The epsilon toxin from Clostridium perfringens stimulates calcium-activated chloride channels, generating extracellular vesicles in Xenopus oocytes.

The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore-forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re-examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin-luciferase reaction. Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca2+ levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca2+ mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx-sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut-like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles.

A highly conserved A-to-I RNA editing event within the glutamate-gated chloride channel GluClα is necessary for olfactory-based behaviors in Drosophila.

A-to-I RNA editing is a cellular mechanism that generates transcriptomic and proteomic diversity, which is essential for neuronal and immune functions. It involves the conversion of specific adenosines in RNA molecules to inosines, which are recognized as guanosines by cellular machinery. Despite the vast number of editing sites observed across the animal kingdom, pinpointing critical sites and understanding their in vivo functions remains challenging. Here, we study the function of an evolutionary conserved editing site in Drosophila, located in glutamate-gated chloride channel (GluClα). Our findings reveal that flies lacking editing at this site exhibit reduced olfactory responses to odors and impaired pheromone-dependent social interactions. Moreover, we demonstrate that editing of this site is crucial for the proper processing of olfactory information in projection neurons. Our results highlight the value of using evolutionary conservation as a criterion for identifying editing events with potential functional significance and paves the way for elucidating the intricate link between RNA modification, neuronal physiology, and behavior.

Regulation of ClC-K/barttin by endocytosis influences distal convoluted tubule hyperplasia.

ClC-K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC-Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline-tyrosine motif in the C-terminus of barttin stimulates ClC-K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co-expressing ClC-K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC-K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock-in mouse. Comparing wild-type (WT) and knock-in mice under a standard diet, we could not observe any difference in ClC-K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high-sodium low-potassium diet, known to induce hyperplasia of distal convoluted tubules, knock-in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. KEY POINTS: It is revealed by mutagenesis and functional experiments that a previously identified proline-tyrosine motif regulating ClC-K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC-K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo. Knock-in mice with a mutation in this motif, under conditions of a high-sodium low-potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild-type animals, indicating a gain of function of the channel in vivo.

[Two cases of Dent disease type 1 with Bartter-like phenotype and literature review].

The clinical presentation, treatment, and follow-up of two boys with type 1 Dent disease who exhibited a Bartter-like phenotype were retropectively analysed. The related literature of pediatric patients with type 1 Dent disease who had hypokalemia and metabolic alkalosis was screened through databases such as PubMed, CNKI, and Wanfang until February 1, 2024, and common features among these patients were summarized through literature review. A total of 7 literatures were included, and 9 children were included in the analysis. All patients were male, presenting with significant low molecular weight proteinuria and hypercalciuria. Other prominent characteristic phenotypes included short stature (7/8), hypophosphatemia (8/9), and rickets (6/8). Seven previously reported patients had missense or nonsense mutations, while 2 patients in this study carried possible pathogenic mutations in the CLCN5 gene, c.315+2T>A (p.?) and c.584dupT (p.I196Yfs*6), respectively. Five patients were able to maintain blood potassium levels around 3 mmol/L with oral potassium chloride solution combined with non-steroidal anti-inflammatory drugs (ibuprofen or indomethacin). The follow-up showed that 2 patients developed chronic kidney disease stage 4 and stage 3 at the age of 13 and 21 years, respectively. The phenotypic overlap between Dent disease and Batter syndrome is considerable,with the distinguishing feature being the presence of significant low molecular weight proteinuria. Patients with type 1 Dent disease presenting with the Bartter-like phenotype have a high prevalence of short stature, hypophosphatemia, and rickets. Non-steroidal anti-inflammatory drugs can be used to correct hypokalemia in patients under periodic renal function assessment.

Structural basis of adenine nucleotides regulation and neurodegenerative pathology in ClC-3 exchanger.

