Structural basis of properties, mechanisms, and channelopathy of cyclic nucleotide-gated channels.

Recent years have seen an outpouring of atomic or near atomic resolution structures of cyclic nucleotide-gated (CNG) channels, captured in closed, transition, pre-open, partially open, and fully open states. These structures provide unprecedented molecular insights into the activation, assembly, architecture, regulation, and channelopathy of CNG channels, as well as mechanistic explanations for CNG channel biophysical and pharmacological properties. This article summarizes recent advances in CNG channel structural biology, describes key structural features and elements, and illuminates a detailed conformational landscape of activation by cyclic nucleotides. The review also correlates structures with findings and properties delineated in functional studies, including nonselective monovalent cation selectivity, Ca2+ permeation and block, block by L-cis-diltiazem, location of the activation gate, lack of voltage-dependent gating, and modulation by lipids and calmodulin. A perspective on future research is also offered.

Discrimination between cyclic nucleotides in a cyclic nucleotide-gated ion channel.

Cyclic nucleotide-gated ion channels are crucial in many physiological processes such as vision and pacemaking in the heart. SthK is a prokaryotic homolog with high sequence and structure similarities to hyperpolarization-activated and cyclic nucleotide-modulated and cyclic nucleotide-gated channels, especially at the level of the cyclic nucleotide binding domains (CNBDs). Functional measurements showed that cyclic adenosine monophosphate (cAMP) is a channel activator while cyclic guanosine monophosphate (cGMP) barely leads to pore opening. Here, using atomic force microscopy single-molecule force spectroscopy and force probe molecular dynamics simulations, we unravel quantitatively and at the atomic level how CNBDs discriminate between cyclic nucleotides. We find that cAMP binds to the SthK CNBD slightly stronger than cGMP and accesses a deep-bound state that a cGMP-bound CNBD cannot reach. We propose that the deep binding of cAMP is the discriminatory state that is essential for cAMP-dependent channel activation.

Structural and functional characterization of an achromatopsia-associated mutation in a phototransduction channel.

Numerous missense mutations in cyclic nucleotide-gated (CNG) channels cause achromatopsia and retinitis pigmentosa, but the underlying pathogenic mechanisms are often unclear. We investigated the structural basis and molecular/cellular effects of R410W, an achromatopsia-associated, presumed loss-of-function mutation in human CNGA3. Cryo-EM structures of the Caenorhabditis elegans TAX-4 CNG channel carrying the analogous mutation, R421W, show that most apo channels are open. R421, located in the gating ring, interacts with the S4 segment in the closed state. R421W disrupts this interaction, destabilizes the closed state, and stabilizes the open state. CNGA3_R410W/CNGB3 and TAX4_R421W channels are spontaneously active without cGMP and induce cell death, suggesting cone degeneration triggered by spontaneous CNG channel activity as a possible cause of achromatopsia. Our study sheds new light on CNG channel allosteric gating, provides an impetus for a reevaluation of reported loss-of-function CNG channel missense disease mutations, and has implications for mutation-specific treatment of retinopathy.

Chemical shift assignments of calmodulin bound to the ß-subunit of a retinal cyclic nucleotide-gated channel (CNGB1).

Rod cyclic nucleotide-gated (CNG) channels are formed by two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to the cytosolic regulatory domain of CNGB1 and decreases the open probability of CNGA1/CNGB1 channels. The CaM binding site within bovine CNGB1 (residues 679-702) binds tightly to Ca2+-bound CaM, which promotes Ca2+-induced inactivation of CNGA1/CNGB1 channels in retinal rods. We report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM-binding domain of CNGB1 (BMRB no. 51222).

Structural basis of the partially open central gate in the human CNGA1/CNGB1 channel explained by additional density for calmodulin in cryo-EM map.

