Atomistic mechanisms of cation permeation in CNGA1 and HCN channels by molecular dynamics simulations

Cyclic nucleotide–gated (CNG) ion channels play essential roles in visual and olfactory signal transduction in vertebrates. Structurally, CNG channels consist of a voltage-sensing domain (VSD), a pore domain, a C-terminal linker (C-linker), and a cyclic nucleotide-binding domain (CNBD). Another member of the cyclic nucleotide–activated ion channel family, hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, is best known for its role in cardiac pacemaking and the regulation of neuronal excitability. Despite their shared evolutionary origin, substantial sequence similarity, and conserved overall architecture, CNG and HCN channels exhibit pronounced differences in voltage dependence, conductance, and ion selectivity. In this thesis, I performed large-scale molecular dynamics (MD) simulations to elucidate the atomistic mechanisms of cation permeation underlying these functional divergences, complemented by experimental single-channel recordings. Because high-resolution structures of native human CNG channels have only recently become available, MD studies of these channels remain limited. To address this gap, microsecond-scale MD simulations of the human CNGA1 channel were carried out to elucidate the cation permeation mechanisms in CNG channels. The results reveal a highly flexible and geometrically wide selectivity filter that supports partially hydrated, multi-ion permeation of both K⁺ and Na⁺. This dynamic filter architecture provides a structural basis for the weak ion selectivity, relatively high unitary conductance, and inward rectification observed in CNG channels. The simulations additionally indicate voltage-dependent conductance behavior arising from intrinsic pore electrostatics coupled with conformational dynamics. To extend this atomistic framework to HCN channels, MD simulations were employed to investigate the molecular mechanism of isoform-specific conductance differences. Single-channel recordings from all four mammalian HCN isoforms show low unitary conductance values (~1 pS) with clear isoform-specific differences. Simulation results, together with mutagenesis data, indicate that these conductance differences primarily arise from negatively charged residues in the outer vestibule rather than from the selectivity filter or inner gate region. Integrating the findings from both channel families enables a direct comparative framework for interpreting their divergent permeation properties in ion conductance and selectivity. Comparative MD simulations of hCNGA1 and mHCN2 channels indicate that differences in selectivity filter geometry and flexibility, together with the associated free-energy barriers and ion hydration states, are key determinants of their distinct conductance and selectivity profiles. A wider and more flexible filter favors high conductance through multi-ion knock-on mechanisms, whereas a narrower and more rigid filter promotes stronger K⁺ selectivity through enhanced ion dehydration. Together, these results establish a unified atomistic framework for ion permeation in cyclic nucleotide–activated channels and provide mechanistic insight into isoform-specific modulation, with potential implications for the rational design of cation channels with tailored functional properties.