Activation Of Anion Channels Research Articles

Activation of osmo-sensitive LRRC8 anion channels in macrophages is important for micro-crystallin joint inflammation

Deposition of monosodium urate and calcium pyrophosphate (MSU and CPP) micro-crystals is responsible for painful and recurrent inflammation flares in gout and chondrocalcinosis. In these pathologies, the inflammatory reactions are due to the activation of macrophages responsible for releasing various cytokines including IL-1β. The maturation of IL-1β is mediated by the multiprotein NLRP3 inflammasome. Here, we find that activation of the NLRP3 inflammasome by crystals and concomitant production of IL-1β depend on cell volume regulation via activation of the osmo-sensitive LRRC8 anion channels. Both pharmacological inhibition and genetic silencing of LRRC8 abolish NLRP3 inflammasome activation by crystals in vitro and in mouse models of crystal-induced inflammation. Activation of LRRC8 upon MSU/CPP crystal exposure induces ATP release, P2Y receptor activation and intracellular calcium increase necessary for NLRP3 inflammasome activation and IL-1β maturation. We identify a function of the LRRC8 osmo-sensitive anion channels with pathophysiological relevance in the context of joint crystal-induced inflammation.

Generation of Astrocyte-specific BEST1 Conditional Knockout Mouse with Reduced Tonic GABA Inhibition in the Brain.

Bestrophin-1 (BEST1) is a Ca2+-activated anion channel known for its role in astrocytes. Best1 is permeable to gliotransmitters, including GABA, to contribute to tonic GABA inhibition and modulate synaptic transmission in neighboring neurons. Despite the crucial functions of astrocytic BEST1, there is an absence of genetically engineered cell-type specific conditional mouse models addressing these roles. In this study, we developed an astrocyte-specific BEST1 conditional knock-out (BEST1 aKO) mouse line. Using the embryonic stem cell (ES cell) targeting method, we developed Best1 floxed mice (C57BL/6JCya-Best1em1flox/Cya), which have exon 3, 4, 5, and 6 of Best1 flanked by two loxP sites. By crossing with hGFAP-CreERT2 mice, we generated Best1 floxed/hGFAP-CreERT2 mice, which allowed for the tamoxifen-inducible deletion of Best1 under the human GFAP promoter. We characterized its features across various brain regions, including the striatum, hippocampal dentate gyrus (HpDG), and Parafascicular thalamic nucleus (Pf). Compared to the Cre-negative control, we observed significantly reduced BEST1 protein expression in immunohistochemistry (IHC) and tonic GABA inhibition in patch clamp recordings. The reduction in tonic GABA inhibition was 66.7% in the striatum, 46.4% in the HpDG, and 49.6% in the Pf. Our findings demonstrate that the BEST1 channel in astrocytes significantly contributes to tonic inhibition in the local brain areas. These mice will be valuable for future studies not only on tonic GABA release but also on tonic release of gliotransmitters mediated by astrocytic BEST1.

Unravelling the mechanisms of PFAS toxicity to submerged macrophytes and epiphytic biofilms at metabolic and molecular levels

Per- and poly-fluoroalkyl substances (PFAS) are an emerging class of persistent organic pollutants that are widespread in aquatic ecosystems and pose a serious threat to aquatic organisms. It is thus crucial to explore the toxicity mechanisms of PFAS to submerged macrophytes and biofilms. In this study, Vallisneria natans (V. natans) was exposed to environmentally relevant concentrations of perfluorooctanoic acid (PFOA) and perfluorooctane sulphonate (PFOS). Results showed that PFAS induced the excessive production of reactive oxygen species, triggering antioxidant responses. V. natans exhibited an improved stress tolerance by altering the biosynthesis of several plant secondary metabolites and the histidine, arginine, proline pathways in response to PFAS exposure. Moreover, PIP1–1, PIP2–2, SLAH1 and SLAH2 genes were upregulated, indicating the activation of aquaporins and slow-type anion channels. The uptake of PFOA and PFOS by V. natans was 41.74 % and 52.31 %, respectively. Notably, PFAS bound to functional proteins (GSTF10), promoting the detoxification of plants. Exposure to PFAS also altered the structure of biofilms by inducing the synthesis of large amounts of polysaccharides and proteins. The diversity and richness of the microbial community within periphytic biofilms changed significantly. These results provide a comprehensive description of the responses of aquatic plants and periphytic biofilms to PFAS and the removal mechanism of PFAS, contributing to the environmental risk assessments and removal of PFAS in aquatic ecosystems.

