Structural basis of isoform-specific cAMP regulation and autoinhibition in hyperpolarization-activated cyclic nucleotide-gated 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 HCN1ΔC 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, providing insights into 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.