HCN channels have a reverse electromechanical coupling mechanism, where hyperpolarized membrane potentials facilitate pore opening through an inward displacement of the S4 segment of the voltage-sensing domain (VSD). This voltage dependence is finely regulated by the binding of cAMP to an intracellular domain (CNBD). Of the four widely studied isoforms of human HCN channels, the HCN3 channel is practically insensitive to cAMP or is even inhibited by it, but the structural determinants underlying this unexpected behavior are still unclear. Here, we evaluated the possible role of flexibility in very specific regions of the VSD that could be determinants for this behavior. Part of the S2-S3L linker is more rigid in HCN3, which correlates with low atomic mobility for this region in proximity to the C-linker subdomain of the opposite subunit. We built structural models using AlphaFold 3 and Swiss-Model and thus reconstructed the disordered regions that connect the transmembrane segments of the VSD and that in some of the structures deposited in the PDB have not been resolved. Besides, in an attempt to reveal the evolutionary trends that this transmembrane domain may have undergone, we conducted a comparative study with phylogenetically distant HCN channels and found an interesting tendency to lose sensitivity to cAMP as VSD flexibility is lost. Our analysis confirms a large body of published experimental findings. Finally, we found that in metazoans, two types of HCN channels clearly diverge: (1) those that are highly sensitive to cAMP with moderate flexibility profiles in protostomes, and (2) those that show less marked sensitivity to this ligand in deuterostomes. We also propose a possible evolutionary scenario for the appearance of cAMP-modulated HCN channels in the last eukaryotic common ancestor (LECA).
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