Impact of CFTR Correctors on the Channel Activity of Rescued F508del CFTR in Airway Epithelial Cells

Cystic fibrosis (CF) is caused by the functional deficiency of a cAMP-activated anion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). Over 90% of CF patients carry at least one copy of the F508del CFTR gene. The F508del mutation causes a global conformational change that prevents the nascent protein from exiting the endoplasmic reticulum, leading to loss of function at the cell surface. CFTR correctors have been developed to bind to the nascent F508del CFTR, change its conformation, and mobilize it to the plasma membrane. Rescued F508del CFTR (rF508del) does not fully regain channel activity. Lumacaftor, the first CFTR corrector approved by the FDA, acutely inhibits rF508del channel opening (Ambrosetti et al. Pediatric Pulmonology 54(S2): 190-191). Our objective is to evaluate the impact of the other two FDA-approved CFTR correctors, tezacaftor and elexacaftor, on rF508del channel activity. We hypothesize that different CFTR correctors impact rF508del channel activity differently. We utilized an electrode-based, real-time iodide efflux assay to evaluate the acute impact of CFTR correctors on the cAMP-dependent anion channel activity of rF508del in a CF bronchial epithelial cell line stably expressing F508del CFTR (CFBE-DF) at 37°C. CFBE-DF cells were incubated with a corrector for over 24 hours before iodide efflux assay was performed in the acute presence or absence of the same corrector. Genistein was used as a potentiator. F508del processing was assessed by quantitative immunoblotting. The lack of acute exposure to 5μM lumacaftor results in a 2% increase in the mean F508del processing (n=3, p=0.94) and a 48% increase in channel activity (n=3, p=0.04). F508del processing peaks at 1μM lumacaftor but the channel activity peaks at a much lower 0.05μM lumacaftor. In contrast, the lack of acute exposure to 5μM tezacaftor results in an 8% reduction in the mean channel activity (n>3, p=0.79) and a 17% reduction in the mean F508del processing (n>3, p=0.43). Both F508del processing and channel activity peak at 5μM tezacaftor. The lack of acute exposure to 2μM elexacaftor results in an 11% decrease in the mean F508del channel activity (n=4. p=0.54). F508del channel activity peaks at 2μM elexacaftor. We conclude that, unlike lumacaftor, tezacaftor and elexacaftor do not inhibit the channel activity of rescued F508del CFTR in CFBE-DF cells. A better understanding of the impact of CFTR correctors on the channel activity of rescued CFTR processing mutants will inform the development of highly effcacious CFTR modulator combinations for the treatment of CF patients. This work is supported by NIH (5P30DK072482), Samford University, and Cystic Fibrosis Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Anion Channel TMEM16a/Ano1 Modulates CFTR Activity, but Does Not Function as an Apical Anion Channel in Colonic Epithelium from Cystic Fibrosis Patients and Healthy Individuals.

Studies in human colonic cell lines and murine intestine suggest the presence of a Ca2+-activated anion channel, presumably TMEM16a. Is there a potential for fluid secretion in patients with severe cystic fibrosis transmembrane conductance regulator (CFTR) mutations by activating this alternative pathway? Two-dimensional nondifferentiated colonoid-myofibroblast cocultures resembling transit amplifying/progenitor (TA/PE) cells, as well as differentiated monolayer (DM) cultures resembling near-surface cells, were established from both healthy controls (HLs) and patients with severe functional defects in the CFTR gene (PwCF). F508del mutant and CFTR knockout (null) mice ileal and colonic mucosa was also studied. HL TA/PE monolayers displayed a robust short-circuit current response (ΔIeq) to UTP (100 µM), forskolin (Fsk, 10 µM) and carbachol (CCH, 100 µM), while ΔIeq was much smaller in differentiated monolayers. The selective TMEM16a inhibitor Ani9 (up to 30 µM) did not alter the response to luminal UTP, significantly decreased Fsk-induced ΔIeq, and significantly increased CCH-induced ΔIeq in HL TA/PE colonoid monolayers. The PwCF TA/PE and the PwCF differentiated monolayers displayed negligible agonist-induced ΔIeq, without a significant effect of Ani9. When TMEM16a was localized in intracellular structures, a staining in the apical membrane was not detected. TMEM16a is highly expressed in human colonoid monolayers resembling transit amplifying cells of the colonic cryptal neck zone, from both HL and PwCF. While it may play a role in modulating agonist-induced CFTR-mediated anion currents, it is not localized in the apical membrane, and it has no function as an apical anion channel in cystic fibrosis (CF) and healthy human colonic epithelium.

