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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 V 0.5 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Neuromuscular blocking agents are used commonly to induce skeletal muscle relaxation during surgery. While muscle relaxation facilitates surgical procedures and tracheal intubation, adequate recovery of muscle function after surgery is required to support pulmonary function, and even mild residual neuromuscular block increases the risk of severe postoperative pulmonary complications. While recovery of muscle function after surgery involving neuromuscular blocking agents can be monitored and, in addition, be accelerated by use of current antagonists (reversal agents), there is a clear clinical need for a safe drug to antagonize all types of neuromuscular blocking agents. Here, we show that inhibition of the skeletal muscle-specific chloride ion (Cl - ) channel, the ClC-1 channel, markedly accelerates recovery of both single contraction (twitch) and, important physiologically, sustained (tetanic) contractions in a rat model mimicking neuromuscular blocking agent-induced muscle block used during surgery. This suggests ClC-1 inhibition as a mechanism for fast and efficacious recovery of neuromuscular function induced by any neuromuscular blocking agents., (© 2024. The Author(s).)

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.

Paramecium exhibits responsive behavior to environmental changes, moving either closer to or further away from stimuli. Electrophysiological experiments have revealed that these behavioral responses are controlled by membrane potentials. Anoctamin, a Ca 2+ -activated Cl - channel, is involved in the regulation of membrane potential in mammals. However, it remains uncertain whether Cl - channels like anoctamin regulate Paramecium behavior. Herein, replacement of external Cl - ions with acetate ion and application of Cl - channel blocker niflumic acid (NFA, 0.1 μM) increased spontaneous avoiding reactions (sARs). Hence, we hypothesized that anoctamin is involved in the stabilization of membrane potential fluctuation. Paramecium cells in which the anoctamin-like protein 1 gene was knocked down displayed frequent sARs in the culture medium without external stimulation. Treatment of anoctamin-like protein 1-knockdown cells with the Ca 2+ chelator BAPTA or Ca-channel blocker nicardipine reversed the increase in sARs. Electrophysiological experiments revealed extension of membrane depolarization when positive currents were applied to anoctamin-like protein 1-knockdown cells. We concluded that anoctamin-like protein 1 works as a Cl-channel and stabilizes the membrane potential oscillation, reducing sARs., (© 2024 International Society of Protistologists.)

TMEM16 proteins, also known as anoctamins, are a family of ten membrane proteins with various tissue expression and subcellular localization. TMEM16A (anoctamin 1) is a plasma membrane protein that acts as a calcium-activated chloride channel. It is expressed in many types of epithelial cells, smooth muscle cells and some neurons. In airway epithelial cells, TMEM16A expression is particularly enhanced by inflammatory stimuli that also promote goblet cell metaplasia and mucus hypersecretion. Therefore, pharmacological modulation of TMEM16A could be beneficial to improve mucociliary clearance in chronic obstructive respiratory diseases. However, the correct approach to modulate TMEM16A activity (activation or inhibition) is still debated. Pharmacological inhibitors of TMEM16A could also be useful as anti-hypertensive agents given the TMEM16A role in smooth muscle contraction. In contrast to TMEM16A, TMEM16F (anoctamin 6) behaves as a calcium-activated phospholipid scramblase, responsible for the externalization of phosphatidylserine on cell surface. Inhibitors of TMEM16F could be useful as anti-coagulants and anti-viral agents. The role of other anoctamins as therapeutic targets is still unclear since their physiological role is still to be defined., Competing Interests: Declaration of competing interest The authors declare that there is a patent application for the compounds ARN7149, ARN4550, and ARN11391 mentioned in the submitted review. There are no other conflicts of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Leucine-rich repeat-containing protein 8 A (LRRC8A) protein is a critical member of volume-regulated anion channels. It plays a critical roles in the regulation of cellular volume and involves in the development of diseases like osteoarthritis. Screening of lead compounds to modulate its function may provide potential therapeutics of related diseases. Here, we employ virtual screening techniques and molecular dynamics (MD) simulation to screen potential inhibitors against LRRC8A. LRRC8A was regarded as the drug target to investigate potential compounds from the ZINC15 database via molecular docking. The final compound was selected among the top 10 Autodock Vina score (-8.8 Kcal/mol) with the ZINC ID ZINC000018195627 after druggability prediction. The docked complex from the virtual screening was subjected to MD simulation to analyze the stability of the LRRC8A protein-ligand complex, with parameters including root mean square deviation, root mean square fluctuation and radius of gyration. Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method was further employed to predict the binding free energies from MD simulation trajectory. Our study provides insightful analysis for the potential compound to modulate LRRC8A and lay the foundation of therapeutics development against osteoarthritis.Communicated by Ramaswamy H. Sarma.

