Cyclic Nucleotide Binding Research Articles

Conformation sensitive antibody reveals an altered cytosolic PAS/CNBH assembly during hERG channel gating

The human ether-à -go-go-related gene (hERG) K+ channel has a crucial function in cardiac repolarization and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain fragment variable (scFv) antibody fragments can modulate hERG function by binding to the PAS domain.

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Harley et al (Proc Natl Acad Sci U S A 2021;118:e2108796118, PMID 34716268) investigated the roles of the cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains of the hERG K+ channel in channel gating by using single-chain variable fragment (scFv) antibodies. The authors mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface. ScFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. Förster resonance energy transfer (FRET) with scFv2.12 can be detected when the channel gate is open but not when it is closed.

Using Optical Tweezers to Monitor Allosteric Signals Through Changes in Folding Energy Landscapes.

Signaling proteins are composed of conserved protein interaction domains that serve as allosteric regulatory elements of enzymatic or binding activities. The ubiquitous, structurally conserved cyclic nucleotide binding (CNB) domain is found covalently linked to proteins with diverse folds that perform multiple biological functions. Given that the structures of cAMP-bound CNB domains in different proteins are very similar, it remains a challenge to determine how this domain allosterically regulates such diverse protein functions and folds. Instead of a structural perspective, we focus our attention on the energy landscapes underlying the CNB domains and their responses to cAMP binding. We show that optical tweezers is an ideal tool to investigate how cAMP binding coupled to interdomain interactions remodel the energy landscape of the regulatory subunit of protein kinase A (PKA), which harbors two CNB domains. We mechanically manipulate and probe the unfolding and refolding behavior of the CNB domains as isolated structures or selectively as part of the PKA regulatory subunit, and in the presence and absence of cAMP. Optical tweezers allows us to dissect the changes in the energy landscape associated with cAMP binding, and to examine the allosteric interdomain interactions triggered by the cyclic nucleotide. This single molecule approach can be used to study other modular, multidomain signaling proteins found in nature.

Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating

The human ERG (hERG) K+ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS-CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.

Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1.

An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149–811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.

Thermodynamic profile of mutual subunit control in a heteromeric receptor

Cyclic nucleotide-gated (CNG) ion channels of olfactory neurons are tetrameric membrane receptors that are composed of two A2 subunits, one A4 subunit, and one B1b subunit. Each subunit carries a cyclic nucleotide-binding domain in the carboxyl terminus, and the channels are activated by the binding of cyclic nucleotides. The mechanism of cooperative channel activation is still elusive. Using a complete set of engineered concatenated olfactory CNG channels, with all combinations of disabled binding sites and fit analyses with systems of allosteric models, the thermodynamics of microscopic cooperativity for ligand binding was subunit- and state-specifically quantified. We show, for the closed channel, that preoccupation of each of the single subunits increases the affinity of each other subunit with a Gibbs free energy (ΔΔG) of ∼-3.5 to ∼-5.5 kJ ⋅ mol-1, depending on the subunit type, with the only exception that a preoccupied opposite A2 subunit has no effect on the other A2 subunit. Preoccupation of two neighbor subunits of a given subunit causes the maximum affinity increase with ΔΔG of ∼-9.6 to ∼-9.9 kJ ⋅ mol-1 Surprisingly, triple preoccupation leads to fewer negative ΔΔG values for a given subunit as compared to double preoccupation. Channel opening increases the affinity of all subunits. The equilibrium constants of closed-open isomerizations systematically increase with progressive liganding. This work demonstrates, on the example of the heterotetrameric olfactory CNG channel, a strategy to derive detailed insights into the specific mutual control of the individual subunits in a multisubunit membrane receptor.