Use of adenine base editing and homology-independent targeted integration strategies to correct the cystic fibrosis causing variant, W1282X.

Small molecule drugs known as modulators can treat ~ 90% of people with cystic fibrosis (CF), but do not work for premature termination codon variants such as W1282X (c.3846G > A). Here we evaluated two gene editing strategies, Adenine Base Editing (ABE) to correct W1282X, and Homology-Independent Targeted Integration (HITI) of a CFTR superexon comprising exons 23-27 (SE23-27) to enable expression of a CFTR mRNA without W1282X. In Flp-In-293 cells stably expressing a CFTR expression minigene bearing W1282X, ABE corrected 24% of W1282X alleles, rescued CFTR mRNA from nonsense mediated decay and restored protein expression. However, bystander editing at the adjacent adenine (c.3847A > G), caused an amino acid change (R1283G) that affects CFTR maturation and ablates ion channel activity. In primary human nasal epithelial cells homozygous for W1282X, ABE corrected 27% of alleles, but with a notably lower level of bystander editing, and CFTR channel function was restored to 16% of wild-type levels. Using the HITI approach, correct integration of a SE23-27 in intron 22 of the CFTR locus in 16HBEge W1282X cells was detected in 5.8% of alleles, resulting in 7.8% of CFTR transcripts containing the SE23-27 sequence. Analysis of a clonal line homozygous for the HITI-SE23-27 produced full-length mature protein and restored CFTR anion channel activity to 10% of wild-type levels, which could be increased three-fold upon treatment with the triple combination of CF modulators. Overall, these data demonstrate two different editing strategies can successfully correct W1282X, the second most common class I variant, with a concomitant restoration of CFTR function.

Apo state pore opening as functional basis of increased EAAT anion channel activity in episodic ataxia 6.

SLC1A2 and SLC1A3 encode the glial glutamate transporters EAAT2 and EAAT1, which are not only the predominant glutamate uptake carriers in our brain, but also function as anion channels. Two homologous mutations, which predict substitutions of prolines in the center of the fifth transmembrane helix by arginine (P289R EAAT2, P290R EAAT1), have been identified in patients with epileptic encephalopathy (SLC1A2) or with episodic ataxia type 6 (SLC1A3). Both mutations have been shown to impair glutamate uptake and to increase anion conduction. The molecular processes that link the disease-causing mutations to two major alterations of glutamate transporter function remain insufficiently understood. The mutated proline is conserved in every EAAT. Since the pathogenic changes mainly affect the anion channel function, we here study the functional consequences of the homologous P312R mutation in the neuronal glutamate transporter EAAT4, a low capacity glutamate transporter with predominant anion channel function. To assess the impact of charge and structure of the inserted amino acid for the observed functional changes, we generated and functionally evaluated not only P312R, but also substitutions of P312 with all other amino acids. However, only exchange of proline by arginine, lysine, histidine and asparagine were functionally tolerated. We compared WT, P312R and P312N EAAT4 using a combination of cellular electrophysiology, fast substrate application and kinetic modelling. We found that WT and mutant EAAT4 anion currents can be described with a 11-state model of the transport cycle, in which several states are connected to branching anion channel states to account for the EAAT anion channel function. Substitutions of P312 modify various transitions describing substrate binding/unbinding, translocation or anion channel opening. Most importantly, P312R generates a new anion conducting state that is accessible in the outward facing apo state and that is the main determinant of the increased anion conduction of EAAT transporters carrying this mutation. Our work provides a quantitative description how a naturally occurring mutation changes glutamate uptake and anion currents in two genetic diseases.

Roles of the RON3 C-terminal fragment in erythrocyte invasion and blood-stage parasite proliferation in Plasmodium falciparum.