The recently reported structure of the human CNGA1/CNGB1 CNG channel in the open state (Xue et al., 2021a) shows that one CNGA1 and one CNGB1 subunit do not open the central hydrophobic gate completely upon cGMP binding. This is different from what has been reported for CNGA homomeric channels (Xue et al., 2021b; Zheng et al., 2020). In seeking to understand how this difference is due to the presence of the CNGB1 subunit, we find that the deposited density map (Xue et al., 2021a) (EMDB 24465) contains an additional density not reported in the images of the original publication. This additional density fits well the structure of calmodulin (CaM), and it unambiguously connects the newly identified D-helix of CNGB1 to one of the CNGA1 helices (A1R) participating in the coiled-coil region. Interestingly, the CNGA1 subunit that engages in the interaction with this additional density is the one that, together with CNGB1, does not open completely the central gate. The sequence of the D-helix of CNGB1 contains a known CaM-binding site of exquisitely high affinity - named CaM2 (Weitz et al., 1998) -, and thus the presence of CaM in that region is not surprising. The mechanism through which CaM reduces currents across the membrane by acting on the native channel (Bauer, 1996; Hsu and Molday, 1993; Weitz et al., 1998) remains unclear. We suggest that the presence of CaM may explain the partially open central gate reported by Xue et al. (2021a). The structure of the open and closed states of the CNGA1/CNGB1 channel may be different with and without CaM present.

The structure of the native CNGA1/CNGB1 CNG channel from bovine retinal rods.

In rod photoreceptors of the retina, the cyclic nucleotide-gated (CNG) channel is composed of three CNGA and one CNGB subunits, and it closes in response to light activation to generate an electrical signal that is conveyed to the brain. Here we report the cryo-EM structure of the closed state of the native rod CNG channel isolated from bovine retina. The structure reveals differences between CNGA1 and CNGB1 subunits. Three CNGA1 subunits are tethered at their C terminus by a coiled-coil region. The C-helix in the cyclic nucleotide-binding domain of CNGB1 features a different orientation from that in the three CNGA1 subunits. The arginine residue R994 of CNGB1 reaches into the ionic pathway and blocks the pore, thus introducing an additional gate, which is different from the central hydrophobic gate known from homomeric CNGA channels. These results address the long-standing question of how CNGB1 subunits contribute to the function of CNG channels in visual and olfactory neurons.

Structure of the human cone photoreceptor cyclic nucleotide-gated channel.

Cyclic nucleotide-gated (CNG) channels transduce light-induced chemical signals into electrical signals in retinal cone and rod photoreceptors. Structures of native CNG channels, which are heterotetramers formed by CNGA and CNGB subunits, have not been obtained. In the present study, we report a high-resolution cryo-electron microscopy structure of the human cone CNG channel in the apo closed state. The channel contains three CNGA3 and one CNGB3 subunits. Arg403 in the pore helix of CNGB3 projects into an asymmetric selectivity filter and forms hydrogen bonds with two pore-lining backbone carbonyl oxygens. Arg442 in S6 of CNGB3 protrudes into and occludes the pore below the hydrophobic cavity gate previously observed in homotetrameric CNGA channels. It is interesting that Arg403Gln is a disease mutation, and Arg442 is replaced by glutamine in some animal species with dichromatic or monochromatic vision. These and other unique structural features and the disease link conferred by CNGB3 indicate a critical role of CNGB3 in shaping cone photoresponses.

Cyclic nucleotide gated channel genes (CNGCs) in Rosaceae: genome-wide annotation, evolution and the roles on Valsa canker resistance.

KEY MESSAGE: In Rosaceae, tandem duplication caused the drastic expansion of CNGC gene family Group I. The members MdCN11 and MdCN19 negatively regulate Valsa canker resistance. Apple (Malus domestica) and pear (Pyrus bretschneideri and P. communis) are important fruit crops in Rosaceae family but are suffering from threats of Valsa canker. Cyclic nucleotide-gated ion channels (CNGCs) take crucial roles in plant immune responses. In the present study, a total of 355 CNGCs was identified from 8 Rosaceae plants. Based on phylogenetic analysis, 540 CNGCs from 18 plants (8 in Rosaceae and 10 others) could be divided into four groups. Group I was greatly expanded in Rosaceae resulted from tandem duplications. A large number of cis-acting regulatory elements (cis-elements) responsive to signals from multiple stresses and hormones were identified in the promoter regions of CNGCs in Malus spp. and Pyrus spp. Expressions of most Group I members were obviously up-regulated in Valsa canker susceptible varieties but not in the resistant ones. Furthermore, overexpression of the MdCN11 and MdCN19 in both apple fruits and 'Duli' (P. betulifolia) suspension cells compromised Valsa canker resistance. Overexpression of MdCN11 induced expression of hypersensitive response (HR)-related genes. In conclusion, tandem duplication resulted in a drastic expansion of CNGC Group I members in Rosaceae. Among these, MdCN11 and MdCN19 negatively regulate the Valsa canker resistance via inducting HR.

CNG channel structure, function, and gating: a tale of conformational flexibility.

