Structural basis for gating inhibition by the cytoplasmic domain in HCN1 channels

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate rhythmic electrical activity in cardiac and neuronal tissues, with isoform-specific cAMP sensitivity remaining poorly understood. While HCN2 exhibits strong cAMP regulation, HCN1 shows minimal response. To investigate the structural basis of this divergence, we analyzed two engineered HCN1 variants using cryo-electron microscopy. One variant (HCN112) incorporates the C-linker and CNBD from HCN2 into the HCN1 backbone and exhibited enhanced cAMP sensitivity, with structural analysis revealing a compressed cytoplasmic domain arrangement that may facilitate regulatory interactions. In contrast, the truncated HCN1{Delta}C variant (lacking the cytoplasmic domain) displayed an intermediately open pore conformation, supporting auto-inhibitory regulation by the CNBD in wild-type channels. These structural insights elucidate how domain-specific interactions modulate cAMP-dependent gating and intrinsic auto-inhibition, resolving long-standing questions about mechanistic divergence among HCN isoforms. Our findings not only shed new light on the structural mechanisms underlying isoform-specific cAMP sensitivity but also have implications for the development of therapeutic strategies targeting HCN channels in neurological and cardiac disorders. SIGNIFICANCE STATEMENTHyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential regulators of rhythmic electrical activity in the heart and brain, and their dysfunction is linked to disorders such as epilepsy, depression, and cardiac arrhythmias. Although HCN channel isoforms display highly divergent responses to cAMP modulation, the structural basis for these differences has remained elusive. This study reveals, through cryo-EM structures, how domain-specific interactions within the cytoplasmic regions of HCN1 channels underlie their unique gating and auto-inhibition properties, as well as their muted cAMP sensitivity compared to HCN2. These insights resolve long-standing mechanistic questions about HCN channel regulation and pave the way for rational design of targeted therapies that selectively modulate HCN isoform activity in neurological and cardiac disease.