Ciliary defects are linked to ciliopathies, but impairments in the sensory cilia of Caenorhabditis elegans neurons extend lifespan, a phenomenon with previously unclear mechanisms. Our study reveals that neuronal cilia defects trigger the unfolded protein response of the endoplasmic reticulum (UPRER) within intestinal cells, a process dependent on the insulin/insulin-like growth factor 1 (IGF-1) signaling transcription factor and the release of neuronal signaling molecules. While inhibiting UPRER doesn\'t alter the lifespan of wild-type worms, it normalizes the extended lifespan of ciliary mutants. Notably, deactivating the cyclic nucleotide-gated (CNG) channel TAX-4 on the ciliary membrane promotes lifespan extension through a UPRER-dependent mechanism. Conversely, constitutive activation of TAX-4 attenuates intestinal UPRER in ciliary mutants. Administering a CNG channel blocker to worm larvae activates intestinal UPRER and increases adult longevity. These findings suggest that ciliary dysfunction in sensory neurons triggers intestinal UPRER, contributing to lifespan extension and implying that transiently inhibiting ciliary channel activity may effectively prolong lifespan.

In treating retinitis pigmentosa, a genetic disorder causing progressive vision loss, selective inhibition of rod cyclic nucleotide-gated (CNG) channels holds promise. Blocking the increased Ca2+-influx in rod photoreceptors through CNG channels can potentially delay disease progression and improve the quality of life for patients. To find inhibitors for rod CNG channels, we investigated the impact of 16 cGMP analogues on both rod and cone CNG channels using the patch-clamp technique. Although modifications at the C8 position of the guanine ring did not change the ligand efficacy, modifications at the N1 and N2 positions rendered cGMP largely ineffective in activating retinal CNG channels. Notably, PET-cGMP displayed selective potential, favoring rod over cone, whereas Rp-cGMPS showed greater efficiency in activating cone over rod CNG channels. Ligand docking and molecular dynamics simulations on cyclic nucleotide-binding domains showed comparable binding energies and binding modes for cGMP and its analogues in both rod and cone CNG channels (CNGA1 vs CNGA3 subunits). Computational experiments on CNGB1a vs CNGB3 subunits showed similar binding modes albeit with fewer amino acid interactions with cGMP due to an inactivated conformation of their C-helix. In addition, no clear correlation could be observed between the computational scores and the CNG channel efficacy values, suggesting additional factors beyond binding strength determining ligand selectivity and potency. This study highlights the importance of looking beyond the cyclic nucleotide-binding domain and toward the gating mechanism when searching for selective modulators. Future efforts in developing selective modulators for CNG channels should prioritize targeting alternative channel domains.

Cyclic AMP controls neuronal ion channel activity. For example hyperpolarization-activated cyclic nucleotide-gated (HCN) and M-type K+ channels are activated by cAMP. These effects have been suggested to be involved in astrocyte control of neuronal activity, for example, by controlling the action potential firing frequency. In cortical neurons, cAMP can induce mixed-mode oscillations (MMOs) consisting of small-amplitude, subthreshold oscillations separating complete action potentials, which lowers the firing frequency greatly. We extend a model of neuronal activity by including HCN and M channels, and show that it can reproduce a series of experimental results under various conditions involving and inferring with cAMP-induced activation of HCN and M channels. In particular, we find that the model can exhibit MMOs as found experimentally, and argue that both HCN and M channels are crucial for reproducing these patterns. To understand how M and HCN channels contribute to produce MMOs, we exploit the fact that the model is a three-time scale dynamical system with one fast, two slow, and two super-slow variables. We show that the MMO mechanism does not rely on the super-slow dynamics of HCN and M channel gating variables, since the model is able to produce MMOs even when HCN and M channel activity is kept constant. In other words, the cAMP-induced increase in the average activity of HCN and M channels allows MMOs to be produced by the slow-fast subsystem alone. We show that the slow-fast subsystem MMOs are due to a folded node singularity, a geometrical structure well known to be involved in the generation of MMOs in slow-fast systems. Besides raising new mathematical questions for multiple-timescale systems, our work is a starting point for future research on how cAMP signalling, for example resulting from interactions between neurons and glial cells, affects neuronal activity via HCN and M channels.

UNASSIGNED: This study aimed to investigate the role of the long non-coding RNA (lncRNA) NEAT1 in corneal epithelial wound healing in mice.
UNASSIGNED: The central corneal epithelium of wild-type (WT), MALAT1 knockout (M-KO), NEAT1 knockout (N-KO), and NEAT1 knockdown (N-KD) mice was scraped to evaluate corneal epithelial and nerve regeneration rates. RNA sequencing of the corneal epithelium from WT and N-KO mice was performed 24 hours after debridement to determine the role of NEAT1. Quantitative PCR (qPCR) and ELISA were used to confirm the bioinformatic analysis. The effects of the cAMP signaling pathway were evaluated in N-KO and N-KD mice using SQ22536, an adenylate cyclase inhibitor.
UNASSIGNED: Central corneal epithelial debridement in N-KO mice significantly promoted epithelial and nerve regeneration rates while suppressing inflammatory cell infiltration. Furthermore, the expression of Atp1a2, Ppp1r1b, Calm4, and Cngb1, which are key components of the cAMP signaling pathway, was upregulated in N-KO mice, indicative of its activation. Furthermore, the cAMP pathway inhibitor SQ22536 reversed the accelerated corneal epithelial wound healing in both N-KO and N-KD mice.
UNASSIGNED: NEAT1 deficiency contributes to epithelial repair during corneal wound healing by activating the cAMP signaling pathway, thereby highlighting a potential therapeutic strategy for corneal epithelial diseases.

