Phenotype chart of HCN-EAG chimeras illustrates the design principles for hyperpolarization-dependent gating. (A) Schematic showing the three structural modules that contribute to hyperpolarization-dependent gating (left). Simplified representations of HCN1 (center) and EAG (right) highlighting the corresponding modules. (B–G) Gating phenotypes of various chimeras and mutants. (B) HHHHE, (C) HHHEH, (D) HHHEE, (E) EEEHH, described in Cowgill et al. (2019); (F) HHHEΔC, described in Lin et al. (2024); and (G) HCN1 C-terminal deletion, from (Wainger et al, 2001) and (Wang et al, 2001). This phenotype chart shows that the HCN-derived VSD is necessary but not sufficient for hyperpolarization-dependent gating. In addition, at least one of the two secondary structural modules—either the PD or the CTD—must also be derived from HCN to confer this gating behavior. When all three modules are appropriately matched, as in wild-type HCN channels, the system exhibits more robust hyperpolarization-dependent gating. VSD, voltage-sensing domain; PD, pore domain; CTD, C-terminal domain.