Regulation of sinoatrial funny channels by cyclic nucleotides: From adrenaline and IK2 to direct binding of ligands to protein subunits

The funny current, and the HCN channels that form it, are affected by the direct binding of cyclic nucleotides. Binding of these second messengers causes a depolarizing shift of the activation curve, which leads to greater availability of current at physiological membrane voltages. This review outlines a brief history on this regulation and provides some evidence that other cyclic nucleotides, especially cGMP, may be important for the regulation of the funny channel in the heart. Current understanding of the molecular mechanism of cyclic nucleotide regulation is also presented, which includes the notions that full and partial agonism occur as a consequence of negatively cooperative binding. Knowledge gaps, including a potential role of cyclic nucleotide-regulation of the funny current under pathophysiological conditions, are included. The work highlighted here is in dedication to Dario DiFrancesco on his retirement.

Molecular Determinants of PKA RIα Driven Liquid‐Liquid Phase Separation

We recently discovered a regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS), which is critical for effective cAMP compartmentation. PKA RIα LLPS is enhanced by cAMP, which results in phase separated bodies that dynamically sequester cAMP. Interestingly, although cAMP-dependent dissociation of the canonical PKA complex, which consists of regulatory (R) and catalytic (C) subunits, allows RIα to undergo LLPS, the PKA C subunit is co-recruited to RIα phase separated bodies, resulting in high PKA activity. In this study, we performed systematic mutations to the dimerization/docking (D/D) domain, inhibitory sequence (IS), and cyclic nucleotide binding (CNB) domains to elucidate the molecular drivers of RIα LLPS. Mutations of key residues within the CNB domains and within the D/D domain and N3A motif of the CNB-A domain disrupt PKA RIα LLPS, indicating cAMP binding and RIα dimerization are required for LLPS. Conversely, mutations within the IS pseudo-substrate increase RIα LLPS, indicating a disrupted R:C complex enhances LLPS. The IS mutations also prevented recruitment of the catalytic subunit to the RIα LLPS, providing opportunities to further probe this non-canonical recruitment mechanism. Importantly, the loss of RIα LLPS leads to disruption of cAMP compartmentation, and increased cell proliferation, DNA synthesis, and anchorage independent growth, suggesting that dysregulated cAMP signaling plays a role in tumorigenesis.

Impacts of OrX and cAMP-insensitive Orco to the insect olfactory heteromer activity.

Insect odorant receptors (ORs) have been suggested to function as ligand-gated cation channels, with OrX/Orco heteromers combining ionotropic and metabotropic activity. The latter is mediated by different G proteins and results in Orco self-activation by cyclic nucleotide binding. In this contribution, we co-express the odor-specific subunits DmOr49b and DmOr59b with either wild-type Orco or an Orco-PKC mutant lacking cAMP activation heterologously in mammalian cells. We show that the characteristics of heteromers strongly depend on both the OrX type and the coreceptor variant. Thus, methyl acetate-sensitive Or59b/Orco demonstrated 25-fold faster response kinetics over o-cresol-specific Or49b/Orco, while the latter required a 10-100 times lower ligand concentration to evoke a similar electrical response. Compared to wild-type Orco, Orco-PKC decreased odorant sensitivity in both heteromers, and blocked an outward current rectification intrinsic to the Or49b/Orco pair. Our observations thus provide an insight into insect OrX/Orco functioning, highlighting their natural and artificial tuning features and laying the groundwork for their application in chemogenetics, drug screening, and repellent design.

Identifying Novel Isoform-Specific HCN Channel Ligands using SPR

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are expressed abundantly in the heart and central nervous system where they control cardiac and neuronal rhythmicity. Four HCN channel isoforms exist in mammals, HCN1-4. While HCN2 is the most abundantly expressed isoform in the brain, HCN4 is the predominant isoform in the heart. Identification of isoform specific regulators of HCN channels is crucial for the development of drugs that can treat cardiovascular diseases without affecting the neuronal functions and vice versa. HCN channels consist of four subunits, each containing six transmembrane segments. In addition, HCN channel subunits contain a cyclic nucleotide-binding (CNB) domain in their C-terminal region, which is linked to the pore-forming transmembrane segment by a C-linker. Upon cyclic nucleotide binding to the CNB domain, the HCN channel undergoes a conformational change that facilitates channel pore opening. The role of CNB domain in regulating channel opening via ligand binding makes it an ideal target for isoform-specific small molecule regulators. To identify isoform specific binders of HCN channel CNB domains, we conducted a surface plasmon resonance (SPR)-based screen. For the SPR screen, the C-linker/CNB domains of HCN2 and HCN4 channels were expressed in BL21 cells and purified using Ni-affinity and size-exclusion chromatography. Then the isolated C-linker/CNB domains were immobilized on the SPR senor chip, and screened for binding to the small molecules in the Spectrum Library using SPR. Using this approach, we identified nine compounds that selectively bind to the CNB domain of HCN4, but not to HCN2 channels. These results indicate that CNB domains of HCN channels harbor binding sites for HCN isoform-specific small molecule ligands.