Cyclic nucleotide-gated (CNG) channels are key to the signal transduction machinery of certain sensory modalities both in vertebrate and invertebrate organisms. They translate a chemical change in cyclic nucleotide concentration into an electrical signal that can spread through sensory cells. Despite CNG and voltage-gated potassium channels sharing a remarkable amino acid sequence homology and basic architectural plan, their functional properties are dramatically different. While voltage-gated potassium channels are highly selective and require membrane depolarization to open, CNG channels have low ion selectivity and are not very sensitive to voltage. In the last few years, many high-resolution structures of intact CNG channels have been released. This wealth of new structural information has provided enormous progress toward the understanding of the molecular mechanisms and driving forces underpinning CNG channel activation. In this review, we report on the current understanding and controversies surrounding the gating mechanism in CNG channels, as well as the deep intertwining existing between gating, the ion permeation process, and its modulation by membrane voltage. While the existence of this powerful coupling was recognized many decades ago, its direct structural demonstration, and ties to the CNG channel inherent pore flexibility, is a recent achievement.

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.

Electromechanical coupling mechanism for activation and inactivation of an HCN channel.

Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET, patch-clamp fluorometry and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removing cAMP produced a largely rigid-body rotation of the C-linker relative to the transmembrane domain, bringing the A' helix of the C-linker in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not the presence of cAMP. These results suggest that - in contrast to electromechanical coupling for channel activation - the A' helix serves to couple the S4-helix movement for channel inactivation, which is likely a conserved mechanism for CNBD-family channels.

The GARP Domain of the Rod CNG Channel's ß1-Subunit Contains Distinct Sites for Outer Segment Targeting and Connecting to the Photoreceptor Disk Rim.

Vision begins when light is captured by the outer segment organelle of photoreceptor cells in the retina. Outer segments are modified cilia filled with hundreds of flattened disk-shaped membranes. Disk membranes are separated from the surrounding plasma membrane, and each membrane type has unique protein components. The mechanisms underlying this protein sorting remain entirely unknown. In this study, we investigated the outer segment delivery of the rod cyclic nucleotide-gated (CNG) channel, which is located in the outer segment plasma membrane, where it mediates the electrical response to light. Using Xenopus and mouse models of both sexes, we now show that the targeted delivery of the CNG channel to the outer segment uses the conventional secretory pathway, including protein processing in both ER and Golgi, and requires preassembly of its constituent α1 and ß1 subunits. We further demonstrate that the N-terminal glutamic acid-rich protein (GARP) domain of CNGß1 contains two distinct functional regions. The glutamic acid-rich region encodes specific information targeting the channel to rod outer segments. The adjacent proline-enriched region connects the CNG channel to photoreceptor disk rims, likely through an interaction with peripherin-2. These data reveal fine functional specializations within the structural domains of the CNG channel and suggest that its sequestration to the outer segment plasma membrane requires an interaction with peripherin-2.SIGNIFICANCE STATEMENT Neurons and other differentiated cells have a remarkable ability to deliver and organize signaling proteins at precise subcellular locations. We now report that the CNG channel, mediating the electrical response to light in rod photoreceptors, contains two specialized regions within the N terminus of its ß-subunit: one responsible for delivery of this channel to the ciliary outer segment organelle and another for subsequent channel sequestration into the outer segment plasma membrane. These findings expand our understanding of the molecular specializations used by neurons to populate their critical functional compartments.

The Complex Story of Plant Cyclic Nucleotide-Gated Channels.

Plant cyclic nucleotide-gated channels (CNGCs) are tetrameric cation channels which may be activated by the cyclic nucleotides (cNMPs) adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). The genome of Arabidopsis thaliana encodes 20 CNGC subunits associated with aspects of development, stress response and immunity. Recently, it has been demonstrated that CNGC subunits form heterotetrameric complexes which behave differently from the homotetramers produced by their constituent subunits. These findings have widespread implications for future signalling research and may help explain how specificity can be achieved by CNGCs that are known to act in disparate pathways. Regulation of complex formation may involve cyclic nucleotide-gated channel-like proteins.

Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel.

