<p>The gating of HCN channels is regulated by both voltage and the binding of cyclic nucleotides to their intracellular domain. However, the molecular determinants underlying this regulation by cyclic nucleotide binding remain unclear and controversial. Here, we combine theoretical and experimental approaches to investigate the binding process in the HCN2 channel. First, molecular dynamics simulations show that the binding of cAMP and cGMP to one HCN2 subunit affects not only the stability of that subunit but also that of neighbouring ones in the absence of any large changes in backbone structure and in a way that is consistent with negative cooperativity. Next, network analysis reveals an inter-subunit communication path that connects cAMP and cGMP binding to the C-linker, which is attached to the pore domain. Finally, experimental analyses confirm that this path is essential for cyclic nucleotide-induced interactions between subunits and high affinity and negatively cooperative binding of ligand that is driven by favourable entropy. Together, these findings provide new insights into the regulatory mechanism of HCN2 gating mediated by cyclic nucleotides and clarify the role of residue E488, which lies on this path and whose mutations are known to cause idiopathic generalized epilepsy.</p>