CLIC1-mediated autophagy confers resistance to DDP in gastric cancer.

Gastric cancer has been a constant concern to researchers as one of the most common malignant tumors worldwide. The treatment options for gastric cancer include surgery, chemotherapy and traditional Chinese medicine. Chemotherapy is an effective treatment for patients with advanced gastric cancer. Cisplatin (DDP) has been approved as a critical chemotherapy drug to treat various kinds of solid tumors. Although DDP is an effective chemotherapeutic agent, many patients develop drug resistance during treatment, which has become a severe problem in clinical chemotherapy. This study aims to investigate the mechanism of DDP resistance in gastric cancer. The results show that intracellular chloride channel 1 (CLIC1) expression was increased in AGS/DDP and MKN28/DDP, and as compared to the parental cells, autophagy was activated. In addition, the sensitivity of gastric cancer cells to DDP was decreased compared to the control group, and autophagy increased after overexpression of CLIC1. On the contrary, gastric cancer cells were more sensitive to cisplatin after transfection of CLIC1siRNA or treatment with autophagy inhibitors. These experiments suggest that CLIC1 could alter the sensitivity of gastric cancer cells to DDP by activating autophagy. Overall, the results of this study recommend a novel mechanism of DDP resistance in gastric cancer.

[Analysis of clinical presentation and genetic characteristics of malignant infantile osteopetrosis].

Objective: To investigate the clinical presentation and genetic characteristics of malignant infantile osteopetrosis. Methods: This was a retrospective case study. Thirty-seven children with malignant infantile osteopetrosis admitted into Beijing Children's Hospital from January 2013 to September 2022 were enrolled in this study. According to the gene mutations, the patients were divided into the CLCN7 group and the TCIRG1 group. Clinical characteristics, laboratory tests, and prognosis were compared between two groups. Wilcoxon test or Fisher exact test were used in inter-group comparison. The survival rate was estimated with the Kaplan-Meier method and the Log-Rank test was used to compare the difference in survival between groups. Results: Among the 37 cases, there were 22 males and 15 females. The age of diagnosis was 0.5 (0.2, 1.0) year. There were 13 patients (35%) and 24 patients (65%) with mutations in CLCN7 and TCIRGI gene respectively. Patients in the CLCN7 group had an older age of diagnosis than those in the TCIRGI group (1.2 (0.4, 3.6) vs. 0.4 (0.2, 0.6) years, Z=-2.60, P=0.008). The levels of serum phosphorus (1.7 (1.3, 1.8) vs. 1.1 (0.8, 1.6) mmol/L, Z=-2.59, P=0.010), creatine kinase isoenzyme (CK-MB) (457 (143, 610) vs. 56 (37, 82) U/L, Z=-3.38, P=0.001) and the level of neutrophils (14.0 (9.9, 18.1) vs. 9.2 (6.7, 11.1) ×109/L, Z=-2.07, P=0.039) at diagnosis were higher in the CLCN7 group than that in the TCIRG1 group. However, the level of D-dimer in the CLCN7 group was lower than that in the TCIRGI group (2.7 (1.0, 3.1) vs. 6.3 (2.5, 9.7) µg/L, Z=2.83, P=0.005). After hematopoietic stem cell transplantation, there was no significant difference in 5-year overall survival rate between the two groups (92.3%±7.4% vs. 83.3%±7.6%, χ²=0.56, P=0.456). Conclusions: TCIRGI gene mutations are more common in children with osteopetrosis. Children with TCIRGI gene mutations have younger age, lower levels of phosphorus, CK-MB, and neutrophils and higher level of D-dimer at the onset. After hematopoietic stem cell transplantation, patients with CLCN7 or TCIRGI gene mutations have similar prognosis.

Hyperbaric oxygen ameliorates neuronal injury and neurological function recovery in rats with intracerebral hemorrhage by silencing microRNA-204-5p-targeted chloride channel protein 3.