Plasmodium species cause malaria, and in the instance of Plasmodium falciparum is responsible for a societal burden of over 600,000 deaths annually. The symptoms and pathology of malaria are due to intraerythocytic parasites. Erythrocyte invasion is mediated by the parasite merozoite stage, and is accompanied by the formation of a parasitophorous vacuolar membrane (PVM), within which the parasite develops. The merozoite apical rhoptry organelle contains various proteins that contribute to erythrocyte attachment and invasion. RON3, a rhoptry bulb membrane protein, undergoes protein processing and is discharged into the PVM during invasion. RON3-deficient parasites fail to develop beyond the intraerythrocytic ring stage, and protein export into erythrocytes by the Plasmodium translocon of exported proteins (PTEX) apparatus is abrogated, as well as glucose uptake into parasites. It is known that truncated N- and C-terminal RON3 fragments are present in rhoptries, but it is unclear which RON3 fragments contribute to protein export by PTEX and glucose uptake through the PVM. To investigate and distinguish the roles of the RON3 C-terminal fragment at distinct developmental stages, we used a C-terminus tag for conditional and post-translational control. We demonstrated that RON3 is essential for blood-stage parasite survival, and knockdown of RON3 C-terminal fragment expression from the early schizont stage induces a defect in erythrocyte invasion and the subsequent development of ring stage parasites. Protein processing of full-length RON3 was partially inhibited in the schizont stage, and the RON3 C-terminal fragment was abolished in subsequent ring-stage parasites compared to the RON3 N-terminal fragment. Protein export and glucose uptake were abrogated specifically in the late ring stage. Plasmodial surface anion channel (PSAC) activity was partially retained, facilitating small molecule traffic across the erythrocyte membrane. The knockdown of the RON3 C-terminal fragment after erythrocyte invasion did not alter parasite growth. These data suggest that the RON3 C-terminal fragment participates in erythrocyte invasion and serves an essential role in the progression of ring-stage parasite growth by the establishment of the nutrient-permeable channel in the PVM, accompanying the transport of ring-stage parasite protein from the plasma membrane to the PVM.

High resolution cryo-EM structures of TRPC5-Gαi3 complexes reveal direct activation of an ion channel by Gαi-GTP

Transient receptor potential canonical 4 and 5 (TRPC4 and TRPC5) are Ca2+-permeable nonselective cation channels known to be activated by Gi/o proteins. Recently, Won et al. (Nat Commun. 2023, 14:2550) reported the cryo-EM structures of TRPC5 in complex with Gαi3. The G protein alpha subunit was found to directly bind to an ankyrin-like repeat domain in the periphery of the cytosolic portion of TRPC5 some 50 Å away from the membrane. This establishes the TRPC4/C5 ion channels as true effectors of Gα subunits, although the channel gating still depends on the coexistence of Ca2+ and phosphatidylinositol 4,5-bisphosphate.

Identification of Inflammatory Gene in the Congenital Heart Surgery Patients following Cardiopulmonary Bypass via the Way of WGCNA and Machine Learning Algorithms.

Performing cardiopulmonary bypass (CPB) to reduce ischemic injury during surgery is a common approach to cardiac surgery. However, this procedure can lead to systemic inflammation and multiorgan dysfunction. Therefore, elucidating the molecular mechanisms of CPB-induced inflammatory cytokine release is essential as a critical first step in identifying new targets for therapeutic intervention. The GSE143780 dataset which is mRNA sequencing from total circulating leukocytes of the neonatorum was downloaded from the Gene Expression Omnibus (GEO) database. A total of 21 key module genes were obtained by analyzing the intersection of differentially expressed gene (DEG) and gene coexpression network analysis (WGCNA), and then, 4 genes (TRAF3IP2-AS1, PPARGC1B, CD4, and PDLIM5) were further confirmed after the least absolute shrinkage and selection operator (LASSO) and support vector machine recursive feature elimination (SVM-RFE) screening and were used as hub genes for CPB-induced inflammatory cytokine release in patients with congenital heart defects. The enrichment analysis revealed 21 key module genes mainly related to the functions of developmental cell growth, regulation of monocyte differentiation, regulation of myeloid leukocyte differentiation, ERK1 and ERK2 cascade, volume-sensitive anion channel activity, and estrogen receptor binding. The result of gene set enrichment analysis (GSEA) showed that the hub genes were related to different physiological functions of cells. The ceRNA network established for hub genes includes 3 hub genes (PPARGC1B, CD4, and PDLIM5), 55 lncRNAs, and 34 miRNAs. In addition, 4 hub genes have 215 potential therapeutic agents. Finally, expression validation of the four hub genes revealed that they were all significantly low expressed in the surgical samples than before.

Identification of LRRC8/VRAC channel structural elements required for regulation by cell volume increase and intracellular ionic strength.

