The structure of the apo cAMP-binding domain of HCN4 - a stepping stone toward understanding the cAMP-dependent modulation of the hyperpolarization-activated cyclic-nucleotide-gated ion channels.

The hyperpolarization-activated cyclic-nucleotide-gated (HCN) ion channels control nerve impulse transmission and cardiac pacemaker activity. The modulation by cAMP is critical for the regulatory function of HCN in both neurons and cardiomyocytes, but the underlying mechanism is not fully understood. Here, we show how the structure of the apo cAMP-binding domain of the HCN4 isoform has contributed to a model for the cAMP-dependent modulation of the HCN ion-channel. This model recapitulates the structural and dynamical changes that occur along the thermodynamic cycle arising from the coupling of cAMP-binding and HCN self-association equilibria. The proposed model addresses some of the questions previously open about the auto-inhibition of HCN and its cAMP-induced activation, while opening new opportunities for selectively targeting HCN through allosteric ligands. A remaining challenge is the investigation of HCN dimers and their regulatory role. Overcoming this challenge will require the integration of crystallography, cryo electron microscopy, NMR, electrophysiology and simulations.

Akimoto M ，VanSchouwen B ，Melacini G 《-》

A mechanism for the auto-inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel opening and its relief by cAMP.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels control neuronal and cardiac electrical rhythmicity. There are four homologous isoforms (HCN1-4) sharing a common multidomain architecture that includes an N-terminal transmembrane tetrameric ion channel followed by a cytoplasmic "C-linker," which connects a more distal cAMP-binding domain (CBD) to the inner pore. Channel opening is primarily stimulated by transmembrane elements that sense membrane hyperpolarization, although cAMP reduces the voltage required for HCN activation by promoting tetramerization of the intracellular C-linker, which in turn relieves auto-inhibition of the inner pore gate. Although binding of cAMP has been proposed to relieve auto-inhibition by affecting the structure of the C-linker and CBD, the nature and extent of these cAMP-dependent changes remain limitedly explored. Here, we used NMR to probe the changes caused by the binding of cAMP and of cCMP, a partial agonist, to the apo-CBD of HCN4. Our data indicate that the CBD exists in a dynamic two-state equilibrium, whose position as gauged by NMR chemical shifts correlates with the V½ voltage measured through electrophysiology. In the absence of cAMP, the most populated CBD state leads to steric clashes with the activated or "tetrameric" C-linker, which becomes energetically unfavored. The steric clashes of the apo tetramer are eliminated either by cAMP binding, which selects for a CBD state devoid of steric clashes with the tetrameric C-linker and facilitates channel opening, or by a transition of apo-HCN to monomers or dimer of dimers, in which the C-linker becomes less structured, and channel opening is not facilitated.

Akimoto M ，Zhang Z ，Boulton S ，Selvaratnam R ，VanSchouwen B ，Gloyd M ，Accili EA ，Lange OF ，Melacini G ... -  《-》

Role of Dynamics in the Autoinhibition and Activation of the 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 allosterically modulates HCN through the cAMP-dependent formation of a tetrameric gating ring spanning the intracellular region (IR) of HCN, to which cAMP binds. Although the apo versus holo conformational changes of the cAMP-binding domain (CBD) have been previously mapped, only limited information is currently available on the HCN IR dynamics, which have been hypothesized to play a critical role in the cAMP-dependent gating of HCN. Here, using molecular dynamics simulations validated and complemented by experimental NMR and CD data, we comparatively analyze HCN IR dynamics in the four states of the thermodynamic cycle arising from the coupling between cAMP binding and tetramerization equilibria. This extensive set of molecular dynamics trajectories captures the active-to-inactive transition that had remained elusive for other CBDs, and it provides unprecedented insight on the role of IR dynamics in HCN autoinhibition and its release by cAMP. Specifically, the IR tetramerization domain becomes more flexible in the monomeric states, removing steric clashes that the apo-CDB structure would otherwise impose. Furthermore, the simulations reveal that the active/inactive structural transition for the apo-monomeric CBD occurs through a manifold of pathways that are more divergent than previously anticipated. Upon cAMP binding, these pathways become disallowed, pre-confining the CBD conformational ensemble to a tetramer-compatible state. This conformational confinement primes the IR for tetramerization and thus provides a model of how cAMP controls HCN channel gating.

VanSchouwen B ，Akimoto M ，Sayadi M ，Fogolari F ，Melacini G ... -  《-》

High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer.

Eukaryotic cyclic nucleotide-modulated channels perform their diverse physiological roles by opening and closing their pores to ions in response to cyclic nucleotide binding. We here present a structural model for the cyclic nucleotide-modulated potassium channel homolog from Mesorhizobium loti, MloK1, determined from 2D crystals in the presence of lipids. Even though crystals diffract electrons to only ∼10 Å, using cryoelectron microscopy (cryo-EM) and recently developed computational methods, we have determined a 3D map of full-length MloK1 in the presence of cyclic AMP (cAMP) at ∼4.5 Å isotropic 3D resolution. The structure provides a clear picture of the arrangement of the cyclic nucleotide-binding domains with respect to both the pore and the putative voltage sensor domains when cAMP is bound, and reveals a potential gating mechanism in the context of the lipid-embedded channel.

Kowal J ，Biyani N ，Chami M ，Scherer S ，Rzepiela AJ ，Baumgartner P ，Upadhyay V ，Nimigean CM ，Stahlberg H ... -  《-》

Free energy landscape remodeling of the cardiac pacemaker channel explains the molecular basis of familial sinus bradycardia.

The hyperpolarization-activated and cyclic nucleotide-modulated ion channel (HCN) drives the pacemaker activity in the heart, and its malfunction can result in heart disorders. One such disorder, familial sinus bradycardia, is caused by the S672R mutation in HCN, whose electrophysiological phenotypes include a negative shift in the channel activation voltage and an accelerated HCN deactivation. The outcomes of these changes are abnormally low resting heart rates. However, the molecular mechanism underlying these electrophysiological changes is currently not fully understood. Crystallographic investigations indicate that the S672R mutation causes limited changes in the structure of the HCN intracellular gating tetramer, but its effects on protein dynamics are unknown. Here, we utilize comparative S672R versus WT NMR analyses to show that the S672R mutation results in extensive perturbations of the dynamics in both apo- and holo-forms of the HCN4 isoform, reflecting how S672R remodels the free energy landscape for the modulation of HCN4 by cAMP, i.e. the primary cyclic nucleotide modulator of HCN channels. We show that the S672R mutation results in a constitutive shift of the dynamic auto-inhibitory equilibrium toward inactive states of HCN4 and broadens the free-energy well of the apo-form, enhancing the millisecond to microsecond dynamics of the holo-form at sites critical for gating cAMP binding. These S672R-induced variations in dynamics provide a molecular basis for the electrophysiological phenotypes of this mutation and demonstrate that the pathogenic effects of the S672R mutation can be rationalized primarily in terms of modulations of protein dynamics.

Boulton S ，Akimoto M ，Akbarizadeh S ，Melacini G ... -  《-》