Cyclic Nucleotide Binding Research Articles

Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine Monophosphate-Dependent Protein Kinase Holoenzyme Reveals Distinct Roles of Tandem Cyclic Nucleotide-Binding Domains.

The cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent protein kinases (PKA and PKG) are key effectors of cyclic nucleotide signaling. Both share structural features that include tandem cyclic nucleotide-binding (CNB) domains, CNB-A and CNB-B, yet their functions are separated through preferential activation by either cAMP or cGMP. Based on structural studies and modeling, key CNB contact residues have been identified for both kinases. In this study, we explored the requirements for conversion of PKA activation from cAMP-dependent to cGMP-dependent. The consequences of the residue substitutions T192R/A212T within CNB-A or G316R/A336T within CNB-B of PKA-RIα on cyclic nucleotide binding and holoenzyme activation were assessed in vitro using purified recombinant proteins, and ex vivo using RIα-deficient mouse embryonic fibroblasts genetically reconstituted with wild-type or mutant PKA-RIα. In vitro, a loss of binding and activation selectivity was observed when residues in either one of the CNB domains were mutated, while mutations in both CNB domains resulted in a complete switch of selectivity from cAMP to cGMP. The switch in selectivity was also recapitulated ex vivo, confirming their functional roles in cells. Our results highlight the importance of key cyclic nucleotide contacts within each CNB domain and suggest that these domains may have evolved from an ancestral gene product to yield two distinct cyclic nucleotide-dependent protein kinases.

Investigating cyclic nucleotide and cyclic dinucleotide binding to HCN channels by surface plasmon resonance.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding domain (CNBD) in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides to the HCN pore opening. Recently, cyclic dinucleotides were shown to antagonize the effect of cyclic nucleotides in HCN4 but not in HCN2 channels. Based on the structural analysis and mutational studies it has been proposed that cyclic dinucleotides affect HCN4 channels by binding to the C-linker pocket (CLP). Here, we first show that surface plasmon resonance (SPR) can be used to accurately measure cyclic nucleotide binding affinity to the C-linker/CNBD of HCN2 and HCN4 channels. We then used SPR to investigate cyclic dinucleotide binding in HCN channels. To our surprise, we detected no binding of cyclic dinucleotides to the isolated monomeric C-linker/CNBDs of HCN4 channels with SPR. The binding of cyclic dinucleotides was further examined with isothermal calorimetry (ITC), which indicated no binding of cyclic dinucleotides to both monomeric and tetrameric C-linker/CNBDs of HCN4 channels. Taken together, our results suggest that interaction of the C-linker/CNBD with other parts of the channel is necessary for cyclic-dinucleotide binding in HCN4 channels.

Structural Basis of Analog Specificity in PKG I and II.

Cyclic GMP analogs, 8-Br, 8-pCPT, and PET-cGMP, have been widely used for characterizing cellular functions of cGMP-dependent protein kinase (PKG) I and II isotypes. However, interpreting results obtained using these analogs has been difficult due to their low isotype specificity. Additionally, each isotype has two binding sites with different cGMP affinities and analog selectivities, making understanding the molecular basis for isotype specificity of these compounds even more challenging. To determine isotype specificity of cGMP analogs and their structural basis, we generated the full-length regulatory domains of PKG I and II isotypes with each binding site disabled, determined their affinities for these analogs, and obtained cocrystal structures of both isotypes bound with cGMP analogs. Our affinity and activation measurements show that PET-cGMP is most selective for PKG I, whereas 8-pCPT-cGMP is most selective for PKG II. Our structures of cyclic nucleotide binding (CNB) domains reveal that the B site of PKG I is more open and forms a unique π/π interaction through Arg285 at β4 with the PET moiety, whereas the A site of PKG II has a larger β5/β6 pocket that can better accommodate the bulky 8-pCPT moiety. Our structural and functional results explain the selectivity of these analogs for each PKG isotype and provide a starting point for the rational design of isotype selective activators.

Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity

Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood. Analysis of isolated cyclic nucleotide-binding (CNB) domains of PKA regulatory subunit type Iα (RIα) reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here, we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RIα CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP specificity. Binding data with the generic purine nucleotide 3',5'-cyclic inosine monophosphate (cIMP) reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding and the molecular basis of cyclic nucleotide specificity.

Recruitment of Epac2A to Insulin Granule Docking Sites Regulates Priming for Exocytosis.