Hyperbaric oxygen (HBO) therapy is of clinical utility in patients with transient cerebral ischemia. The investigatory study was to identify the potential regulatory mechanism of HBO treatment on neuronal injury and neurological function recovery in rats with intracerebral hemorrhage (ICH). Firstly, the rat model of ICH was established by collagenase, and the experimental rats were treated with HBO at 2.5 absolute atmospheres for 60 min each time. Next, lentivirus interfering with microRNA (miR)-204-5p or chloride channel protein 3 (CLCN3) expression was injected via the tail vein. Afterward, neurological function assessment was conducted, serum S100ß and NSE contents were detected by enzymer-linked immunosorbent assay, and pathological conditions of brain tissue were observed by hematoxylin-eosin staining. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining was used to detect neuronal apoptosis. The results showed that HBO alleviated neuronal injury and neurological function recovery in ICH rats and reduced serum S100ß and NSE content (all P<0.05). At the same time, overexpressing miR-204-5p or depleting CLCN3 further promoted the therapeutic effect of HBO on ICH rats (all P<0.05), while silencing miR-204-5p or elevating CLCN3 did oppositely (all P<0.05). In conclusion, HBO alleviates neuronal injury and neurological function recovery in ICH rats by silencing miR-204-5p-targeted CLCN3.

Neutral lipid storage disease with myopathy and myotonia associated to pathogenic variants on PNPLA2 and CLCN1 genes: case report.

BACKGROUND: Neutral lipid storage disease with myopathy (NLSD-M) is an autosomal recessive disease that manifests itself around the 3rd to 4th decade with chronic myopathy predominantly proximal in the shoulder girdle. Clinical myotonia is uncommon. We will report a rare case of association of pathogenic variants on PNPLA2 and CLCN1 genes with a mixed phenotype of NLSD-M and a subclinical form of Thomsen's congenital myotonia. CASE PRESENTATION: We describe a patient with chronic proximal myopathy, subtle clinical myotonia and electrical myotonia on electromyography (EMG). Serum laboratory analysis disclosure hyperCKemia (CK 1280 mg/dL). A blood smear analysis showed Jordan's anomaly, a hallmark of NLSD-M. A genetic panel was collected using next-generation sequencing (NGS) technique, which identified two pathogenic variants on genes supporting two different diagnosis: NLSD-M and Thomsen congenital myotonia, whose association has not been previously described. CONCLUSIONS: Although uncommon, it is important to remember the possibility of association of pathogenic variants to explain a specific neuromuscular disease phenotype. The use of a range of complementary methods, including myopathy genetic panels, may be essential to diagnostic definition in such cases.

Lysosomal chloride transporter CLH-6 protects lysosome membrane integrity via cathepsin activation.

Lysosomal integrity is vital for cell homeostasis, but the underlying mechanisms are poorly understood. Here, we identify CLH-6, the C. elegans ortholog of the lysosomal Cl-/H+ antiporter ClC-7, as an important factor for protecting lysosomal integrity. Loss of CLH-6 affects lysosomal degradation, causing cargo accumulation and membrane rupture. Reducing cargo delivery or increasing CPL-1/cathepsin L or CPR-2/cathepsin B expression suppresses these lysosomal defects. Inactivation of CPL-1 or CPR-2, like CLH-6 inactivation, affects cargo digestion and causes lysosomal membrane rupture. Thus, loss of CLH-6 impairs cargo degradation, leading to membrane damage of lysosomes. In clh-6(lf) mutants, lysosomes are acidified as in wild type but contain lower chloride levels, and cathepsin B and L activities are significantly reduced. Cl- binds to CPL-1 and CPR-2 in vitro, and Cl- supplementation increases lysosomal cathepsin B and L activities. Altogether, these findings suggest that CLH-6 maintains the luminal chloride levels required for cathepsin activity, thus facilitating substrate digestion to protect lysosomal membrane integrity.