VRACs mediate volume regulatory Cl- and organic solute efflux and are encoded by Lrrc8a-e genes. Native LRRC8/VRACs are heteromers with unknown stoichiometry comprising the essential LRRC8A subunit and another paralog. We utilize a chimeric protein approach to define LRRC8/VRAC structure-function relationships. Functional chimeric channels with physiologically relevant properties require the presence of the first intracellular loop (IL1) of LRRC8A and the first extracellular loop of another paralog. Replacement of the C-terminus of LRRC8C or LRRC8E with the C-terminus of LRRC8A (8A(C-term)) gives rise to robust plasma membrane anion channel activity. 8C-8A(C-term) and 8E-8A(C-term) chimeric channels exhibit greatly reduced sensitivity to reduced intracellular ionic strength (IS) and cannot be activated by hypotonicity-induced cell volume increases of up to 4-fold. However, both channels could be activated by rapid, stepwise volume increases of 2-2.5-fold induced by positive pressure applied to the patch pipette. Addition of the LRRC8A IL1 to both chimeras restored normal sensitivity to hypotonicity-induced volume increases and IS. A cryo-EM structure of a LRRC8 chimeric channel comprising LRRC8C with 25 amino acids unique to the LRRC8A IL1, 8C-8A(IL125), suggested a possible functional interaction between 8A(IL125) and leucine-rich repeat motif 5, LRR5. Deletion of 9 cytoplasm-facing LRR5 amino acids or substitution with alanine greatly reduced volume and IS sensitivity of 8C-8A(IL125) channels. Point mutations in the 8A(IL125) region identified two unique lysine residues required for channel regulation. We conclude that the LRRC8 C-terminus is 1) required for channel volume and IS sensing, 2) that channel regulation requires a functional interaction between the unique 8A(IL125) region and LRR5 and 3) that LRR5 and the 8A(IL125) region are key components of the LRRC8/VRAC volume and IS sensing machinery.

The CIPK23 protein kinase represses SLAC1-type anion channels in Arabidopsis guard cells and stimulates stomatal opening.

Guard cells control the opening of stomatal pores in the leaf surface, with the use of a network of protein kinases and phosphatases. Loss of function of the CBL-interacting protein kinase 23 (CIPK23) was previously shown to decrease the stomatal conductance, but the molecular mechanisms underlying this response still need to be clarified. CIPK23 was specifically expressed in Arabidopsis guard cells, using an estrogen-inducible system. Stomatal movements were linked to changes in ion channel activity, determined with double-barreled intracellular electrodes in guard cells and with the two-electrode voltage clamp technique in Xenopus oocytes. Expression of the phosphomimetic variant CIPK23T190D enhanced stomatal opening, while the natural CIPK23 and a kinase-inactive CIPK23K60N variant did not affect stomatal movements. Overexpression of CIPK23T190D repressed the activity of S-type anion channels, while their steady-state activity was unchanged by CIPK23 and CIPK23K60N . We suggest that CIPK23 enhances the stomatal conductance at favorable growth conditions, via the regulation of several ion transport proteins in guard cells. The inhibition of SLAC1-type anion channels is an important facet of this response.

Calcium Channels, OST1 and Stomatal Defence: Current Status and Beyond

Stomatal immunity is regulated by pathogen-associated molecular patterns (PAMPs)- and abscisic acid (ABA)-triggered signalling in different ways. Cytoplasmic Ca2+ signature in the guard cells plays a vital function in stomatal immunity, but the mechanism of Ca2+ import is unknown. It has been very recently established that the hyperosmolality-gated calcium-permeable channels (OSCAs) and cyclic nucleotide-gated channels (CNGCs) are responsible for the influx of Ca2+ in the cytoplasm, which are activated after BIK1-mediated phosphorylation and ABA interaction during PAMPs- and ABA-triggered stomatal immunity in plants, respectively. Further, ABA-triggered OPEN STOMATA1 (OST1) causes the disassembly of microtubules in the guard cells besides activation of S-type anion channels (SLAC1) for the efflux of cytoplasmic anions that leads to stomata closure.

Impaired Bestrophin Channel Activity in an iPSC-RPE Model of Best Vitelliform Macular Dystrophy (BVMD) from an Early Onset Patient Carrying the P77S Dominant Mutation.