Epac is a cAMP-activated guanine nucleotide exchange factor that mediates cAMP signaling in various types of cells, including β-cells, where it is involved in the control of insulin secretion. Upon activation, the protein redistributes to the plasma membrane, but the underlying molecular mechanisms and functional consequences are unclear. Using quantitative high-resolution microscopy, we found that cAMP elevation caused rapid binding of Epac2A to the β-cell plasma membrane, where it accumulated specifically at secretory granules and rendered them more prone to undergo exocytosis. cAMP-dependent membrane binding required the high-affinity cyclic nucleotide-binding (CNB) and Ras association domains, but not the disheveled-Egl-10-pleckstrin domain. Although the N-terminal low-affinity CNB domain (CNB-A) was dispensable for the translocation to the membrane, it was critical for directing Epac2A to the granule sites. Epac1, which lacks the CNB-A domain, was recruited to the plasma membrane but did not accumulate at granules. We conclude that Epac2A controls secretory granule release by binding to the exocytosis machinery, an effect that is enhanced by prior cAMP-dependent accumulation of the protein at the plasma membrane.

CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from Leptospira licerasiae-which shares sequence similarity to eukaryotic CNG and HCN channels-in the presence of a saturating concentration of cAMP. A short S4-S5 linker connects nearby voltage-sensing and pore domains to produce a non-domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.

Spectroscopic Studies of a Bacterial Cyclic Nucleotide-Gated Ion Channel

Cyclic nucleotide-gated (CNG) channels are members of the Kv channel superfamily and play a crucial role in the phototransduction and olfactory transduction pathways, where they generate the initial electrical signal following sensory transduction. CNG channels are directly regulated by cyclic nucleotides, which bind to a C-terminal cyclic nucleotide-binding domain (CNBD) connected to the pore by an intervening C-linker domain. Mutagenesis and crosslinking experiments indicate that the C-terminal region (C-linker/CNBD) undergoes a conformation change to transduce cyclic nucleotide binding into channel activation, though the exact nature of this change remains unclear. To address this question, we first sought a stable and well-behaved bacterial CNG channel suitable for spectroscopic studies. We used fluorescence-detection size-exclusion chromatography to screen a library of bacterial orthologs, which identified several candidates that could be expressed and purified at high levels. Flux studies of liposome-reconstituted channels demonstrated that these orthologs are fully functional and are activated by cyclic nucleotides. We then mutated the endogenous Cys residues in these channels before introducing Cys residues throughout the C-terminal region for spin label and fluorophore attachment. Inter-subunit double electron-electron resonance (DEER) measurements will be used to detect large-scale structural changes in these channels induced by cyclic nucleotide binding, while transition metal FRET (tmFRET) will be used to characterize subtle, localized changes to channel structure following activation. Our ultimate goal is to use these two spectroscopic techniques to characterize small- and large-scale rearrangements induced by cyclic nucleotide binding, allowing us to generate a complete structural model for CNG channel activation.

Single-Molecule Fluorescence Imaging of low Affinity Binding Interactions in Pacemaker ion Channels

Molecular recognition is a cornerstone of cellular signaling processes. However, most observations of binding rely on ensemble measurements that average over and thereby obscure the individual underlying steps. Single-molecule fluorescence can reveal these dynamic events, but is traditionally limited to low concentrations of fluorophores only suitable for studying high affinity reactions. Here, we show how a combination of fluorescence resonance energy transfer (FRET) and zero-mode waveguide nanofabricated devices can be used to study single-molecule association dynamics at the micromolar to millimolar concentrations necessary for many relevant signaling pathways. As an exemplar system, we apply this approach to cyclic nucleotide binding at its receptor domain from hyperpolarization cyclic nucleotide-gated (HCN) ion channels. Our observations reveal the underlying dynamic events during cyclic nucleotide binding: an initial encounter complex when the binding site is in its receptive state, followed by an isomerization of the bound complex that traps the ligand.

Investigating Ligand Binding to HCN Channels by Surface Plasmon Resonance

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding (CNB) domain in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides in the CNB domain to the pore opening. Surface plasmon resonance (SPR) is a powerful biophysical tool for quantitatively investigating ligand-protein and protein-protein interactions. Here we used SPR to detect ligand binding to the isolated C-linker/CNB domain of HCN channels. The isolated C-linker/CNB domains of wild-type (WT) and L586W mutant HCN2 channels were immobilized on a NTA sensor chip and cAMP was injected over the protein coated surface. The mutant C-linker/CNB domain has been used before for ligand binding studies based on the changes in the fluorescence of the introduced tryptophan upon ligand binding. From the cyclic nucleotide concentration dependent SPR responses, we determined the binding affinity for cAMP to be 6.3 + 2.6 μM for the WT and 10.8 + 2.3μM for the mutant C-linker/CNB domains. The binding affinity determined for the mutant L586W C-linker-CNB domains determined with SPR is in agreement with the binding affinity of 13 + 2 μM determined with the fluorescence-based method. These results indicate that SPR is well suited for the detection of binding of known HCN channel ligands. Therefore, SPR can be used to identify novel HCN channel regulators for treatment of diseases associated with abnormal functions of these channels, such as epilepsy and cardiac arrhythmias.