Identification of CLIC5 as a Prognostic Biomarker and Correlated Immunomodulator for Lung Adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is one of the most common pathological types of lung cancer. The gene Chloride Intracellular Channel 5 (CLIC5) has an important role in neurophysiology, cardiovascular biology, and tumour biology. Here, we explored the prognostic value and immune infiltration of CLIC5 expression in LUAD patients. METHODS: We extracted transcriptional LUAD data from The Cancer Genome Atlas (TCGA) and the University of Alabama Cancer Database to explore CLIC5 expression profiles and their relation to CLIC5 and clinicopathological parameters. The relationship between CLIC5 and survival time was explored using Kaplan-Meier Plotter. Then, we integrated the data from TCGA and the Gene Expression Omnibus (GEO) database to perform univariate and multivariate Cox regression. We performed CLIC5 immunohistochemical staining on 167 lung adenocarcinoma samples for further verification. In addition, we analysed the Gene Ontology (GO) database, Kyoto Encyclopaedia of Genes and Genomes pathways and network analysis of protein-protein interactions in lung tissue, to explore the potential mechanism of CLIC5. To analyse the correlation between immune infiltration and CLIC5 expression, we first compared the expression of immune cells in tumour tissues and normal tissues based on the TCGA and GEO databases. We found 51 immunomodulators related to CLIC5 and structured their enrichment pathways as well as those of 50 correlated genes. We used a Cox regression model to identify multiple-gene risk prediction signatures. Finally, we assessed the prognostic accuracy of the risk scores via receiver operating characteristic curves. RESULTS: CLIC5 expression levels were significantly lower in LUAD tissue than in normal tissue. Lower CLIC5 expression was negatively correlated to the overall survival of LUAD patients based on survival analysis. We identified CLIC5 as an independent prognosis predictor. Functional network analysis suggested that CLIC5 is related to multiple pathways. CLIC5 expression is closely related to infiltration levels of many immune cells and immune marker sets in LUAD patients. Furthermore, the risk score based on immunomodulators related to CLIC5 was an independent prognosis predictor in the TCGA lung cohorts. CONCLUSION: Our findings suggest that CLIC5 is a promising molecular marker for the prognosis and immune infiltration of LUAD patients.

Intracellular Chloride Channels Regulate Endothelial Metabolic Reprogramming in Pulmonary Arterial Hypertension.

Mitochondrial fission and a metabolic switch from oxidative phosphorylation to glycolysis are key features of vascular pathology in pulmonary arterial hypertension (PAH) and are associated with exuberant endothelial proliferation and apoptosis. The underlying mechanisms are poorly understood. We describe the contribution of two intracellular chloride channel proteins, CLIC1 and CLIC4, both highly expressed in PAH and cancer, to mitochondrial dysfunction and energy metabolism in PAH endothelium. Pathological overexpression of CLIC proteins induces mitochondrial fragmentation, inhibits mitochondrial cristae formation, and induces metabolic shift toward glycolysis in human pulmonary artery endothelial cells, consistent with changes observed in patient-derived cells. Interactions of CLIC proteins with structural components of the inner mitochondrial membrane offer mechanistic insights. Endothelial CLIC4 excision and mitofusin 2 supplementation have protective effects in human PAH cells and preclinical PAH. This study is the first to demonstrate the key role of endothelial intracellular chloride channels in the regulation of mitochondrial structure, biogenesis, and metabolic reprogramming in expression of the PAH phenotype.

Hericium caput-medusae (Bull.:Fr.) Pers. fermentation concentrate polysaccharides improves intestinal bacteria by activating chloride channels and mucus secretion.

ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional edible fungus in China and many other Asian countries, Hericium caput-medusae (Bull. Fr.) Pers. is widely used to improve the health of the gastrointestinal tract. For example, the drug "Weilexin Granules" is mainly composed of H. caput-medusae (Bull. Fr.) Pers. fermentation concentrate. However, the mechanism of action remains to be elucidated. AIMS OF THE STUDY: The purpose of this study was to assess whether polysaccharides from H. caput-medusae (Bull. Fr.) Pers. fermentation concentrate (HFP) exerts a gut protective effect and a regulatory effect on the intestinal microbiota through the chloride channels and mucus secretion. MATERIALS AND METHODS: HFP was extracted, characterized and different concentrations of HFP (100, 200, 400 mg/kg) were administered to mice for 14 days. The changes in gut microbiota were observed via 16S high throughput sequencing. Short-chain fatty acids (SCFAs) was detected by GC-MS. AB-PAS staining was used to observe the secretion of mucus. The chloride channel activity and protein expression were verified by short-circuit current measurement and Western blot. RESULTS: HFP regulated the abundance of gut microbiota in mice, with increased levels of Ruminococcaceae and Lachnospiraceae and reduced proportions of Staphylococcus and Enterobacter. HFP enhanced mucus volume as well as increased intestinal fluid secretion by activating the chloride channels. In addition, short-circuit current experiments also proved that HFP activates Cl⁻ currents targeting cystic fibrosis transmembrane conductance regulator (CFTR) and Anoamin1 (ANO1). CONCLUSION: In conclusion, HFP might increase intestinal fluid secretion by promoting Cl⁻ secretion, which in turn advanced mucus hydration as well as regulated gut microbiota to improve intestinal health. Therefore, H. caput-medusae (Bull. Fr.) Pers. could be potentially used in the regulation of intestinal secretion and microbes.

