Structural Basis of Tonic Inhibition by Dimers of Dimers in Hyperpolarization-Activated Cyclic-Nucleotide-Modulated (HCN) Ion Channels

The hyperpolarization-activated cyclic-nucleotide-modulated (HCN) ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic AMP (cAMP) modulates HCN activity through the cAMP-dependent formation of a tetrameric gating ring spanning the intracellular region (IR) of HCN. In the absence of cAMP, the IR cAMP-binding domain (CBD) mainly samples its inactive conformation, resulting in steric clashes that destabilize the IR tetramer. Although these clashes with the inactive CBD are released through tetramer dissociation into monomers, functional mutagenesis suggests that the apo IR is not fully monomeric. To investigate the inhibitory nonmonomeric IR species, we performed molecular dynamics simulations starting from "hybrid" structures that are tetrameric but contain inactive apo-state CBD conformations. The ensemble of simulated trajectories reveals that full dissociation of the tetramer into monomers is not necessary to release the steric hindrance with the inactive CBD. Specifically, we found that partial dissociation of the tetramer into dimers is sufficient to accommodate four inactive CBDs, while reduction of the quaternary symmetry of the nondissociated tetramer from 4- to 2-fold permits accommodation of two inactive CBDs. Our findings not only rationalize available electrophysiological, fluorometry, and sedimentation equilibrium data, but also provide unprecedented structural insight into previously elusive nonmonomeric autoinhibitory HCN species.