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Previous studies have shown that xenon reduces hyperpolarization-activated cyclic nucleotide-gated channels type-2 (HCN2) channel-mediated current (Ih) amplitude and shifts the half-maximal activation voltage (V1/2) in thalamocortical circuits of acute brain slices to more hyperpolarized potentials. HCN2 channels are dually gated by the membrane voltage and via cyclic nucleotides binding to the cyclic nucleotide-binding domain (CNBD) on the channel. In this study, we hypothesize that xenon interferes with the HCN2 CNBD to mediate its effect. Using the transgenic mice model HCN2EA, in which the binding of cAMP to HCN2 was abolished by two amino acid mutations (R591E, T592A), we performed ex-vivo patch-clamp recordings and in-vivo open-field test to prove this hypothesis. Our data showed that xenon (1.9 mM) application to brain slices shifts the V1/2 of Ih to more hyperpolarized potentials in wild-type thalamocortical neurons (TC) (V1/2: −97.09 [−99.56–−95.04] mV compared to control −85.67 [−94.47–−82.10] mV; p = 0.0005). These effects were abolished in HCN2EA neurons (TC), whereby the V1/2 reached only −92.56 [−93.16– −89.68] mV with xenon compared to −90.03 [−98.99–−84.59] mV in the control (p = 0.84). After application of a xenon mixture (70% xenon, 30% O2), wild-type mice activity in the open-field test decreased to 5 [2–10] while in HCN2EA mice it remained at 30 [15–42]%, (p = 0.0006). In conclusion, we show that xenon impairs HCN2 channel function by interfering with the HCN2 CNBD site and provide in-vivo evidence that this mechanism contributes to xenon-mediated hypnotic properties.

Nuclear-associated Ca2+ oscillationsmediate plant responses to beneficial microbial partners—namely, nitrogen-fixing rhizobial bacteria that colonize roots of legumes and arbuscular mycorrhizal fungi that colonize roots of the majority of plant species. A potassium-permeable channel is known to be required for symbiotic Ca2+ oscillations, but the calcium channels themselves have been unknown until now.We show that three cyclic nucleotide–gated channels in Medicago truncatula are required for nuclear Ca2+ oscillations and subsequent symbiotic responses.These cyclic nucleotide–gated channels are located at the nuclear envelope and are permeable to Ca2+.We demonstrate that the cyclic nucleotide–gated channels form a complex with the postassium-permeable channel, which modulates nuclear Ca2+ release. These channels, like their counterparts in animal cells, might regulate multiple nuclear Ca2+ responses to developmental and environmental conditions.

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels, essential for visual and olfactory sensory transduction. Although the channels include voltage-sensor domains (VSDs), their conductance is thought to be independent of the membrane potential, and their gating regulated by cytosolic cyclic nucleotide-binding domains. Mutations in these channels result in severe, degenerative retinal diseases, which remain untreatable. The lack of structural information on CNG channels has prevented mechanistic understanding of disease-causing mutations, precluded structure-based drug design, and hampered in silico investigation of the gating mechanism. To address this, we built a 3D model of the cone tetrameric CNG channel, based on homology to two distinct templates with known structures: the transmembrane (TM) domain of a bacterial channel, and the cyclic nucleotide-binding domain of the mouse HCN2 channel. Since the TM-domain template had low sequence-similarity to the TM domains of the CNG channels, and to reconcile conflicts between the two templates, we developed a novel, hybrid approach, combining homology modeling with evolutionary coupling constraints. Next, we used elastic network analysis of the model structure to investigate global motions of the channel and to elucidate its gating mechanism. We found the following: (i) In the main mode of motion, the TM and cytosolic domains counter-rotated around the membrane normal. We related this motion to gating, a proposition that is supported by previous experimental data, and by comparison to the known gating mechanism of the bacterial KirBac channel. (ii) The VSDs could facilitate gating (supplementing the pore gate), explaining their presence in such 'voltage-insensitive' channels. (iii) Our elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently, but are coupled to each other allosterically.

Background Cyclic nucleotide-gated ion channels (CNGCs) are nonselective cation channels that are ubiquitous in eukaryotic organisms. As Ca 2+ channels, some CNGCs have also proven to be K + -permeable and involved in plant development and responses to environmental stimuli. Sugarcane is an important sugar and energy crop worldwide. However, reports on CNGC genes in sugarcane are limited. Results In this study, 16 CNGC genes and their alleles were identified from Saccharum spontaneum and classified into 5 groups based on phylogenetic analysis. Investigation of gene duplication and syntenic relationships between S . spontaneum and both rice and Arabidopsis demonstrated that the CNGC gene family in S. spontaneum expanded primarily by segmental duplication events. Many SsCNGC s showed variable expression during growth and development as well as in tissues, suggesting functional divergence. Light-responsive cis -acting elements were discovered in the promoters of all the identified SsCNGC s, and the expression of most of the SsCNGC s showed a diurnal rhythm. In sugarcane, the expression of some SsCNGC s was regulated by low-K + treatment. Notably, SsCNGC13 may be involved in both sugarcane development and its response to environmental stimuli, including response to low-K + stress. Conclusion This study identified the CNGC genes in S . spontaneum and provided insights into the transcriptional regulation of these SsCNGC s during development, circadian rhythm and under low-K + stress. These findings lay a theoretical foundation for future investigations of the CNGC gene family in sugarcane.

Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.

Memory consolidation is defined by the stabilization of a memory trace after acquisition, and consists of numerous molecular cascades that mediate synaptic plasticity. Commonly, a distinction is made between an early and a late consolidation phase, in which early refers to the first hours in which labile synaptic changes occur, whereas late consolidation relates to stable and long-lasting synaptic changes induced by de novo protein synthesis. How these phases are linked at a molecular level is not yet clear. Here we studied the interaction of the cyclic nucleotide-mediated pathways during the different phases of memory consolidation in rodents. In addition, the same pathways were studied in a model of neuronal plasticity, long-term potentiation (LTP). We demonstrated that cGMP/protein kinase G (PKG) signaling mediates early memory consolidation as well as early-phase LTP, whereas cAMP/protein kinase A (PKA) signaling mediates late consolidation and late-phase-like LTP. In addition, we show for the first time that early-phase cGMP/PKG signaling requires late-phase cAMP/PKA-signaling in both LTP and long-term memory formation.

Endogenous cyclic diadenylate phosphodiesterase activity was accidentally detected in lysates of Escherichia coli BL21. Since this kind of activity is uncommon in Gram-negative bacteria, its identification was undertaken. After partial purification and analysis by denaturing gel electrophoresis, renatured activity correlated with a protein identified by fingerprinting as CpdB (cpdB gene product), which is annotated as 3´-nucleotidase / 2´,3´-cyclic-mononucleotide phosphodiesterase, and it is synthesized as a precursor protein with a signal sequence removable upon export to the periplasm. It has never been studied as a recombinant protein. The coding sequence of mature CpdB was cloned and expressed as a GST fusion protein. The study of the purified recombinant protein, separated from GST, confirmed CpdB annotation. The assay of catalytic efficiencies (kcat/Km) for a large substrate set revealed novel CpdB features, including very high efficiencies for 3´-AMP and 2´,3´-cyclic mononucleotides, and previously unknown activities on cyclic and linear dinucleotides. The catalytic efficiencies of the latter activities, though low in relative terms when compared to the major ones, are far from negligible. Actually, they are perfectly comparable to those of the 'average' enzyme and the known, bona fide cyclic dinucleotide phosphodiesterases. On the other hand, CpdB differs from these enzymes in its extracytoplasmic location and in the absence of EAL, HD and DHH domains. Instead, it contains the domains of the 5´-nucleotidase family pertaining to the metallophosphoesterase superfamily, although CpdB lacks 5´-nucleotidase activity. The possibility that the extracytoplasmic activity of CpdB on cyclic dinucleotides could have physiological meaning is discussed.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) detects infections or tissue damage by binding to microbial or self DNA in the cytoplasm 1 . Upon binding DNA, cGAS produces cGAMP that binds to and activates the adaptor protein STING, which then activates the kinases IKK and TBK1 to induce interferons and other cytokines 2 – 6 . Here we report that STING also activates autophagy through a mechanism that is independent of TBK1 activation and interferon induction. Upon binding cGAMP, STING translocates to the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) and the Golgi in a process that is dependent on the COP-II complex and ARF GTPases. STING-containing ERGIC serves as a membrane source for LC3 lipidation, which is a key step in autophagosome biogenesis. cGAMP induced LC3 lipidation through a pathway that is dependent on WIPI2 and ATG5 but independent of the ULK and VPS34–beclin kinase complexes. Furthermore, we show that cGAMP-induced autophagy is important for the clearance of DNA and viruses in the cytosol. Interestingly, STING from the sea anemone Nematostella vectensis induces autophagy but not interferons in response to stimulation by cGAMP, which suggests that induction of autophagy is a primordial function of the cGAS–STING pathway. The authors report that the cGAS–STING pathway drives a form of autophagy that is independent of interferon induction and distinct from the conventional autophagy.

The cGAS–STING signalling pathway has emerged as a key mediator of inflammation in the settings of infection, cellular stress and tissue damage. Underlying this broad involvement of the cGAS–STING pathway is its capacity to sense and regulate the cellular response towards microbial and host-derived DNAs, which serve as ubiquitous danger-associated molecules. Insights into the structural and molecular biology of the cGAS–STING pathway have enabled the development of selective small-molecule inhibitors with the potential to target the cGAS–STING axis in a number of inflammatory diseases in humans. Here, we outline the principal elements of the cGAS–STING signalling cascade and discuss the general mechanisms underlying the association of cGAS–STING activity with various autoinflammatory, autoimmune and degenerative diseases. Finally, we outline the chemical nature of recently developed cGAS and STING antagonists and summarize their potential clinical applications.The cGAS–STING pathway drives innate immune activation in response to cytosolic DNA. This is important for immunity to bacteria and viruses, but aberrant cGAS–STING activity is also linked to inflammatory disease. Here, Ablasser and colleagues discuss how cGAS–STING signalling contributes to various autoimmune, inflammatory and degenerative diseases and describe the novel therapeutics targeting this pathway.

Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in non-native lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. Kinetic modeling suggests that binding of the second ligand is either independent of the first ligand or exhibits up to ~10-fold positive binding cooperativity. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane. Here the authors use nanovesicles, microfluidics and single-molecule methods to observe individual ligand binding events that stimulate activation of CNG ion channels. They find that binding is followed by a conformational change of either independent or weakly cooperative binding domains.