ClC-3 silencing mediates lysosomal acidification arrest and autophagy inhibition to sensitize chemo-photothermal therapy.

Protective autophagy can be activated by external stimuli such as chemotherapy (CT) and photothermal therapy (PTT), leading to tumour resistance. As a key subcellular for autophagy, lysosomal dysfunction is crucial for autophagy suppression. Furthermore, lysosomal drug sequestration enhances basic drug resistance such as doxorubicin (DOX), which is trapped away from its target site, namely, the nucleus. Moreover, most of nanodrug delivery systems are internalised to lysosome for degradation, which further leads to DOX resistance. Lysosome serves as an essential organelle in drug resistance mechanisms, whose acidification arrest provides a potential strategy to inhibit autophagy and lysosomal drug sequestration simultaneously. The chloride channel-3 (ClC-3) protein is known as an important Cl--H+ transporter to maintain lysosomal pH at low values of various human cells. Herein, a black phosphorus-based theranostic nanoplatform of BP-A-S@D is constructed, and HeLa cells are used as a model to verify the effect of ClC-3 on tumour lysosomal acidification and autophagy regulation. Consequently, ClC-3 silencing inhibits not only protective autophagy to sensitise chemo-photothermal therapy, but also DOX resistance by suppressing lysosomal acidification. Therefore, ClC-3 silencing could simultaneously inhibit autophagy and lysosomal drug sequestration to improve anti-tumour efficiency.

High-salt diet accelerates bone loss accompanied by activation of ion channels related to kidney and bone tissue in ovariectomized rats.

Excessive salt intake can induce a variety of diseases, such as hypertension, cardiovascular disease, kidney disease and so on，it is also one of the factors promoting bone resorption. The mechanism of osteoporosis-induced exacerbations of high salt diet is not well-defined. In this study, we used ovariectomized 6-month-old Sprague Dawley rats to construct a high bone turnover model, and then administrated with high sodium chloride diet (2.0% w/w NaCl, 8.0% w/w NaCl) for 12 weeks to observe the effect of high salt diet on bone metabolism. The results showed that high salt diet could lead to the destruction of bone microstructure, promote the excretion of urinary calcium and phosphorus and accelerate the bone turnover, as well as cause the pathologic structural abnormalities in renal tubular. At the same time, it was accompanied by the up-regulated expression of the epithelial sodium channel (ENaCα), voltage-gated chloride channels (ClC)- 3 and the down-regulated expression of Na-Cl cotransporter (NCC), sodium calcium exchanger (NCX1) in femoral tissue and renal tubules. These findings confirm that high salt diet can destroy the microstructure of bone by increasing bone resorption and affect some ion channels of bone tissue and renal tubule in ovariectomized rats.

Transcriptome Sequencing and Comparison of Venom Glands Revealed Intraspecific Differentiation and Expression Characteristics of Toxin and Defensin Genes in Mesobuthus martensii Populations.