Chemical or drug treatments are successfully used to treat parasitic nematode infections that impact human, animal and plant health. Many of these exert their effects through modifying neural function underpinning behaviours essential for parasite viability. Selectivity against the parasite may be achieved through distinct pharmacological properties of the parasite nervous system, as exemplified by the success of the ivermectin which target a glutamate-gated chloride channel found only in invertebrates. Despite the success of the ivermectins, emerging resistance and concerns around eco-toxicity are driving the search for new nematocidal chemicals or drugs. Here, we describe the potential of a 5-HT-gated chloride channel MOD-1, which is involved in vital parasite behaviours with constrained distribution in the invertebrate phyla. This ion channel has potential pharmacophores that could be targeted by new nematocidal chemicals and drugs. We have developed a microtiter based bioassay for MOD-1 pharmacology based on its ectopic expression in the Caenorhabditis elegans essential neuron M4. We have termed this technology 'PhaGeM4' for 'Pharmacogenetic targeting of M4 neuron'. Exposure of transgenic worms harbouring ectopically expressed MOD-1 to 5-HT results in developmental arrest. By additional expression of a fluorescence marker in body wall muscle to monitor growth we demonstrate that this assay is suitable for the identification of receptor agonists and antagonists. Indeed, the developmental progression is a robustly quantifiable bioassay that resolves MOD-1 activation by quipazine, 5-carboxyamidotryptamine and fluoxetine and highlight methiothepin as a potent antagonist. This assay has the intrinsic ability to highlight compounds with optimal bioavailability and furthermore to filter out off-target effects. It can be extended to the investigation of other classes of membrane receptors and modulators of neuronal excitation. This approach based on heterologous modulation of the essential M4 neuron function offers a route to discover new effective and selective anthelmintics potentially less confounded by disruptive environmental impact., (Copyright © 2022. Published by Elsevier Inc.)

Background/aim: Chloride intracellular channel protein 1 (CLIC1) is known as a promoter of cancer progression, metastasis, and angiogenesis. Thus, CLIC1 could be a future therapeutic target. This study aimed to evaluate the effect of anti-CLIC1 antibodies on tumour cells and vessels of human renal cell carcinoma (RCC) in rabbit cornea and chick embryo chorioallantoic membrane (CAM) models., Materials and Methods: Human cc-RCC xenografts on rabbit cornea and CAM surface were performed. Anti-CLIC1 antibodies were applied for 5 consecutive days on both tumor models. We comparatively evaluated treated and untreated tumors by combining ultrasonography with microscopic techniques., Results: RCC implants rapidly recruited blood vessels and had an exponential growth rate on both tumor models. Anti-CLIC1 antibodies suppressed tumor growth by inducing tumor cell necrosis. Tumor vessels regressed rapidly but not completely during anti-CLIC1 antibodies based therapy., Conclusion: Anti-CLIC1 antibodies induced tumor necrosis and tumor vasculature regression in human cc-RCC xenografts in both in vivo experimental models., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background and Purpose: Nematode glutamate-gated chloride channels (GluCls) are targets of ivermectin (IVM) and moxidectin (MOX), structurally dissimilar macrocyclic lactone (ML) anthelmintics. IVM and MOX possess different pharmacokinetics and efficacy profiles but are thought to have the same binding site, through which they allosterically activate GluCls, apart from the GLC-2 receptor, which is antagonized by IVM. Our goal was to determine GLC-2 sensitivity to MOX, investigate residues involved in antagonism of GLC-2, and to identify differences in receptor-level pharmacology between IVM and MOX., Experimental Approach: Two-electrode voltage clamp electrophysiology was used to study the pharmacology of Caenorhabditis elegans GLC-2 receptors heterologously expressed in Xenopus laevis oocytes. In silico homology modeling identified Cel-GLC-2 residues Met291 and Gln292 at the IVM binding site that differ from other GluCls; we mutated these residues to those found in ML-sensitive GluCls, and those of filarial nematode GLC-2., Key Results: We discovered that MOX inhibits wild-type C. elegans GLC-2 receptors roughly 10-fold more potently than IVM, and with greater maximal inhibition of glutamate activation (MOX = 86.9 ± 2.5%; IVM = 57.8 ± 5.9%). IVM was converted into an agonist in the Met291Gln mutant, but MOX remained an antagonist. Glutamate responses were abrogated in a Met291Leu Gln292Thr double mutant (mimicking filarial nematode GLC-2), but MOX and IVM were converted into positive allosteric modulators of glutamate at this construct., Conclusions and Implications: Our data provides new insights into differences in receptor-level pharmacology between IVM and MOX and identify residues responsible for ML antagonism of GLC-2., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Transmembrane protein 206 (TMEM206), a proton-activated chloride channel, has been implicated in various biochemical processes, including bone metabolism, and has emerged as a novel cancer-related protein in multiple tumor types. However, its role in primary malignant bone tumors, particularly in osteosarcoma (OS), remains unclear. This study is aimed at exploring the effects of TMEM206 gene silencing on the proliferation, migration, invasion, and metastasis of human OS cells in vitro and in vivo using an shRNA-knockdown strategy. We found that TMEM206 is frequently overexpressed and that high levels of TMEM206 correlated with clinical stage and pulmonary metastasis in patients with OS. We provided evidence that TMEM206-silenced OS cancer cells exhibit decreased proliferation, migration, and invasion in vitro . Mechanistically, we identified β -catenin, a key member of Wnt/ β -catenin signaling, as a downstream effector of TMEM206. TMEM206 silencing inhibits the Wnt/ β -catenin signaling pathway in expression rescue experiments, confirming that TMEM206 silencing attenuates OS cell tumorigenic behavior, at least in part, via the β -catenin mediated downregulation of Wnt/ β -catenin signaling. More importantly, TMEM206 knockdown-related phenotype changes were replicated in a xenograft nude mouse model where pulmonary metastases of OS cells were suppressed. Together, our results demonstrate that silencing TMEM206 negatively modulates the Wnt/ β -catenin signaling pathway via β -catenin to suppress proliferation, migration, invasion, and metastasis in OS carcinogenesis, suggesting TMEM206 as a potential oncogenic biomarker and a potential target for OS treatment., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Fei Peng et al.)