Investigation of Cyclic AMP Binding Interactions with Isolated Cyclic Nucleotide Binding Domain of HCN1 Channel using Atomistic Molecular Dynamics Simulations at Microsecond Timescale

Hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) is a homotetrameric, voltage-gated potassium/sodium channel whose activity is modulated by cyclic AMP. The binding of cAMP to an intracellular cyclic nucleotide binding domain (CNBD) promotes an increase in channel activity through a depolarizing shift in the voltage threshold necessary for channel activation, as well as kinetically favoring the active channel conformation, leading to improved conductance. While the precise mechanisms of this change are still being investigated, the shift in the CNDB region, which is induced by the binding of cAMP, is able to be propagated to the remainder of the channel through an adjacent C-linker, causing an alteration in the channel that leads to increased conductance. Although static structures of both the apo and holo channel states have been solved and compared, there is still a need to understand the precise binding interactions which occur between the cyclic nucleotide and the binding pocket, as well as the dynamic changes to the local protein structure that might lead to the aforementioned changes in activity. Using atomistic molecular dynamics simulations, we have simulated both the apo and holo forms of the isolated CNBD region at a microsecond timescale, and have performed a variety of analyses to relate the two conformations and investigate the interactions of the ligand with the binding pocket. We have also used enhanced sampling techniques to calculate the binding free energy of cAMP. This study provides a computational framework for the study of HCN1-ligand interactions.

A Hydrophobic Patch in the PAS-cap Alpha-Helix Is Critical for Slow Deactivation in HERG Channels

Slow deactivation is a critical biophysical and physiological property of human ether-à-go-go-related gene (hERG) channels mediating the cardiac repolarizing current IKr. Disruption of direct interactions between the intracellular N-terminal eag domain and the C-terminal cyclic nucleotide binding homology domain (CNBhD) can impair slow repolarization and are associated with disease. The eag domain is multipartite: it comprises an N-terminal Per-Arnt-Sim (PAS) cap (residues 1-25) and globular PAS domain (residues 26-135).

Allosteric signaling in C-linker and cyclic nucleotide-binding domain of HCN2 channels

Opening of hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels is controlled by membrane hyperpolarization and binding of cyclic nucleotides to the tetrameric cyclic nucleotide-binding domain (CNBD), attached to the C-linker (CL) disk. Confocal patch-clamp fluorometry revealed pronounced cooperativity of ligand binding among protomers. However, by which pathways allosteric signal transmission occurs remained elusive. Here, we investigate how changes in the structural dynamics of the CL-CNBD of mouse HCN2 upon cAMP binding relate to inter- and intrasubunit signal transmission. Applying a rigidity-theory-based approach, we identify two intersubunit and one intrasubunit pathways that differ in allosteric coupling strength between cAMP-binding sites or toward the CL. These predictions agree with results from electrophysiological and patch-clamp fluorometry experiments. Our results map out distinct routes within the CL-CNBD that modulate different cAMP-binding responses in HCN2 channels. They signify that functionally relevant submodules may exist within and across structurally discernable subunits in HCN channels.

Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma.

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.

Cyclic nucleotide selectivity of protein kinase G isozymes.