Mesobuthus martensii, a famous and important Traditional Chinese Medicine has a long medical history and unique functions. It is the first scorpion species whose whole genome was sequenced worldwide. In addition, it is the most widespread and infamous poisonous animal in northern China with complex habitats. It possesses several kinds of toxins that can regulate different ion channels and serve as crucial natural drug resources. Extensive and in-depth studies have been performed on the structures and functions of toxins of M. martensii. In this research, we compared the morphology of M. martensii populations from different localities and calculated the COI genetic distance to determine intraspecific variations. Transcriptome sequencing by RNA-sequencing of the venom glands of M. martensii from ten localities and M. eupeus from one locality was analyzed. The results revealed intraspecific variation in the expression of sodium channel toxin genes, potassium channel toxin genes, calcium channel toxin genes, chloride channel toxin genes, and defensin genes that could be related to the habitats in which these populations are distributed, except the genetic relationships. However, it is not the same in different toxin families. M. martensii and M. eupeus exhibit sexual dimorphism under the expression of toxin genes, which also vary in different toxin families. The following order was recorded in the difference of expression of sodium channel toxin genes: interspecific difference; differences among different populations of the same species; differences between sexes in the same population, whereas the order in the difference of expression of potassium channel toxin genes was interspecific difference; differences between both sexes of same populations; differences among the same sex in different populations of the same species. In addition, there existed fewer expressed genes of calcium channel toxins, chloride channel toxins, and defensins (no more than four members in each family), and their expression differences were not distinct. Interestingly, the expression of two calcium channel toxin genes showed a preference for males and certain populations. We found a difference in the expression of sodium channel toxin genes, potassium channel toxin genes, and chloride channel toxin genes between M. martensii and M. eupeus. In most cases, the expression of one member of the toxin gene clusters distributed in series on the genome were close in different populations and genders, and the members of most clusters expressed in same population and gender tended to be the different. Twenty-one toxin genes were found with the MS/MS identification evidence of M. martensii venom. Since scorpions were not subjected to electrical stimulation or other special treatments before conducting the transcriptome extraction experiment, the results suggested the presence of intraspecific variation and sexual dimorphism of toxin components which revealed the expression characteristics of toxin and defensin genes in M. martensii. We believe this study will promote further in-depth research and use of scorpions and their toxin resources, which in turn will be helpful in standardizing the identification and medical applications of Quanxie in traditional Chinese medicine.

Improved growth and tuber quality of transgenic potato plants overexpressing either NHX antiporter, CLC chloride channel, or both.

The nutritional enhancement of potato plants (Solanum tuberosum L.,) is highly critical. As it is considered a worldwide basic vegetarian nutrition to maintain health. S. tuberosum is one of the foremost staples and the world's fourth-largest food crop. In advance, its need is increasing because of its high-industrial value and population blast. To improve both potato growth and behavior under harsh environmental conditions, we produced transgenic potato plants overexpressing either VvNHX (a sodium proton antiporter from Vitis vinifera), VvCLC (a chloride channel from Vitis vinifera), or both. Control and transgenic plants were grown in greenhouse and field under non-stressed conditions for 85 days in order to characterize their phenotype and evaluate their agronomical performance. To this aim, the evaluation of plant growth parameters, tuber yields and characteristics (calibers, eye number and color), the chemical composition of tubers, was conducted and compared between the different lines. The obtained results showed that transgenic plants displayed an improved growth (flowering precocity, gain of vigor and better vegetative growth) along with enhanced tuber yields and quality (increased protein and starch contents). Our findings provide then insight into the role played by the VvNHX antiport and the VvCLC channel and a greater understanding of the effect of their overexpression in potato plants.

Chronic facial inflammatory pain-induced anxiety is associated with bilateral deactivation of the rostral anterior cingulate cortex.

Patients with chronic pain, especially orofacial pain, often suffer from affective disorders, including anxiety. Previous studies largely focused on the role of the caudal anterior cingulate cortex (cACC) in affective responses to pain, long-term potentiation (LTP) in cACC being thought to mediate the interaction between anxiety and chronic pain. But recent evidence indicates that the rostral ACC (rACC), too, is implicated in processing affective pain. However, whether such processing is associated with neuronal and/or synaptic plasticity is still unknown. We addressed this issue in a chronic facial inflammatory pain model (complete Freund's adjuvant model) in rats, by combining behavior, Fos protein immunochemistry and ex vivo intracellular recordings in rACC slices prepared from these animals. Facial mechanical allodynia occurs immediately after CFA injection, peaks at post-injection day 3 and progressively recovers until post-injection days 10-11, whereas anxiety is delayed, being present at post-injection day 10, when sensory hypersensitivity is relieved, but, notably, not at post-injection day 3. Fos expression reveals that neuronal activity follows a bi-phasic time course in bilateral rACC: first enhanced at post-injection day 3, it gets strongly depressed at post-injection day 10. Ex vivo recordings from lamina V pyramidal neurons, the rACC projecting neurons, show that both their intrinsic excitability and excitatory synaptic inputs have undergone long-term depression (LTD) at post-injection day 10. Thus chronic pain processing is associated with dynamic changes in rACC activity: first enhanced and subsequently decreased, at the time of anxiety-like behavior. Chronic pain-induced anxiety might thus result from a rACC deactivation-cACC hyperactivation interplay.

Chloride intracellular channel 4 (CLIC4) expression profile in the mouse medial prefrontal cortex and its regulation by ethanol.