The ClC-3 chloride/proton exchanger is both physiologically and pathologically critical, as it is potentiated by ATP to detect metabolic energy level and point mutations in ClC-3 lead to severe neurodegenerative diseases in human. However, why this exchanger is differentially modulated by ATP, ADP or AMP and how mutations caused gain-of-function remains largely unknow. Here we determine the high-resolution structures of dimeric wildtype ClC-3 in the apo state and in complex with ATP, ADP and AMP, and the disease-causing I607T mutant in the apo and ATP-bounded state by cryo-electron microscopy. In combination with patch-clamp recordings and molecular dynamic simulations, we reveal how the adenine nucleotides binds to ClC-3 and changes in ion occupancy between apo and ATP-bounded state. We further observe I607T mutation induced conformational changes and augments in current. Therefore, our study not only lays the structural basis of adenine nucleotides regulation in ClC-3, but also clearly indicates the target region for drug discovery against ClC-3 mediated neurodegenerative diseases.

A New Case Report of a CLCNKB Complex Heterozygous Mutation in Adult-Onset Type III Bartter Syndrome.

BACKGROUND: Type III Bartter syndrome (BS) is an autosomal recessive renal tubular disease caused by the mutation of the chloride voltage-gated channel Kb (CLCNKB) gene. This condition is characterized by renal sodium loss, hypokalemia, metabolic alkaliosis, high renin, and high aldosterone levels. METHODS: We report a case of adult type III BS caused by a novel complex heterozygous mutation of the CLCNKB gene. The peripheral blood was extracted for whole genome DNA extraction, and the genome exon region of BS- related genes, was predicted by high-throughput sequencing and protein function prediction software. The selected mutation sites were verified by sequencing with Sanger method. RESULTS: The new complex heterozygous mutations of CLCNKB include heterozygous deletion of exon 2 - 20 of CLCNKB and nonsense mutation of exon 19, c.2010G>A (p.W670X). This complex heterozygous mutation has not been reported in humans. CONCLUSIONS: For patients with high clinical suspicion of BS, a clear diagnosis should be made through genetic test-ing to improve patients' quality of life and provide genetic guidance.

A CLIC1 network coordinates matrix stiffness and the Warburg effect to promote tumor growth in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) features substantial matrix stiffening and reprogrammed glucose metabolism, particularly the Warburg effect. However, the complex interplay between these traits and their impact on tumor advancement remains inadequately explored. Here, we integrated clinical, cellular, and bioinformatics approaches to explore the connection between matrix stiffness and the Warburg effect in PDAC, identifying CLIC1 as a key mediator. Elevated CLIC1 expression, induced by matrix stiffness through Wnt/ß-catenin/TCF4 signaling, signifies poorer prognostic outcomes in PDAC. Functionally, CLIC1 serves as a catalyst for glycolytic metabolism, propelling tumor proliferation. Mechanistically, CLIC1 fortifies HIF1α stability by curbing hydroxylation via reactive oxygen species (ROS). Collectively, PDAC cells elevate CLIC1 levels in a matrix-stiffness-responsive manner, bolstering the Warburg effect to drive tumor growth via ROS/HIF1α signaling. Our insights highlight opportunities for targeted therapies that concurrently address matrix properties and metabolic rewiring, with CLIC1 emerging as a promising intervention point.

Endocytosed dsRNAs induce lysosomal membrane permeabilization that allows cytosolic dsRNA translocation for Drosophila RNAi responses.

RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl-/H+ antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.

The plasma membrane inner leaflet PI(4,5)P2 is essential for the activation of proton-activated chloride channels.

Proton-activated chloride (PAC) channels, ubiquitously expressed in tissues, regulate intracellular Cl- levels and cell death following acidosis. However, molecular mechanisms and signaling pathways involved in PAC channel modulation are largely unknown. Herein, we determine that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] of the plasma membrane inner leaflet is essential for the proton activation of PAC channels. PI(4,5)P2 depletion by activating phosphatidylinositol 5-phosphatases or Gq protein-coupled muscarinic receptors substantially inhibits human PAC currents. In excised inside-out patches, PI(4,5)P2 application to the cytoplasmic side increases the currents. Structural simulation reveals that the putative PI(4,5)P2-binding site is localized within the cytosol in resting state but shifts to the cell membrane's inner surface in an activated state and interacts with inner leaflet PI(4,5)P2. Alanine neutralization of basic residues near the membrane-cytosol interface of the transmembrane helice 2 significantly attenuates PAC currents. Overall, our study uncovers a modulatory mechanism of PAC channel through inner membrane PI(4,5)P2.