Best Vitelliform Macular dystrophy (BVMD) is the most prevalent of the distinctive retinal dystrophies caused by mutations in the BEST1 gene. This gene, which encodes for a homopentameric calcium-activated ion channel, is crucial for the homeostasis and function of the retinal pigment epithelia (RPE), the cell type responsible for recycling the visual pigments generated by photoreceptor cells. In BVMD patients, mutations in this gene induce functional problems in the RPE cell layer with an accumulation of lipofucsin that evolves into cell death and loss of sight. In this work, we employ iPSC-RPE cells derived from a patient with the p.Pro77Ser dominant mutation to determine the correlation between this variant and the ocular phenotype. To this purpose, gene and protein expression and localization are evaluated in iPSC-RPE cells along with functional assays like phagocytosis and anion channel activity. Our cell model shows no differences in gene expression, protein expression/localization, or phagocytosis capacity, but presents an increased chloride entrance, indicating that the p.Pro77Ser variant might be a gain-of-function mutation. We hypothesize that this variant disturbs the neck region of the BEST1 channel, affecting channel function but maintaining cell homeostasis in the short term. This data shed new light on the different phenotypes of dominant mutations in BEST1, and emphasize the importance of understanding its molecular mechanisms. Furthermore, the data widen the knowledge of this pathology and open the door for a better diagnosis and prognosis of the disease.

Phytocytokine signalling reopens stomata in plant immunity and water loss.

Stomata exert considerable effects on global carbon and water cycles by mediating gas exchange and water vapour1,2. Stomatal closure prevents water loss in response to dehydration and limits pathogen entry3,4. However, prolonged stomatal closure reduces photosynthesis and transpiration and creates aqueous apoplasts that promote colonization by pathogens. How plants dynamically regulate stomatal reopening in a changing climate is unclear. Here we show that the secreted peptides SMALL PHYTOCYTOKINES REGULATING DEFENSE AND WATER LOSS (SCREWs) and the cognate receptor kinase PLANT SCREW UNRESPONSIVE RECEPTOR (NUT) counter-regulate phytohormone abscisic acid (ABA)- and microbe-associated molecular pattern (MAMP)-induced stomatal closure. SCREWs sensed by NUT function as immunomodulatory phytocytokines and recruit SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors to relay immune signalling. SCREWs trigger the NUT-dependent phosphorylation of ABA INSENSITIVE 1 (ABI1) and ABI2, which leads to an increase in the activity of ABI phosphatases towards OPEN STOMATA 1 (OST1)-a key kinase that mediates ABA- and MAMP-induced stomatal closure5,6-and a reduction in the activity of S-type anion channels. After induction by dehydration and pathogen infection, SCREW-NUT signalling promotes apoplastic water loss and disrupts microorganism-rich aqueous habitats to limit pathogen colonization. The SCREW-NUT system is widely distributed across land plants, which suggests that it has an important role in preventing uncontrolled stomatal closure caused by abiotic and biotic stresses to optimize plant fitness.

Select Amino Acid Formulation Influenced Anion Secretion Induced by Th2 Cytokines in Human Bronchial Epithelial Cells (HBECs)

