The HCN domain is required for HCN channel cell-surface expression and couples voltage- and cAMP-dependent gating mechanisms.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD), and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane segment via the C-linker. Previous functional analysis of HCN channels has suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of this coupling remain unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed the HCN domain (HCND), forms a direct structural link between the VSD and C-linker-CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified Arg237 and Gly239 residues on the S2 of the VSD that form direct interactions with Ile135 on the HCND. Disrupting these interactions abolished HCN2 currents. We also identified three residues on the C-linker-CNBD (Glu478, Gln482, and His559) that form direct interactions with residues Arg154 and Ser158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the cell-surface expression of HCN channels and provides a functional link between voltage- and cAMP-dependent mechanisms of HCN channel gating.