Prognosis poor, immune infiltration of colon adenocarcinoma associated with low expression levels of calcium-activated chloride channel.

The calcium-activated chloride channel (CLCA4) in colon adenocarcinoma (COAD) and immunological infiltration have not been extensively studied. This work thoroughly employed several datasets to assess the expression, prognosis, and association between immune infiltration and clinicopathological characteristics of CLCA4 in cancer, as well as look into potential signalling pathways. The human protein atlas (HPA), TIMER, UALCAN, TISIDB, GSCA, SangerBox, GeneMANIA, and LinkedOmics were among the datasets that were used. The findings demonstrated that, in comparison to normal tissues, COAD tissues had lower levels of CLCA4 expression. The prognosis was worse for those whose levels of CLCA4 expression were lower. For validation, immunohistochemistry (HPA) was used. Positive correlations between CLCA4 mRNA expression and its copy number variation (CNV) were observed, and CLCA4 CNV was linked to immunological infiltration. Subsequent investigation demonstrated the association between immune cell markers, immune checkpoint genes, and immunological infiltration with CLCA4. The overall survival and disease-free survival of M0 patients were considerably better than those of M1 patients, and the groups with tumour stages M0 and M1 had notably different levels of CLCA4 expression. Its substantial enrichment in ion channel activity, transmembrane transporter activity, digestion, and other biological processes was revealed by gene ontology analysis. Oxidative phosphorylation, pancreatic secretion, Parkinson's and Alzheimer's diseases, renin secretion, and other signalling pathways were the primary associations found for CLCA4. It is evident that the immunological microenvironment and functions like ion transport, metabolism, and intestinal digestion are all impacted by CLCA4 expression.

Fitness costs, realized heritability, and mechanism of resistance to tenvermectin B in Plutella xylostella.

Tenvermectin B (TVM-B) and five TVM-B analogs were produced by fermentation of a genetically engineered strain Streptomyces avermitilis HU02, and TVM-B is being developed as a new insecticide. Through 11 generations of resistance selection against TVM-B in the diamondback moth, Plutella xylostella, the median lethal concentration (LC50) was increased from 14.84 to 1213.73 mg L-1. The resistance to TVM-B in P. xylostella developed fast and its realized heritability was high (h2 = 0.2901 (F7), h2 = 0.4070 (F11)). However, the relative fitness was 0.6916 suggesting a fitness cost in the resistant strains. The fitness cost was partially explained by the upregulation of the detoxification enzyme activity by 2.15 folds in carboxylate esterase (CarE) and the gene expressions of ATP-binding cassette transporter gene (ABCC2) and the alpha subunit of the glutamate-gated chloride channel (GluCl) by 1.70- and 2.32 folds, respectively. The resistance was also explained by two points of mutations at the alpha subunit of the glutamate-gated chloride channel in the P. xylostella (PxGluClα) subunit in F11. However, there was little change in the binding affinity. These results provided helpful information for the mechanism study of TVM-B resistance and will be conducive to designing rational resistance management strategies in P. xylostella.

The effects of Cl- channel inhibitors and pyrethroid insecticides on calcium-activated chloride channels in neurons of Helicoverpa armigera.

TMEM16A, a member of the Transmembrane protein 16 family, serves as the molecular basis for calcium activated chloride channels (CaCCs). We use RT-PCR to demonstrate the expression of TMEM16A in the neurons of Helicoverpa armigera, and record the CaCCs current of acute isolated neurons of H. armigera for the first time using patch clamp technology. In order to screen effective inhibitors of calcium-activated chloride channels, the inhibitory effects of four chloride channel inhibitors, CaCCinh-A01, NPPB, DIDS, and SITS, on CaCCs were compared. The inhibitory effects of the four inhibitors on the outward current of CaCCs were CaCCinh-A01 (10 µM, 56.31 %), NPPB (200 µM, 43.69 %), SITS (1 mM, 12.41 %) and DIDS (1 mM, 13.29 %). Among these inhibitors, CaCCinh-A01 demonstrated the highest efficacy as a blocker. To further explore whether calcium channel proteins can serve as potential targets of pyrethroids, we compared the effects of (type I) tefluthrin and (type II) deltamethrin on CaCCs. 10 µM and 100 µM tefluthrin can stimulate a large tail current in CaCCs, prolonging their deactivation time by 10.44 ms and 31.49 ms, and the V0.5 shifted in the hyperpolarization by 2-8 mV. Then, deltamethrin had no obvious effect on the deactivation and activation of CaCCs. Therefore, CaCCs of H. armigera can be used as a potential target of pyrethroids, but type I and type II pyrethroids have different effects on CaCCs.