Phylogenetic Diversity of NTT Nucleotide Transport Proteins in Free-Living and Parasitic Bacteria and Eukaryotes.

Plasma membrane-located nucleotide transport proteins (NTTs) underpin the lifestyle of important obligate intracellular bacterial and eukaryotic pathogens by importing energy and nucleotides from infected host cells that the pathogens can no longer make for themselves. As such their presence is often seen as a hallmark of an intracellular lifestyle associated with reductive genome evolution and loss of primary biosynthetic pathways. Here, we investigate the phylogenetic distribution of NTT sequences across the domains of cellular life. Our analysis reveals an unexpectedly broad distribution of NTT genes in both host-associated and free-living prokaryotes and eukaryotes. We also identify cases of within-bacteria and bacteria-to-eukaryote horizontal NTT transfer, including into the base of the oomycetes, a major clade of parasitic eukaryotes. In addition to identifying sequences that retain the canonical NTT structure, we detected NTT gene fusions with HEAT-repeat and cyclic nucleotide binding domains in Cyanobacteria, pathogenic Chlamydiae and Oomycetes. Our results suggest that NTTs are versatile functional modules with a much wider distribution and a broader range of potential roles than has previously been appreciated.

Fine Tuning HCN Channel Activity

HCN (hyperopolarization-activated cyclic-nucleotide gated) channels are activated by membrane hyperopolarization and further modulated by binding of cAMP to the CNBD (cyclic nucleotide binding domain). Understanding the molecular mechanisms of regulation of this family of ion channels is critical as it pertains to the physiological processes as well as to diseases associated dysfunctions in the cardiac and neuronal If/Ih currents. In the brain, cAMP modulation of HCN isoforms 1 and 2 is controlled and fine tuned by the auxillary protein TRIP8b while, in the heart, cAMP regulation of the cardiac isoform HCN4 is prevented by cyclic dinucleotides, an emerging class of second messengers in mammals.

The intrinsically liganded cyclic nucleotide-binding homology domain promotes KCNH channel activation.

Channels in the ether-à-go-go or KCNH family of potassium channels are characterized by a conserved, C-terminal domain with homology to cyclic nucleotide-binding homology domains (CNBhDs). Instead of cyclic nucleotides, two amino acid residues, Y699 and L701, occupy the binding pocket, forming an "intrinsic ligand." The role of the CNBhD in KCNH channel gating is still unclear, however, and a detailed characterization of the intrinsic ligand is lacking. In this study, we show that mutating both Y699 and L701 to alanine, serine, aspartate, or glycine impairs human EAG1 channel function. These mutants slow channel activation and shift the conductance-voltage (G-V) relation to more depolarized potentials. The mutations affect activation and the G-V relation progressively, indicating that the gating machinery is sensitive to multiple conformations of the CNBhD. Substitution with glycine at both sites (GG), which eliminates the side chains that interact with the binding pocket, also reduces the ability of voltage prepulses to populate more preactivated states along the activation pathway (i.e., the Cole-Moore effect), as if stabilizing the voltage sensor in deep resting states. Notably, deletion of the entire CNBhD (577-708, ΔCNBhD) phenocopies the GG mutant, suggesting that GG is a loss-of-function mutation and the CNBhD requires an intrinsic ligand to exert its functional effects. We developed a kinetic model for both wild-type and ΔCNBhD mutant channels that describes all our observations on activation kinetics, the Cole-Moore shift, and G-V relations. These findings support a model in which the CNBhD both promotes voltage sensor activation and stabilizes the open pore. The intrinsic ligand is critical for these functional effects.

Structure of a eukaryotic cyclic-nucleotide-gated channel

Cyclic-nucleotide-gated channels are essential for vision and olfaction. They belong to the voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage. Here we report a 3.5-Å-resolution single-particle electron cryo-microscopy structure of a cyclic-nucleotide-gated channel from Caenorhabditis elegans in the cyclic guanosine monophosphate (cGMP)-bound open state. The channel has an unusual voltage-sensor-like domain, accounting for its deficient voltage dependence. A carboxy-terminal linker connecting S6 and the cyclic-nucleotide-binding domain interacts directly with both the voltage-sensor-like domain and the pore domain, forming a gating ring that couples conformational changes triggered by cyclic nucleotide binding to the gate. The selectivity filter is lined by the carboxylate side chains of a functionally important glutamate and three rings of backbone carbonyls. This structure provides a new framework for understanding mechanisms of ion permeation, gating and channelopathy of cyclic-nucleotide-gated channels and cyclic nucleotide modulation of related channels.