Multiple cyclic nucleotide-gated channels (CNGCs) are abscisic acid (ABA)-activated Ca2+ channels in Arabidopsis (Arabidopsis thaliana) guard cells. In particular, CNGC5, CNGC6, CNGC9, and CNGC12 are essential for ABA-specific cytosolic Ca2+ signaling and stomatal movements. However, the mechanisms underlying ABA-mediated regulation of CNGCs and Ca2+ signaling are still unknown. In this study, we identified the Ca2+-independent protein kinase OPEN STOMATA 1 (OST1) as a CNGC activator in Arabidopsis. OST1-targeted phosphorylation sites were identified in CNGC5, CNGC6, CNGC9, and CNGC12. These CNGCs were strongly inhibited by Ser-to-Ala mutations and fully activated by Ser-to-Asp mutations at the OST1-targeted sites. The overexpression of individual inactive CNGCs (iCNGCs) under the UBIQUITIN10 promoter in wild-type Arabidopsis conferred a strong dominant-negative-like ABA-insensitive stomatal closure phenotype. In contrast, expressing active CNGCs (aCNGCs) under their respective native promoters in the cngc5-1 cngc6-2 cngc9-1 cngc12-1 quadruple mutant fully restored ABA-activated cytosolic Ca2+ oscillations and Ca2+ currents in guard cells, and rescued the ABA-insensitive stomatal movement mutant phenotypes. Thus, we uncovered that ABA elicits cytosolic Ca2+ signaling via an OST1-CNGC module, in which OST1 functions as a convergence point of the Ca2+-dependent and -independent pathways in Arabidopsis guard cells.

The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.

Cyclic nucleotide-gated ion channels (CNGCs) remain poorly studied in crop plants, most of which are polyploid. In allotetraploid Upland cotton (Gossypium hirsutum), silencing GhCNGC13 and 32 impaired plant growth and shoot apical meristem (SAM) development, while triggering plant autoimmunity. Both growth hormones (indole-3-acetic acid and gibberellin) and stress hormones (abscisic acid, salicylic acid, and jasmonate) increased, while leaf photosynthesis decreased. The silenced plants exhibited an enhanced resistance to Botrytis cinerea; however, Verticillium wilt resistance was weakened, which was associated with LIPOXYGENASE2 (LOX2) downregulation. Transcriptomic analysis of silenced plants revealed 4835 differentially expressed genes (DEGs) with functional enrichment in immunity and photosynthesis. These DEGs included a set of transcription factors with significant over-representation in the HSF, NAC, and WRKY families. Moreover, numerous members of the GhCNGC family were identified among the DEGs, which may indicate a coordinated action. Collectively, our results suggested that GhCNGC13 and 32 functionally link to photosynthesis, plant growth, and plant immunity. We proposed that GhCNGC13 and 32 play a critical role in the \"growth-defense tradeoff\" widely observed in crops.

An estimated 1 in 10 000 people are born without the ability to smell, a condition known as congenital anosmia, and about one third of those people have non-syndromic, or isolated congenital anosmia (ICA). Despite the significant impact of olfaction for our quality of life, the underlying causes of ICA remain largely unknown. Using whole exome sequencing (WES) in 10 families and 141 individuals with ICA, we identified a candidate list of 162 rare, segregating, deleterious variants in 158 genes. We confirmed the involvement of CNGA2, a previously implicated ICA gene that is an essential component of the olfactory transduction pathway. Furthermore, we found a loss-of-function variant in SREK1IP1 from the family gene candidate list, which was also observed in 5% of individuals in an additional non-family cohort with ICA. Although SREK1IP1 has not been previously associated with olfaction, its role in zinc ion binding suggests a potential influence on olfactory signaling. This study provides a more comprehensive understanding of the spectrum of genetic alterations and their etiology in ICA patients, which may improve the diagnosis, prognosis, and treatment of this disorder and lead to better understanding of the mechanisms governing basic olfactory function.

In a family with inappropriate sinus tachycardia (IST), we identified a mutation (p.V240M) of the hyperpolarization-activated cyclic nucleotide-gated type 4 (HCN4) channel, which contributes to the pacemaker current (If) in human sinoatrial node cells. Here, we clinically study fifteen family members and functionally analyze the p.V240M variant. Macroscopic (IHCN4) and single-channel currents were recorded using patch-clamp in cells expressing human native (WT) and/or p.V240M HCN4 channels. All p.V240M mutation carriers exhibited IST that was accompanied by cardiomyopathy in adults. IHCN4 generated by p.V240M channels either alone or in combination with WT was significantly greater than that generated by WT channels alone. The variant, which lies in the N-terminal HCN domain, increased the single-channel conductance and opening frequency and probability of HCN4 channels. Conversely, it did not modify the channel sensitivity for cAMP and ivabradine or the level of expression at the membrane. Treatment with ivabradine based on functional data reversed the IST and the cardiomyopathy of the carriers. In computer simulations, the p.V240M gain-of-function variant increases If and beating rate and thus explains the IST of the carriers. The results demonstrate the importance of the unique HCN domain in HCN4, which stabilizes the channels in the closed state.