BACKGROUND: Chloride intracellular channel 4 (CLIC4) is a multifunctional metamorphic protein for which a growing body of evidence supports a major role in the brain's molecular and behavioral responses to ethanol (EtOH). Although key to understanding the functional biology underlying this role, little is known about the cellular and subcellular expression patterns of CLIC4 in brain and how they are affected by EtOH. METHODS: We used qRT-PCR to assess Clic4 mRNA expression in the medial prefrontal cortex (mPFC) of C57BL/6J mice in the absence and presence of acute EtOH exposure. Two complementary immunohistochemical techniques were employed to assess the subcellular localization of the CLIC4 protein and its pattern of expression across brain cell types in the mPFC in the absence and presence of acute EtOH. RESULTS: Through immunohistochemical and stereological techniques, we show that CLIC4 protein is robustly expressed by oligodendrocytes (most abundant), microglia, and astrocytes, with minimal expression in neurons. Following acute EtOH exposure, we observed a rapid increase in Clic4 mRNA expression in female but not male mice and an overall increase in the number of oligodendrocytes and astrocytes expressing the CLIC4 protein. CONCLUSIONS: These findings suggest that Clic4 functions as an early response gene for acute EtOH in brain, which likely underlies its ability to modulate EtOH behavior. Our results also suggest that the role of CLIC4 in the brain's response to EtOH is mediated through oligodendrocytes.

ß-eudesmol but not atractylodin exerts an inhibitory effect on CFTR-mediated chloride transport in human intestinal epithelial cells.

Oriental herbal medicine with the two bioactive constituents, ß-eudesmol (BE) and atractylodin (AT), has been used as a remedy for gastrointestinal disorders. There was no scientific evidence reporting their antidiarrheal effect and underpinning mechanisms. Therefore, we aimed to investigate the anti-secretory activity of these two compounds in vitro. The inhibitory effect of BE and AT on cAMP-induced Cl- secretion was evaluated by Ussing chamber in human intestinal epithelial (T84) cells. Short-circuit current (ISC) and apical Cl- current (ICl-) were measured after adding indirect and direct cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activator. MTT assay was used to determine cellular cytotoxicity. Protein-ligand interaction was investigated by in silico molecular docking analysis. BE, but not AT concentration-dependently (IC50 of ~1.05 µM) reduced cAMP-mediated, CFTRinh-172 inhibitable Cl- secretion as determined by transepithelial ISC across a monolayer of T84 cells. Potency of CFTR-mediated ICl- inhibition by BE did not change with the use of different CFTR activators suggesting a direct blockage of the channel active site(s). Pretreatment with BE completely prevented cAMP-induced ICl-. Furthermore, BE at concentrations up to 200 µM (24 h) had no effect on T84 cell viability. In silico studies indicated that BE could best dock onto dephosphorylated structure of CFTR at ATP-binding pockets in nucleotide-binding domain (NBD) 2 region. These findings provide the first evidence for the anti-secretory effect of BE involving inhibition of CFTR function. BE represents a promising candidate for the therapeutic or prophylactic intervention of diarrhea resulted from intestinal hypersecretion of Cl.

Calcium-activated chloride channel is involved in the onset of diarrhea triggered by EGFR tyrosine kinase inhibitor treatment in rats.

EGFR tyrosine kinase inhibitors (TKIs) are mainly used to treat non-small cell lung cancer; however, adverse effects such as severe diarrhea represent a major obstacle towards the continuation of EGFR-TKIs therapy. Chloride channels, which control the fluid flow in the intestinal lumen, are proposed as an important target to remediate EGFR-TKIs-induced diarrhea, but the mechanism remains unclear. The aim of this study was to clarify the mechanism underlying EGFR-TKIs-induced diarrhea with a particular focus on the role of intestinal chloride channels. Here, we show that osimertinib-treated rats exhibit diarrhea and an increase in fecal water content without showing any severe histopathological changes. This diarrhea was attenuated by intraperitoneal treatment with the calcium-activated chloride channel (CaCC) inhibitor CaCCinh-A01. These findings were confirmed in afatinib-treated rats with diarrhea. Moreover, treatment with the Japanese traditional herbal medicine, hangeshashinto (HST), decreased fecal water content and improved fecal appearance in rats treated with EGFR-TKIs. HST inhibited the ionomycin-induced CaCC activation in HEK293 cells in patch-clamp current experiments and its active ingredients were identified. In conclusion, secretory diarrhea induced by treatment with EGFR-TKIs might be partially mediated by the activation of CaCC. Therefore, blocking the CaCC could be a potential new treatment for EGFR-TKI-induced diarrhea.

