HCN channel regulation by the cAMP-binding fold is facilitated by interdomain interactions

Hyperpolarization and Cyclic Nucleotide-gated (HCN) channels regulate electrical activity in rhythmically firing cells of the brain and heart. Their voltage-gating is mediated by a transmembrane (TM) region and also by direct binding of cAMP to a cyclic nucleotide-binding (CNB) fold in the cytoplasmic C-terminal region. In its unliganded form, the CNB fold mediates autoinhibition, a mechanism that thermodynamically and kinetically stabilizes the closed channel state; cAMP binding relieves this inhibition. The CNB fold mediates two additional regulatory mechanisms called Open-State Trapping (OST) and Quick-Activation (QA), which can govern the kinetic stability of the open and closed states, respectively. Structural studies have shown direct interactions of the TM region with the cytoplasmic C-linker which connects the TM region to the CNB fold, but the functional role of these interdomain interactions is poorly understood. In this work I found functional evidence for interdomain interactions facilitating autoinhibition, OST and QA, using domain replacements and site directed mutagenesis combined with voltage-clamp assays of channel gating. First, I replaced the TM region of HCN2 with that of HCN4, which augmented the magnitude of autoinhibition by the unliganded CNB fold. This implies that HCN2 channels have an interdomain interaction that limits the magnitude of autoinhibition. The augmentation of autoinhibition was also associated with disruption of the OST and QA mechanisms which are characteristic of HCN2. This resulted in autoinhibition becoming the dominant mechanism contributed by the C-terminal region determining kinetics for both activation and deactivation. Second, I used site-directed mutagenesis to identify a C-linker residue (E457) which stabilizes the open state but exerts different effects on thermodynamics and kinetics, most likely through an electrostatic interaction with the HCN4 TM region. A charge reversal mutation (E457R) limited the thermodynamic effect of autoinhibition while augmenting the kinetic effect of autoinhibition. Notably, in full-length autoinhibited channels E457 undergoes a conformational change such that it no longer stabilizes the open state. Overall, my work suggests that the molecular mechanisms of autoinhibition, OST, and QA must include participation of TM region structures and provides functional evidence for interdomain interactions that facilitate HCN channel regulation by CNB fold-mediated mechanisms.