A reduced mechanical model for cAMP-modulated gating in HCN channels

We developed an in silico mechanical model to analyze the process of cAMP-induced conformational modulations in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which conduct cations across the membrane of mammalian heart and brain cells. The structural analysis reveals a quaternary twist in the cytosolic parts of the four subunits in the channel tetramer. This motion augments the intrinsic dynamics of the very same protein structure. The pronounced differences between the cAMP bound and unbound form include a mutual interaction between the C-linker of the cyclic nucleotide binding domain (CNBD) and the linker between the S4 and S5 transmembrane domain of the channel. This allows a mechanistic annotation of the twisting motion in relation to the allosteric modulation of voltage-dependent gating of this channel by cAMP.

A Quantitative Model for cAMP Binding to the Binding Domain of MloK1

Ligand-protein binding processes are essential in biological systems. A well-studied system is the binding of cyclic adenosine monophosphate to the cyclic nucleotide binding domain of the bacterial potassium channel MloK1. Strikingly, the measured on-rate for cyclic adenosine monophosphate binding is two orders of magnitude slower than a simple Smoluchowski diffusion model would suggest. To resolve this discrepancy and to characterize the ligand-binding path in structural and energetic terms, we calculated 1100 ligand-binding molecular dynamics trajectories and tested two scenarios: In the first scenario, the ligand transiently binds to the protein surface and then diffuses along the surface into the binding site. In the second scenario, only ligands that reach the protein surface in the vicinity of the binding site proceed into the binding site. Here, a binding funnel, which increasingly confines the translational as well as the rotational degrees of freedom, determines the binding pathways and limits the on-rate. From the simulations, we identified five surface binding states and calculated the rates between these surface binding states, the binding site, and the bulk. We find that the transient binding of the ligands to the surface binding states does not affect the on-rate, such that this effect alone cannot explain the observed low on-rate. Rather, by quantifying the translational and rotational degrees of freedom and by calculating the binding committor, our simulations confirmed the existence of a binding funnel as the main bottleneck. Direct binding via the binding funnel dominates the binding kinetics, and only ∼10% of all ligands proceed via the surface into the binding site. The simulations further predict an on-rate between 15 and 40μs−1(mol/l)−1, which agrees with the measured on-rate.

Calmodulin Regulates Human Ether à Go-Go 1 (hEAG1) Potassium Channels through Interactions of the Eag Domain with the Cyclic Nucleotide Binding Homology Domain

The ether à go-go family of voltage-gated potassium channels is structurally distinct. The N terminus contains an eag domain (eagD) that contains a Per-Arnt-Sim (PAS) domain that is preceded by a conserved sequence of 25–27 amino acids known as the PAS-cap. The C terminus contains a region with homology to cyclic nucleotide binding domains (cNBHD), which is directly linked to the channel pore. The human EAG1 (hEAG1) channel is remarkably sensitive to inhibition by intracellular calcium (Ca2+i) through binding of Ca2+-calmodulin to three sites adjacent to the eagD and cNBHD. Here, we show that the eagD and cNBHD interact to modulate Ca2+-calmodulin as well as voltage-dependent gating. Sustained elevation of Ca2+i resulted in an initial profound inhibition of hEAG1 currents, which was followed by a phase when current amplitudes partially recovered, but activation gating was slowed and shifted to depolarized potentials. Deletion of either the eagD or cNBHD abolished the inhibition by Ca2+i. However, deletion of just the PAS-cap resulted in a >15-fold potentiation in response to elevated Ca2+i. Mutations of residues at the interface between the eagD and cNBHD have been linked to human cancer. Glu-600 on the cNBHD, when substituted with residues with a larger volume, resulted in hEAG1 currents that were profoundly potentiated by Ca2+i in a manner similar to the ΔPAS-cap mutant. These findings provide the first evidence that eagD and cNBHD interactions are regulating Ca2+-dependent gating and indicate that the binding of the PAS-cap with the cNBHD is required for the closure of the channels upon CaM binding.

Genetic biomarkers for the risk of seizures in long QT syndrome.