N-acetylcysteine, xCT and suppression of Maxi-chloride channel activity in human placenta.

INTRODUCTION: Placental oxidative stress features in pregnancy pathologies but in clinical trials antioxidant supplementation has not improved outcomes. N-acetylcysteine (NAC) stimulates glutathione production and is proposed as a therapeutic agent in pregnancy. However, key elements of N-acetylcysteine biology, including its cellular uptake mechanism, remains unclear. This study explores how the cystine/glutamate transporter xCT may mediate N-acetylcysteine uptake and how N-acetylcysteine alters placental redox status. METHODS: The involvement of xCT in NAC uptake by the human placenta was studied in perfused placenta and Xenopus oocytes. The effect of short-term N-acetylcysteine exposure on the placental villous proteome was determined using LC-MS. The effect of N-acetylcysteine on Maxi-chloride channel activity was investigated in perfused placenta, villous fragments and cell culture. RESULTS: Maternoplacental N-acetylcysteine administration stimulated intracellular glutamate efflux suggesting a role of the exchange transporter xCT, which was localised to the microvillous membrane of the placental syncytiotrophoblast. Placental exposure to a bolus of N-acetylcysteine inhibited subsequent activation of the redox sensitive Maxi-chloride channel independently of glutathione synthesis. Stable isotope quantitative proteomics of placental villi treated with N-acetylcysteine demonstrated changes in pathways associated with oxidative stress, apoptosis and the acute phase response. DISCUSSION: This study suggests that xCT mediates N-acetylcysteine uptake into the placenta and that N-acetylcysteine treatment of placental tissue alters the placental proteome while regulating the redox sensitive Maxi-chloride channel. Interestingly N-acetylcysteine had antioxidant effects independent of the glutathione pathway. Effective placental antioxidant therapy in pregnancy may require maintaining the balance between normalising redox status without inhibiting physiological redox signalling.

Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia.

COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin 6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.

Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel.

CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC50 = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.

TMEM16A-encoded anoctamin 1 inhibition contributes to chrysin-induced coronary relaxation.

BACKGROUND: Chrysin, a natural flavonoid available in honey, propolis and medicinal plants, has been shown to be vasorelaxant in some vascular beds. Proper intake of an alimental vasodilator as a food additive may be a promising strategy for prevention and treatment of coronary spasmodic disorders. PURPOSE: TMEM16A-encoded anoctamin 1 (ANO1), a Ca2+ activated Cl- channel (CaCC), plays an important role in the modulation of vascular tone. We tested the possibility that inhibition of CaCCs contributes to chrysin-induced coronary arterial relaxation. METHODS: The vascular tone of the rat coronary artery (RCA) was recorded with a wire myograph. CaCC currents were assessed using whole-cell patch clamp in arterial smooth muscle cell (ASMC) freshly isolated from RCAs. An inhibitor study was performed to explore the mechanisms underlying the vasomotor and electrophysiological effects of chrysin. RESULTS: Pre-incubation with chrysin depressed the contractions elicited by thromboxane A2 analog U46619, vasopressin (VP), depolarization and extracellular Ca2+ elevation/depolarization without significant preference among these vasoconstrictors. Besides, chrysin inhibited both intracellular Ca2+ release-dependent and extracellular Ca2+ influx-dependent components of contractions induced by U46619 or VP. In RCAs pre-contracted with U46619, VP or KCl, chrysin elicited concentration-dependent relaxations, which were weakened by Cl- -deprivation. The electrophysiological study showed that chrysin reduced ANO1-antibody-sensitive CaCC currents and depressed CaCC increments induced by U46619. Inhibitor study showed that both the vasorelaxation and the CaCC current reduction induced by chrysin were attenuated by blocking CaCCs and inhibiting cAMP/PKA and NO/PKG pathways. CONCLUSION: The present findings indicate that inhibition of RCA ASMC CaCC currents, which may be consequential following intracellular Ca2+ availability reduction and activation of cAMP/PKA and NO/cGMP signaling pathways, contributes to chrysin-induced RCA relaxation.