The intrinsic activity of the C-terminal catalytic (C) domain of cyclic guanosine monophosphate (cGMP)-dependent protein kinases (PKG) is inhibited by interactions with the N-terminal regulatory (R) domain. Selective binding of cGMP to cyclic nucleotide binding (CNB) domains within the R-domain disrupts the inhibitory R-C interaction, leading to the release and activation of the C-domain. Affinity measurements of mammalian and plasmodium PKG CNB domains reveal different degrees of cyclic nucleotide affinity and selectivity; the CNB domains adjacent to the C-domain are more cGMP selective and therefore critical for cGMP-dependent activation. Crystal structures of isolated CNB domains in the presence and absence of cyclic nucleotides reveal isozyme-specific contacts that explain cyclic nucleotide selectivity and conformational changes that accompany CNB. Crystal structures of tandem CNB domains identify two types of CNB-mediated dimeric contacts that indicate cGMP-driven reorganization of domain-domain interfaces that include large conformational changes. Here, we review the available structural and functional information of PKG CNB domains that further advance our understanding of cGMP mediated regulation and activation of PKG isozymes.

External Cd2+ and protons activate the hyperpolarization-gated K+ channel KAT1 at the voltage sensor.

The functionally diverse cyclic nucleotide binding domain (CNBD) superfamily of cation channels contains both depolarization-gated (e.g., metazoan EAG family K+ channels) and hyperpolarization-gated channels (e.g., metazoan HCN pacemaker cation channels and the plant K+ channel KAT1). In both types of CNBD channels, the S4 transmembrane helix of the voltage sensor domain (VSD) moves outward in response to depolarization. This movement opens depolarization-gated channels and closes hyperpolarization-gated channels. External divalent cations and protons prevent or slow movement of S4 by binding to a cluster of acidic charges on the S2 and S3 transmembrane domains of the VSD and therefore inhibit activation of EAG family channels. However, a similar divalent ion/proton binding pocket has not been described for hyperpolarization-gated CNBD family channels. We examined the effects of external Cd2+ and protons on Arabidopsisthaliana KAT1 expressed in Xenopus oocytes and found that these ions strongly potentiate voltage activation. Cd2+ at 300 µM depolarizes the V50 of KAT1 by 150 mV, while acidification from pH 7.0 to 4.0 depolarizes the V50 by 49 mV. Regulation of KAT1 by Cd2+ is state dependent and consistent with Cd2+ binding to an S4-down state of the VSD. Neutralization of a conserved acidic charge in the S2 helix in KAT1 (D95N) eliminates Cd2+ and pH sensitivity. Conversely, introduction of acidic residues into KAT1 at additional S2 and S3 cluster positions that are charged in EAG family channels (N99D and Q149E in KAT1) decreases Cd2+ sensitivity and increases proton potentiation. These results suggest that KAT1, and presumably other hyperpolarization-gated plant CNBD channels, can open from an S4-down VSD conformation homologous to the divalent/proton-inhibited conformation of EAG family K+ channels.

Toward in vivo-relevant hERG safety assessment and mitigation strategies based on relationships between non-equilibrium blocker binding, three-dimensional channel-blocker interactions, dynamic occupancy, dynamic exposure, and cellular arrhythmia.

The human ether-a-go-go-related voltage-gated cardiac ion channel (commonly known as hERG) conducts the rapid outward repolarizing potassium current in cardiomyocytes (IKr). Inadvertent blockade of this channel by drug-like molecules represents a key challenge in pharmaceutical R&D due to frequent overlap between the structure-activity relationships of hERG and many primary targets. Building on our previous work, together with recent cryo-EM structures of hERG, we set about to better understand the energetic and structural basis of promiscuous blocker-hERG binding in the context of Biodynamics theory. We propose a two-step blocker binding process consisting of: The initial capture step: diffusion of a single fully solvated blocker copy into a large cavity lined by the intra-cellular cyclic nucleotide binding homology domain (CNBHD). Occupation of this cavity is a necessary but insufficient condition for ion current disruption.The IKr disruption step: translocation of the captured blocker along the channel axis, such that: The head group, consisting of a quasi-rod-shaped moiety, projects into the open pore, accompanied by partial de-solvation of the binding interface.One tail moiety packs along a kink between the S6 helix and proximal C-linker helix adjacent to the intra-cellular entrance of the pore, likewise accompanied by mutual de-solvation of the binding interface (noting that the association barrier is comprised largely of the total head + tail group de-solvation cost).Blockers containing a highly planar moiety that projects into a putative constriction zone within the closed channel become trapped upon closing, as do blockers terminating prior to this region.A single captured blocker copy may conceivably associate and dissociate to/from the pore many times before exiting the CNBHD cavity. Lastly, we highlight possible flaws in the current hERG safety index (SI), and propose an alternate in vivo-relevant strategy factoring in: Benefit/risk.The predicted arrhythmogenic fractional hERG occupancy (based on action potential (AP) simulations of the undiseased human ventricular cardiomyocyte).Alteration of the safety threshold due to underlying disease.Risk of exposure escalation toward the predicted arrhythmic limit due to patient-to-patient pharmacokinetic (PK) variability, drug-drug interactions, overdose, and use for off-label indications in which the hERG safety parameters may differ from their on-label counterparts.

