamic Pharmacophore Modeling Reveals New Insights into Cyclic Nucleotide Binding in Hyperpolarization-Activated and Cyclic Nucleotide-Gated Ion Channel 4 (HCN4).

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.

Different fluorescent labels report distinct components of spHCN channel voltage sensor movement.

We used voltage clamp fluorometry to probe the movement of the S4 helix in the voltage-sensing domain of the sea urchin HCN channel (spHCN) expressed in Xenopus oocytes. We obtained markedly different fluorescence responses with either ALEXA-488 or MTS-TAMRA covalently linked to N-terminal Cys332 of the S4 helix. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly, consistent with it reporting the initial inward movement of S4, as previously described.