4-Phenylbutyric Acid Treatment Reduces Low-Molecular-Weight Proteinuria in a Clcn5 Knock-in Mouse Model for Dent Disease-1.

Dent disease-1 (DD-1) is a rare X-linked tubular disorder characterized by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrolithiasis and nephrocalcinosis. This disease is caused by inactivating mutations in the CLCN5 gene which encodes the voltage-gated ClC-5 chloride/proton antiporter. Currently, the treatment of DD-1 is only supportive and focused on delaying the progression of the disease. Here, we generated and characterized a Clcn5 knock-in mouse model that carries a pathogenic CLCN5 variant, c. 1566_1568delTGT; p.Val523del, which has been previously detected in several DD-1 unrelated patients, and presents the main clinical manifestations of DD-1 such as high levels of urinary b2-microglobulin, phosphate and calcium. Mutation p.Val523del causes partial ClC-5 retention in the endoplasmic reticulum. Additionally, we assessed the ability of sodium 4-phenylbutyrate, a small chemical chaperone, to ameliorate DD-1 symptoms in this mouse model. The proposed model would be of significant value in the investigation of the fundamental pathological processes underlying DD-1 and in the development of effective therapeutic strategies for this rare condition.

Chloride intracellular channel 4 blockade improves cognition in mice with Alzheimer's disease: CLIC4 protein expression and tau protein hyperphosphorylation.

Numerous academic literature suggests that amyloid-ß (Aß) deposition, tau protein phosphorylation, and irreversible neuronal death are the three major causes of AD. The chloride intracellular channel (CLIC) protein family not only regulates the polarisation of neurons, but also has important implications for neuronal survival. Chloride intracellular channel 4 (CLIC4) can be pathologically activated by cyclin-dependent kinase 5 (Cdk5), which causes a significant increase in the expression of CLIC4 and mediates neuronal apoptosis. CLIC4 knockdown inhibits H2O2-induced neuronal apoptosis; however, the relationship between CLIC4 and AD remains unknown. In the present study, we showed that CLIC4 expression was elevated in the hippocampus of AD mice; knockdown of hippocampal CLIC4 alleviated Aß25-35-induced cognitive impairment in mice; overexpression of hippocampal CLIC4 accelerated Aß deposition and tau protein hyperphosphorylation in young AD mice (APP/PS1 mice at three months of age). CLIC4 overexpressing mice had a longer escape latency compared to controls in behavioural testing (Morris water maze and T-maze tests). By Co-immunoprecipitation/mass spectrometry (Co-IP/MS) of HT22 cells to identify proteins that specifically bind to CLIC4, we found interactions with CCAAT enhancer binding protein (C/EBPß); a critical pathway involved in the development of various neurodegenerative diseases. In addition, the knockdown of hippocampal CLIC4 alleviated AD-like pathology by inhibiting the C/EBPß/AEP signaling pathway. These data suggest an essential role for high CLIC4 expression in the pathophysiology of AD and reveal that inhibition of CLIC4 expression may provide an opportunity for treatment.

TMEM16 proteins: Ca2+­activated chloride channels and phospholipid scramblases as potential drug targets (Review).

TMEM16 proteins, which function as Ca2+­activated Cl­ channels are involved in regulating a wide variety of cellular pathways and functions. The modulators of Cl­ channels can be used for the molecule­based treatment of respiratory diseases, cystic fibrosis, tumors, cancer, osteoporosis and coronavirus disease 2019. The TMEM16 proteins link Ca2+ signaling, cellular electrical activity and lipid transport. Thus, deciphering these complex regulatory mechanisms may enable a more comprehensive understanding of the physiological functions of the TMEM16 proteins and assist in ascertaining the applicability of these proteins as potential pharmacological targets for the treatment of a range of diseases. The present review examined the structures, functions and characteristics of the different types of TMEM16 proteins, their association with the pathogenesis of various diseases and the applicability of TMEM16 modulator­based treatment methods.