Structure and function of the juxtamembrane GAF domain of potassium biosensor KdpD.

KdpD/KdpE two-component signaling system regulates expression of a high affinity potassium transporter responsible for potassium homeostasis. The C-terminal module of KdpD consists of a GAF domain linked to a histidine kinase domain. Whereas certain GAF domains act as regulators by binding cyclic nucleotides, the role of the juxtamembrane GAF domain in KdpD is unknown. We report the high-resolution crystal structure of KdpD GAF domain (KdpDG ) consisting of five α-helices, four β-sheets and two large loops. KdpDG forms a symmetry-related dimer, wherein parallelly arranged monomers contribute to a four-helix bundle at the dimer-interface, SAXS analysis of KdpD C-terminal module reveals an elongated structure that is a dimer in solution. Substitution of conserved residues with various residues that disrupt the dimer interface produce a range of effects on gene expression demonstrating the importance of the interface in inactive to active transitions during signaling. Comparison of ligand binding site of the classic cyclic nucleotide-binding GAF domains to KdpDG reveals structural differences arising from naturally occurring substitutions in primary sequence of KdpDG that modifies the canonical NKFDE sequence motif required for cyclic nucleotide binding. Together these results suggest a structural role for KdpDG in dimerization and transmission of signal to the kinase domain.

Structural investigation of cyclic nucleotide binding proteins from Trypanosoma cruzi

Fora targeted therapy of Trypanosomiasis, new antiparasitic drugs should be specifically directed against essential pathways in the parasite life cycle. Among these potential targets are signal transduction pathways, which have remained largely unexplored in Trypanosoma species. Of special interest is cAMP-mediated signaling, since cAMP has been shown to play critical roles in the life cycle of T. cruzi and in host cell during invasion. The presented research focuses on the identification and characterisation of novel cAMP response proteins (CARPs) in T. cruzi by using a multi disciplinary approach involving the parasitology group of Dr Martin Edreira (University of Buenos Aires, Argentina) and the structural biology group of Dr Ivan Campeotto (University of Leicester, UK). The aim of the project is not only to increase our knowledge about T. cruzi biology but also to target CARPs for the design and development of novel therapeutic agents against Chagas disease. To date, protein crystals of one of the members of the CARP family have been obtained, paving the way for structure determination and for a structure-based drug design approach.

Mechanism of ligand activation of a eukaryotic cyclic nucleotide-gated channel.

Cyclic nucleotide-gated (CNG) channels convert cyclic nucleotide binding and unbinding into electrical signals in sensory receptors and neurons. The molecular conformational changes underpinning ligand activation are largely undefined. We report both closed- and open-state atomic cryo-EM structures of a full-length C. elegans cGMP-activated channel TAX-4 reconstituted in lipid nanodiscs. These structures, together with computational and functional analyses and a mutant channel structure, reveal a double-barrier hydrophobic gate formed by two S6 amino acids in the central cavity. cGMP binding produces global conformational changes that open the cavity gate located ~52 Å away but do not alter the structure of the selectivity filter – the commonly presumed activation gate. Our work provides mechanistic insights into the allosteric gating and regulation of cyclic nucleotide-gated and -modulated channels and CNG channel-related channelopathies.