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SummaryHyperpolarization-activated, cAMP-gated (HCN) channels sense membrane potential and intracellular cAMP levels. A mutation identified in the cAMP binding domain (CNBD) of the human HCN4 channel, S672R, severely reduces the heart rate, but the molecular mechanism has been unclear. Our biochemical binding assays on isolated CNBD and patch-clamp recordings on the functional channel show that S672R reduces cAMP binding. The crystal structure of the mutant CNBD revealed no global changes except a disordered loop on the cAMP entry path. To address this localized structural perturbation at a whole protein level, we studied the activity-dependent dynamic interaction between cAMP and the functional channel using the patch-clamp fluorometry technique. S672R reduces the binding of cAMP to the channels in the resting state and significantly increases the unbinding rate during channel deactivation. This study on a disease-causing mutation illustrates the important roles played by the structural elements on the ligand entry-exit path in stabilizing the bound ligand in the binding pocket.

Protein structures define a complex network of atomic interactions in three dimensions. Direct visualization of the structure and analysis of the interaction potential energy are not straightforward approaches to pinpoint the atomic contacts that are crucial for protein function. We used the tetrameric hyperpolarization-activated cAMP-regulated (HCN) channel as a model system to study the intersubunit contacts in cAMP-dependent gating. To obtain a systematic survey of the contacts between each pair of residues, we used normal-mode analysis, a computational approach for studying protein dynamics, and constructed the covariance matrix for C-α atoms. The significant contacts revealed by covariance analysis were further investigated by means of mutagenesis and functional assays. Among the mutant channels that show phenotypes different from those of the wild-type, we focused on two mutant channels that express opposite changes in cAMP-dependent gating. Subsequent biochemical assays on isolated C-terminal fragments, including the cAMP binding domain, revealed only minimal effects on cAMP binding, suggesting the necessity of interpreting the cAMP-dependent allosteric regulation at the whole-channel level. For this purpose, we applied the patch-clamp fluorometry technique and observed correlated changes in the dynamic, state-dependent cAMP binding in the mutant channels. This study not only provides further understanding of the intersubunit contacts in allosteric coupling in the HCN channel, it also illustrates an effective strategy for delineating important atomic contacts within a structure.