SthK, a cyclic nucleotide-modulated ion channel from Spirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the hyperpolarization-activated and cyclic nucleotide-gated channel HCN2 in the family of so-called pacemaker channels. Here, we investigate slow cAMP-induced activation in purified SthK channels using stopped-flow assays, mutagenesis, enzymatic catalysis and inhibition assays revealing that the cis/trans conformation of a conserved proline in the cyclic nucleotide-binding domain determines the activation kinetics of SthK. We propose that SthK exists in two forms: trans Pro300 SthK with high ligand binding affinity and fast activation, and cis Pro300 SthK with low affinity and slow activation. Following channel activation, the cis/trans equilibrium, catalyzed by prolyl isomerases, is shifted towards trans, while steady-state channel activity is unaffected. Our results reveal prolyl isomerization as a regulatory mechanism for SthK, and potentially eukaryotic HCN channels. This mechanism could contribute to electrical rhythmicity in cells.

Genotypes and phenotypes of genes associated with achromatopsia: A reference for clinical genetic testing.

Purpose: Achromatopsia is a congenital autosomal recessive cone disorder, and it has been found to be associated with six genes. However, pathogenic variants in these six genes have been identified in patients with various retinal dystrophies with the exception of achromatopsia. Thus, this study aims to investigate the contribution of these genes in hereditary retinal diseases and the potential genotype-phenotype correlations. Methods: Biallelic variants in six achromatopsia-related genes, namely, CNGA3, CNGB3, GNAT2, ATF6, PDE6C, and PDE6H, were analyzed based on data obtained from 7,195 probands with different eye conditions. A systematic genotype-phenotype analysis of these genes was performed based on these data, along with the data reported in the literature. Results: Biallelic potential pathogenic variants (PPVs) in five of the six genes were identified in 119 probands with genetic eye diseases. The variants in CNGA3 were the most common and accounted for 81.5% (97/119). Of the 119 probands, 62.2% (74/119) have cone-rod dystrophy, whereas only 25.2% (30/119) have achromatopsia. No biallelic pathogenic variants in these genes were identified in patients with rod-dominant degeneration. A systematic review of genotypes and phenotypes revealed certain characteristics of each of the six genes, providing clues for the pathogenicity evaluation of the variants of the genes. Conclusions: PPVs in the six genes were identified in various inherited retinal degeneration diseases, most of which are cone-dominant diseases but no rod-dominant diseases based on the data from a cohort of 7,195 probands with different eye conditions. The systematic genotype-phenotype analysis of these genes will be useful in drafting guidelines for the clinical genetic diagnostic application for the investigated genes.

Allosteric conformational change of a cyclic nucleotide-gated ion channel revealed by DEER spectroscopy.

Cyclic nucleotide-gated (CNG) ion channels are essential components of mammalian visual and olfactory signal transduction. CNG channels open upon direct binding of cyclic nucleotides (cAMP and/or cGMP), but the allosteric mechanism by which this occurs is incompletely understood. Here, we employed double electron-electron resonance (DEER) spectroscopy to measure intersubunit distance distributions in SthK, a bacterial CNG channel from Spirochaeta thermophila Spin labels were introduced into the SthK C-linker, a domain that is essential for coupling cyclic nucleotide binding to channel opening. DEER revealed an agonist-dependent conformational change in which residues of the B'-helix displayed outward movement with respect to the symmetry axis of the channel in the presence of the full agonist cAMP, but not with the partial agonist cGMP. This conformational rearrangement was observed both in detergent-solubilized SthK and in channels reconstituted into lipid nanodiscs. In addition to outward movement of the B'-helix, DEER-constrained Rosetta structural models suggest that channel activation involves upward translation of the cytoplasmic domain and formation of state-dependent interactions between the C-linker and the transmembrane domain. Our results demonstrate a previously unrecognized structural transition in a CNG channel and suggest key interactions that may be responsible for allosteric gating in these channels.

Genome-wide identification of CNGC genes in Chinese jujube (Ziziphus jujuba Mill.) and ZjCNGC2 mediated signalling cascades in response to cold stress.

