Predicting Binding Affinities for the Binding Domain of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Isoforms Using Free-Energy Perturbation

Q: What is the clinical evidence from this study?

Population: HCN channel binding affinities (in silico). Intervention: cAMP vs. Different HCN channel isoforms (HCN1-4). Primary outcome: Absolute binding free energy of cAMP to the cyclic nucleotide-binding domain (CNBD).

Q: What does this research mean for the field?

All-atom molecular dynamics simulations and free-energy perturbation successfully determine the absolute binding free energy of cAMP to the cyclic nucleotide-binding domain of HCN isoforms 1-4, providing a mechanistic explanation for differences in channel sensitivity across these isoforms. Novelty: ClaimNovelty.INCREMENTAL. Consensus alignment: ConsensusAlignment.NEUTRAL.

Matthew Brownd; Stephanie Sauve; Hope Woods; Mahmoud Moradi

doi:10.64898/2026.03.04.709733

March 6, 2026Open Access

Predicting Binding Affinities for the Binding Domain of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Isoforms Using Free-Energy Perturbation

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Key result

HCN1 and HCN3 show the highest cAMP binding affinity in simulations, while HCN2 shows the lowest.

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Why the study?

Understanding differences in cAMP modulation responsiveness between HCN isoforms would provide insight into the binding interactions driving channel activation.

Structured PICO

P

Population

Computational models of the cyclic nucleotide-binding domain (CNBD) of monomeric HCN isoforms 1-4 (using structures from PDB and AlphaFold).

E

Exposure

Cyclic adenosine monophosphate (cAMP) binding simulated using all-atom molecular dynamics (MD) and free-energy perturbation (FEP).

O

Outcome

Absolute binding free energy of cAMP to the CNBDs of HCN isoforms 1-4.

Molecular dynamics simulations reveal that HCN1 and HCN3 possess the highest binding affinities for cAMP among HCN isoforms, with specific conserved arginine and glutamate residues driving these interactions, offering insights for isoform-selective drug design.

Limitations

HCN3 initial structure was based on an AlphaFold model because no experimental structure has been determined
The cAMP ligand had to be manually inserted into the HCN3 model using HCN1 as a template
The study relies on in silico molecular dynamics simulations and truncated protein structures rather than full in vivo channels

Implication

Hypothesis-generating for isoform-selective HCN targeting; requires experimental validation before any therapeutic consideration.

Abstract

channels that regulate spontaneous rhythmic electrical activity in both the heart and the brain. Understanding differences in the responsiveness to cyclic adenosine monophosphate (cAMP) modulation between HCN isoforms would offer insight into the specific binding interactions that drive channel activation. Using all-atom molecular dynamics (MD) simulations and the free-energy perturbation (FEP) approach, we determined the absolute binding free energy of cAMP to the the cyclicnucleotide-binding domain (CNBD) of HCN isoforms 1-4. By studying the free-energy of ligand binding to the various isoforms of HCN, our study advances the understanding of HCN channel activation and modulation mechanisms. Overall, our work offers insight into explaining differences in channel sensitivity across the isoforms of HCN.

Discussion

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Cite This Study

Brownd et al. (2026) studied HCN channel binding affinities (in silico). cAMP vs. Different HCN channel isoforms (HCN1-4) was evaluated on Absolute binding free energy of cAMP to the cyclic nucleotide-binding domain (CNBD). Free-energy perturbation simulations demonstrated that HCN1 and HCN3 have the highest comparable binding affinity for cAMP, followed by HCN4, with HCN2 exhibiting the lowest binding affinity.

synapsesocial.com/papers/6a10c6898102eb4b66ee5f99 https://doi.org/10.64898/2026.03.04.709733

Topics

Molecular Mechanisms of Cardiac Remodeling

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Predicting Binding Affinities for the Binding Domain of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Isoforms Using Free-Energy Perturbation

Figures

Structured PICO

Limitations

Implication

Abstract

Discussion

Cite This Study

Topics

Predicting Binding Affinities for the Binding Domain of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Isoforms Using Free-Energy Perturbation

Figures

Structured PICO

Limitations

Implication

Abstract

Discussion

Cite This Study

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Topics

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