IntroductionAirway surface liquid is tightly regulated by ion channels and/or transporters, and plays a critical role in inflammatory airway diseases, such as ARDS, and in edema fluid clearance during lung injury. Although beta‐adrenergic agonists have been shown to improve alveolar fluid clearance, they have limited effect in late stages of ARDS due to reduced epithelial beta‐adrenergic receptor function or agonist delivery to target regions. CFTR, CLCA2, TMEM16A, and alternative Cl‐/HCO3‐ exchangers (e.g., SLC4 family) are known to be expressed in lung epithelia. Their relative contribution during late stages of lung injury where TH2 cytokines such as IL13 are elevated is not fully understood. The expression pattern of these anion channels are dysregulated in ARDS, and restoration of channel activities could improve fluid accumulation within the lung. In the present study, various anion channel and/or exchanger activities were measured in HBECs exposed to IL13. We have shown that select amino acids influence anion channel activity. Therefore, the effect of a proprietary amino acid formulation on specific anion channels were studied in IL13‐treated HBECs.MethodsTransepithelial short‐circuit current (Isc) was sequentially blocked using inhibitors for ENaC (benzamil), CFTR CFTRInh172), TMEM16A (CaCCInhA01) and Na+/K+/2Cl‐ co‐transporter (bumetanide) in differentiated HBECs mounted in Ussing chambers. Patch‐clamp whole‐cell recordings were performed to determine anion channel activity after IL13 treatment, and channel and/or exchanger expression was analyzed by immunofluorescence.ResultsIL13 increased benzamil‐insensitive (anion) Isc (87.5 ± 4.7 µA.cm‐2 vs 21.9 ± 1.8 µA.cm‐2; P <0.001) and decreased CFTR Isc (1.4 ± 0.52 µA.cm‐2 vs 12.2 ± 0.83 µA.cm‐2; P <0.001) compared to controls. Amino acids reduced benzamil‐insensitive Isc (55.9 ± 4.8 µA.cm‐2; P <0.001) and increased CFTR Isc (6.7 ± 0.75 µA.cm‐2; P <0.001). There was no change in TMEM16A Isc with IL13 treatment. Bumetanide‐sensitive Isc was higher in IL13‐treated cells compared to controls (52.5 ± 3.6 µA.cm‐2 vs 5.5 ± 0.62 µA.cm‐2; P <0.001) suggesting anion secretion via alternate mechanisms, and amino acids decreased bumetanide‐sensitive anion secretion (20.5 ± 2.9 µA.cm‐2; P <0.001). Similarly, whole‐cell patch clamp recordings showed that amino acids restored the CFTR response in IL13‐treated cells. Immunofluorescence imaging demonstrated decreased CFTR expression with IL13, while amino acids recovered CFTR at the apical membrane.ConclusionTreatment with IL13 increased anion secretion while reducing CFTR channel activity in HBECs. Amino acid formulation restored CFTR activity and decreased total anion current in the presence of IL13. These studies suggest anion secretion via alternate anion secretory pathways. Future studies should be directed at understanding the link between CFTR and alternate anion channels in conditions associated with elevated IL13, and how amino acids regulate these channel and/or exchanger functions

Smooth Muscle LRRC8A Anion Channel Knockout Promotes Vasodilation and Protects Against TNFα‐induced Vascular Dysfunction

Tumor necrosis factor‐α (TNFα), a pro‐inflammatory cytokine, is associated with cardiovascular risk. Volume‐regulated anion channels (VRACs) are activated by TNFα and are composed of Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E). LRRC8A is required for all VRACs. We previously determined that VRAC inhibitors or LRRC8A knockdown suppress the inflammatory response to TNFα in cultured vascular smooth muscle cells, however, the role of these channels in vasomotor function has not been studied. We hypothesized that loss of LRRC8A would prevent the impairment of vascular function by TNFα. We created vascular smooth muscle‐specific (Sm22alpha‐Cre) LRRC8A knockout (KO) mice and isolated mesenteric arteries from male mice for wire myograph recording. Vessel segments were incubated with TNFα (10 ng/mL) or vehicle for 2 days in tissue culture. Contractile responses to norepinephrine (NE) were unaltered in LRRC8A KO mice but enhanced by TNFα in wild type (WT) than in KO rings (WT+TNFα 284 ± 18.9 %, KO+TNFα 222 ± 7.1 % of KCl contraction, *p<0.05, n = 5‐6). In the vehicle‐treated group both endothelium‐dependent (acetylcholine, ACh) and ‐independent (sodium nitroprusside, SNP) relaxation were increased in LRRC8A KO compared to WT rings contracted with phenylephrine (PE). Exposure to TNFα impaired relaxation to both ACh and SNP in WT vessels but had no effect on relaxation of LRRC8A KO vessels (black *p<0.05 compared to WT control, red *p<0.05 compared to TNFα‐treated, n = 6‐8). The enhanced relaxation responses of KO vessels were recapitulated in WT rings by 20 min of exposure to a VRAC inhibitor, carbenoxolone (CBX; 100 μM). Thus, in addition to its established role in TNFα signaling, LRRC8A anion channel activity modulates vascular reactivity with loss of function enhancing vasodilator function. VRAC inhibition may provide a novel therapeutic approach to the prevention and treatment of inflammation‐induced vascular dysfunction.