The coprevalence, severity, and biomarkers for seizures and arrhythmias in long QT syndrome (LQTS) remain incompletely understood. Using the Rochester-based LQTS Registry, this study included large cohorts of LQTS1-3 participants (LQTS+, n = 965) and those without a LQTS mutation (LQTS-, n = 936). Compared to LQTS- participants, there was a higher prevalence of LQTS1, LQTS2, and LQTS+ participants classified as having seizures (p < 0.001, i.e., history of seizures/epilepsy or antiseizure medication). LQTS+ participants with longer corrected QT interval (QTc) durations were more likely to have seizures. LQTS2 mutations in the KCNH2 pore domain were positive predictors for both arrhythmias and seizures. In contrast, mutations in the cyclic nucleotide binding domain (cNBD) of KCNH2 conferred a negative risk of seizures, but not arrhythmias. LQTS2, KCNH2-pore, KCNH2-cNBD, QTc duration, and sex were independent predictors of seizures. LQTS+ participants with seizures had significantly longer QTc durations, and a history of seizures was the strongest independent predictor of arrhythmias (hazard ratio 4.09, 95% confidence interval 2.63-6.36, p < 0.001). This study highlights potential biomarkers for neurocardiac electrical abnormalities in LQTS.

Abstract 394: Multiple Cellular Mechanisms Underlie the Trafficking-deficient Phenotype for Kv11.1 (hERG) Mutations Linked to Long QT Syndrome

Type 2 Long QT syndrome (LQT2) is commonly caused by missense mutations that disrupt the trafficking of Kv11.1 channels. The goal of this study is to determine the cellular mechanisms that underlie the trafficking-deficient phenotype for LQT2-linked mutations located in different channel domains. We expressed the pore-domain mutation G601S and the cyclic nucleotide binding domain mutation F805C in human embryonic kidney 293 (HEK293) cells or human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). HEK293 cells or hiPSC-CMs expressing G601S showed a diffuse intracellular anti-Kv11.1 staining pattern that colocalized with the transitional endoplasmic reticulum (ER) marker BAP31, but not markers for the rough ER (calnexin), ER-associated degradation compartment (derlin1), ER exit sites (Sec31), or the ER-Golgi intermediate compartment (ERGIC53). Culturing cells in the drug E-4031 (10 μM, 24 hrs), which corrects the G601s trafficking-deficient phenotype, decreased its colocalization with BAP31. Unlike G601S, the trafficking-deficient phenotype for F805C is not corrected by culturing cells in E-4031. Expressing F805C in HEK293 cells or hiPSC-CMs showed a very different anti-Kv11.1 staining pattern that consisted of several discrete aggregates. In hiPSC-CMs, the F805C aggregates partially overlapped with derlin1 staining and co-transfected green fluorescent protein (gfp) in the cytosol. Incubating cells in the proteasome inhibitor bortezomib (10 μM, 4 hrs) dispersed the F805C aggregates to generate a diffuse intracellular staining pattern. We conclude that G601S and F805C localize to distinct ER subcompartments: G601S is sequestered in the transitional ER and F805C localizes to an ER associated degradation subcompartment that is sensitive to proteasome inhibition.

Multiple Calmodulin-Binding Sites Positively and Negatively Regulate Arabidopsis CYCLIC NUCLEOTIDE-GATED CHANNEL12.

Ca(2+) signaling is critical to plant immunity; however, the channels involved are poorly characterized. Cyclic nucleotide-gated channels (CNGCs) are nonspecific, Ca(2+)-permeable cation channels. Plant CNGCs are hypothesized to be negatively regulated by the Ca(2+) sensor calmodulin (CaM), and previous work has focused on a C-terminal CaM-binding domain (CaMBD) overlapping with the cyclic nucleotide binding domain of plant CNGCs. However, we show that the Arabidopsis thaliana isoform CNGC12 possesses multiple CaMBDs at cytosolic N and C termini, which is reminiscent of animal CNGCs and unlike any plant channel studied to date. Biophysical characterizations of these sites suggest that apoCaM interacts with a conserved isoleucine-glutamine (IQ) motif in the C terminus of the channel, while Ca(2+)/CaM binds additional N- and C-terminal motifs with different affinities. Expression of CNGC12 with a nonfunctional N-terminal CaMBD constitutively induced programmed cell death, providing in planta evidence of allosteric CNGC regulation by CaM. Furthermore, we determined that CaM binding to the IQ motif was required for channel function, indicating that CaM can both positively and negatively regulate CNGC12. These data indicate a complex mode of plant CNGC regulation by CaM, in contrast to the previously proposed competitive ligand model, and suggest exciting parallels between plant and animal channels.

The arabidopsis cyclic nucleotide interactome.

BackgroundCyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals.MethodsAn affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling.ResultsA total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment.ConclusionsWe propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0133-2) contains supplementary material, which is available to authorized users.