LAUSR

Significance Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels show an inverted voltage response compared with virtually all other voltage-gated channels, opening on hyperpolarization rather than depolarization. Although the structure of the HCN1 channel was recently solved, the structural element(s) responsible for the inverted gating polarity of HCN is not known. Here, we use a hierarchical approach, by first characterizing the functional contribution of each structural element to channel gating, and then identifying the critical interactions between these elements. Our studies reveal that the HCN voltage sensor can gate the same pore open on both depolarization and hyperpolarization, thereby acting as a bipolar switch. Elements in the pore domain shut off the depolarization-activation pathway in wild-type channels. Despite sharing a common architecture with archetypal voltage-gated ion channels (VGICs), hyperpolarization- and cAMP-activated ion (HCN) channels open upon hyperpolarization rather than depolarization. The basic motions of the voltage sensor and pore gates are conserved, implying that these domains are inversely coupled in HCN channels. Using structure-guided protein engineering, we systematically assembled an array of mosaic channels that display the full complement of voltage-activation phenotypes observed in the VGIC superfamily. Our studies reveal that the voltage sensor of the HCN channel has an intrinsic ability to drive pore opening in either direction and that the extra length of the HCN S4 is not the primary determinant for hyperpolarization activation. Tight interactions at the HCN voltage sensor–pore interface drive the channel into an hERG-like inactivated state, thereby obscuring its opening upon depolarization. This structural element in synergy with the HCN cyclic nucleotide-binding domain and specific interactions near the pore gate biases the channel toward hyperpolarization-dependent opening. Our findings reveal an unexpected common principle underpinning voltage gating in the VGIC superfamily and identify the essential determinants of gating polarity.