Insights into CLC-0's Slow-Gating from Intracellular Proton Inhibition.

The opening of the Torpedo CLC-0 chloride (Cl-) channel is known to be regulated by two gating mechanisms: fast gating and slow (common) gating. The structural basis underlying the fast-gating mechanism is better understood than that of the slow-gating mechanism, which is still largely a mystery. Our previous study on the intracellular proton (H+i)-induced inhibition of the CLC-0 anionic current led to the conclusion that the inhibition results from the slow-gate closure (also called inactivation). The conclusion was made based on substantial evidence such as a large temperature dependence of the H+i inhibition similar to that of the channel inactivation, a resistance to the H+i inhibition in the inactivation-suppressed C212S mutant, and a similar voltage dependence between the current recovery from the H+i inhibition and the recovery from the channel inactivation. In this work, we further examine the mechanism of the H+i inhibition of wild-type CLC-0 and several mutants. We observe that an anion efflux through the pore of CLC-0 accelerates the recovery from the H+i-induced inhibition, a process corresponding to the slow-gate opening. Furthermore, various inactivation-suppressed mutants exhibit different current recovery kinetics, suggesting the existence of multiple inactivated states (namely, slow-gate closed states). We speculate that protonation of the pore of CLC-0 increases the binding affinity of permeant anions in the pore, thereby generating a pore blockage of ion flow as the first step of inactivation. Subsequent complex protein conformational changes further transition the CLC-0 channel to deeper inactivated states.

Cytosolic acidification and oxidation are the toxic mechanisms of SO2 in Arabidopsis guard cells.

SO2/H2SO3 can damage plants. However, its toxic mechanism has still been controversial. Two models have been proposed, cytosolic acidification model and cellular oxidation model. Here, we assessed the toxic mechanism of H2SO3 in three cell types of Arabidopsis thaliana, mesophyll cells, guard cells (GCs), and petal cells. The sensitivity of GCs of Chloride channel a (CLCa)-knockout mutants to H2SO3 was significantly lower than those of wildtype plants. Expression of other CLC genes in mesophyll cells and petal cells were different from GCs. Treatment with antioxidant, disodium 4,5-dihydroxy-1,3-benzenedisulfonate (tiron), increased the median lethal concentration (LC50) of H2SO3 in GCs indicating the involvement of cellular oxidation, while the effect was negligible in mesophyll cells and petal cells. These results indicate that there are two toxic mechanisms of SO2 to Arabidopsis cells: cytosolic acidification and cellular oxidation, and the toxic mechanism may vary among cell types.

Multisystem disorder associated with a pathogenic variant in CLCN7 in the absence of osteopetrosis.

BACKGROUND: We clinically and genetically evaluated a Taiwanese boy presenting with developmental delay, organomegaly, hypogammaglobulinemia and hypopigmentation without osteopetrosis. Whole-exome sequencing revealed a de novo gain-of-function variant, p.Tyr715Cys, in the C-terminal domain of ClC-7 encoded by CLCN7. METHODS: Nicoli et al. (2019) assessed the functional impact of p.Tyr715Cys by heterologous expression in Xenopus oocytes and evaluating resulting currents. RESULTS: The variant led to increased outward currents, indicating it underlies the patient's phenotype of lysosomal hyperacidity, storage defects and vacuolization. This demonstrates the crucial physiological role of ClC-7 antiporter activity in maintaining appropriate lysosomal pH. CONCLUSION: Elucidating mechanisms by which CLCN7 variants lead to lysosomal dysfunction will advance understanding of genotype-phenotype correlations. Identifying modifier genes and compensatory pathways may reveal therapeutic targets. Ongoing functional characterization of variants along with longitudinal clinical evaluations will continue advancing knowledge of ClC-7's critical roles and disease mechanisms resulting from its dysfunction. Expanded cohort studies are warranted to delineate the full spectrum of associated phenotypes.