BACKGROUNDS: Cyclic nucleotide gated channels (CNGCs) play multifaceted roles in plant physiological processes, especially with respect to signalling processes, plant development, and responses to environmental stresses. However, little information is known about the CNGC family in the large cosmopolitan family Rhamnaceae, which has strong tolerance to biotic and abiotic stresses. RESULTS: In the current study, a total of 15 ZjCNGCs which located on 7 chromosomes were firstly identified in Chinese jujube (Ziziphus jujuba Mill.), the most important species of Rhamnaceae in terms of economic and ecological values. Phylogenetic analysis showed that these ZjCNGCs could be classified into four groups, ZjCNGC12 belonged to group IVA, and ZjCNGC13, 14, 15 belonged to group IVB. In addition, the paralogous and orthologous homology duplication of ZjCNGC15 occurred during the evolutionary process. The characteristics of ZjCNGCs regarding to exon-intron numbers and post-translational modifications showed diversified structures and functions. Motif composition and protein sequence analysis revealed that the phosphate-binding cassette and hinge regions were conserved among ZjCNGCs. Prediction of the cis-acting regulatory elements and expression profiles by real-time quantitative PCR analysis showed that some of the ZjCNGCs responded to environmental changes, especially ZjCNGC2, which was significantly downregulated in response to cold stress, and ZjCNGC4 was highly induced in response to cold, salt and alkaline stresses. ZjCNGC13 and 14 were highly induced in the phytoplasma-resistant cultivar and downregulated in the susceptible cultivar. Furthermore, ZjCNGC2 could be regulated by cAMP treatment, microtubule changes and interact with ZjMAPKK4, which suggested that cAMP and microtubule might play important roles in ZjCNGC2 mediated ZjMAPKK4 signalling transduction involved in cold stress. CONCLUSIONS: The identification and classification analysis of ZjCNGCs were firstly reported, and some key individual ZjCNGCs might play essential roles in the response to biotic and abiotic stresses, especially ZjCNGC2 mediated ZjMAPKK4 signalling transduction involved in cold stress. This systematic analysis could provide important information for further functional characterization of ZjCNGCs with the aim of breeding stress-resistant cultivars.

The interaction of CaM7 and CNGC14 regulates root hair growth in Arabidopsis.

Oscillations in cytosolic free calcium determine the polarity of tip-growing root hairs. The Ca2+ channel cyclic nucleotide gated channel 14 (CNGC14) contributes to the dynamic changes in Ca2+ concentration gradient at the root hair tip. However, the mechanisms that regulate CNGC14 are unknown. In this study, we detected a direct interaction between calmodulin 7 (CaM7) and CNGC14 through yeast two-hybrid and bimolecular fluorescence complementation assays. We demonstrated that the third EF-hand domain of CaM7 specifically interacts with the cytosolic C-terminal domain of CNGC14. A two-electrode voltage clamp assay showed that CaM7 completely inhibits CNGC14-mediated Ca2+ influx, suggesting that CaM7 negatively regulates CNGC14-mediated calcium signaling. Furthermore, CaM7 overexpressing lines phenocopy the short root hair phenotype of a cngc14 mutant and this phenotype is insensitive to changes in external Ca2+ concentrations. We, thus, identified CaM7-CNGC14 as a novel interacting module that regulates polar growth in root hairs by controlling the tip-focused Ca2+ signal.

Voltage vs. Ligand II: Structural insights of the intrinsic flexibility in cyclic nucleotide-gated channels.

In the preceding article, we present a flexibility analysis of the voltage-gated ion channel (VGIC) superfamily. In this study, we describe in detail the flexibility profile of the voltage-sensor domain (VSD) and the pore domain (PD) concerning the evolution of 6TM ion channels. In particular, we highlight the role of flexibility in the emergence of CNG channels and describe a significant level of sequence similarity between the archetypical VSD and the TolQ proteins. A highly flexible S4-like segment exhibiting Lys instead Arg for these membrane proteins is reported. Sequence analysis indicates that, in addition to this S4-like segment, TolQ proteins also show similarity with specific motifs in S2 and S3 from typical V-sensors. Notably, S3 flexibility profiles from typical VSDs and S3-like in TolQ proteins are also similar. Interestingly, TolQ from early divergent prokaryotes are comparatively more flexible than those in modern counterparts or true V-sensors. Regarding the PD, we also found that 2TM K+-channels in early prokaryotes are considerably more flexible than the ones in modern microbes, and such flexibility is comparable to the one present in CNG channels. Voltage dependence is mainly exhibited in prokaryotic CNG channels whose VSD is rigid whereas the eukaryotic CNG channels are considerably more flexible and poorly V-dependent. The implication of the flexibility present in CNG channels, their sensitivity to cyclic nucleotides and the cation selectivity are discussed. Finally, we generated a structural model of the putative cyclic nucleotide-modulated ion channel, which we coined here as AqK, from the thermophilic bacteria Aquifex aeolicus, one of the earliest diverging prokaryotes known. Overall, our analysis suggests that V-sensors in CNG-like channels were essentially rigid in early prokaryotes but raises the possibility that this module was probably part of a very flexible stator protein of the bacterial flagellum motor complex.