Ether anesthetics prevents touch-induced trigger hair calcium-electrical signals excite the Venus flytrap

Plants do not have neurons but operate transmembrane ion channels and can get electrical excited by physical and chemical clues. Among them the Venus flytrap is characterized by its peculiar hapto-electric signaling. When insects collide with trigger hairs emerging the trap inner surface, the mechanical stimulus within the mechanosensory organ is translated into a calcium signal and an action potential (AP). Here we asked how the Ca2+ wave and AP is initiated in the trigger hair and how it is feed into systemic trap calcium-electrical networks. When Dionaea muscipula trigger hairs matures and develop hapto-electric excitability the mechanosensitive anion channel DmMSL10/FLYC1 and voltage dependent SKOR type Shaker K+ channel are expressed in the sheering stress sensitive podium. The podium of the trigger hair is interface to the flytrap’s prey capture and processing networks. In the excitable state touch stimulation of the trigger hair evokes a rise in the podium Ca2+ first and before the calcium signal together with an action potential travel all over the trap surface. In search for podium ion channels and pumps mediating touch induced Ca2+ transients, we, in mature trigger hairs firing fast Ca2+ signals and APs, found OSCA1.7 and GLR3.6 type Ca2+ channels and ACA2/10 Ca2+ pumps specifically expressed in the podium. Like trigger hair stimulation, glutamate application to the trap directly evoked a propagating Ca2+ and electrical event. Given that anesthetics affect K+ channels and glutamate receptors in the animal system we exposed flytraps to an ether atmosphere. As result propagation of touch and glutamate induced Ca2+ and AP long-distance signaling got suppressed, while the trap completely recovered excitability when ether was replaced by fresh air. In line with ether targeting a calcium channel addressing a Ca2+ activated anion channel the AP amplitude declined before the electrical signal ceased completely. Ether in the mechanosensory organ did neither prevent the touch induction of a calcium signal nor this post stimulus decay. This finding indicates that ether prevents the touch activated, glr3.6 expressing base of the trigger hair to excite the capture organ.

Identifying Novel Genes and Proteins Involved in Salt Stress Perception and Signaling of Rice Seedlings.

Rice is one of the most important crops worldwide. Crop production is constrained markedly, however, by abiotic stresses such as salinity. To elucidate early stress response signaling networks involved in rice, we report in this study an original quantitative proteomic analysis of the rice seedlings subjected to short-term salt stress. We detected 570 differentially regulated proteins (DRPs) in the root sample. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis demonstrated that DRPs of the root were mainly involved in membrane trafficking, kinase activity, and ion toxicity responses. Interactome analysis revealed the central role of root proteins involved in membrane trafficking in the early response to salinity, such as cell surface receptor-like kinases (RLKs), phosphatidylinositols (PIs), calcium-dependent protein kinases 1 and 5, calcineurin B-like protein-interacting proteins, protein phosphatase 2C (PP2C) inhibitors, and abscisic acid receptors (PYL5/10), indicating activation of S-type anion channel. Furthermore, the proteogenomic analysis revealed 128 unique genome search-specific peptides with high-quality mass spectromety (MS/MS) spectra. We identified 38 novel protein-coding genes, refined the annotation of 17 existing gene models, and suggested several novel stress-responsive proteins, such as RLK5, peroxidase 27, and growth-regulating factor 2. Novel peptides had an ortholog match in the curated protein sequence set of other plant species. In conclusion, this study identifies novel stress-responsive proteins and genes of rice, thus warrant future consideration as candidates for molecular breeding of stress-tolerant crop varieties.

The PYR‐PP2C‐CKL2 module regulates ABA‐mediated actin reorganization during stomatal closure

Limiting water loss by reducing transpiration helps plants survive when water is limited. Under drought stress, abscisic acid (ABA)-mediated gene expression and anion channel activation regulate stomatal closure and stress responses. ABA-induced actin reorganization also affects stomatal closure, but the underlying molecular mechanism remains unclear. In this study, we discovered that under nonstress conditions, the clade A PP2C phosphatases, such as ABI1 and ABI2, interact with CKL2 and inhibit its kinase activity in Arabidopsis. Under drought stress, CKL2 kinase activity was released through the formation of a complex containing ABA, PP2C and a PYR1/PYL/RCAR family (PYL) receptor. The activated CKL2 regulating actin reorganization is another important process to maintain stomatal closure besides ABA-activated SnRK2 signaling. Moreover, CKL2 phosphorylated PYR1-LIKE 1, ABI1 and ABI2 at amino acid residues conserved among PYLs and PP2Cs, and stabilized ABI1 protein. Our results reveal that ABA signaling regulates actin reorganization to maintain stomatal closure during drought stress, and the feedback regulation of PYL1, ABI1 and ABI2 by the CKL2 kinase might fine-tune ABA signaling and affect plant ABA responses.