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., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Pentameric ligand-gated ion channels (pLGICs) activated by the inhibitory neurotransmitter γ-aminobutyric acid (GABA) are expressed widely in both vertebrate and invertebrate species. One of the best characterised insect GABA-gated chloride channels is RDL, an abbreviation of 'resistance to dieldrin', that was originally identified by genetic screening in Drosophila melanogaster. Here we have cloned the analogous gene from the bumblebee Bombus terrestris audax (BtRDL) and examined its pharmacological properties by functional expression in Xenopus oocytes. Somewhat unexpectedly, the sensitivity of BtRDL to GABA, as measured by its apparent affinity (EC50), was influenced by heterologous expression conditions. This phenomenon was observed in response to alterations in the amount of cRNA injected; the length of time that oocytes were incubated before functional analysis; and by the presence or absence of a 3' untranslated region. In contrast, similar changes in expression conditions were not associated with changes in apparent affinity with RDL cloned from D. melanogaster (DmRDL). Changes in apparent affinity with BtRDL were also observed following co-expression of a chaperone protein (NACHO). Similar changes in apparent affinity were observed with an allosteric agonist (propofol) and a non-competitive antagonist (picrotoxinin), indicating that expression-depended changes are not restricted to the orthosteric agonist binding site. Interestingly, instances of expression-dependent changes in apparent affinity have been reported previously for vertebrate glycine receptors, which are also members of the pLGIC super-family. Our observations with BtRDL are consistent with previous data obtained with vertebrate glycine receptors and indicates that agonist and antagonist apparent affinity can be influenced by the level of functional expression in a variety of pLGICs., Competing Interests: Syngenta provided support in the form of salary for JG. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Chloride intracellular channel 1 (CLIC1) is a sensor of oxidative stress in endothelial cells (EC). However, the mechanism by which CLIC1 mediate the regulation of endothelial dysfunction has not been established. In this study, overexpressed CLIC1 impaired the ability of the vascular cells to resist oxidative damage and promoted cellular senescence. Besides, suppressed CLIC1 protected against cellular senescence and dysfunction in Human Umbilical Vein Endothelial Cells (HUVECs) through the Nrf2/HO-1 pathway. We also found that ROS-activated CLIC1-induced oxidative stress in HUVECs. Nrf2 nuclear translocation was inhibited by CLIC1 overexpression, but was enhanced by IAA94 (CLICs inhibitor) treatment or knockdown of CLIC1. The Nrf2/HO-1 pathway plays a critical role in the anti-oxidative effect of suppressing CLIC1. And inhibition of CLIC1 decreases oxidative stress injury by downregulating the levels of ROS, MDA, and the expression of EC effectors (ICAM1 and VCAM1) protein expression and promotes the activity of superoxide dismutase (SOD). The AMPK-mediated signaling pathway activates Nrf2 through Nrf2 phosphorylation and nuclear translocation, which is also regulated by CLIC1. Moreover, the activation of CLIC1 contributes to H 2 O 2 -induced mitochondrial dysfunction and activation of mitochondrial fission. Therefore, elucidation of the mechanisms by which CLIC1 is involved in these pivotal pathways may uncover its therapeutic potential in alleviating ECs oxidative stress and age-related cardiovascular disease development.

Oral squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a high incidence of recurrence and distant metastasis. However, the mechanism of epithelial to mesenchymal transition (EMT) during tumor progression and metastasis in OSCC has not yet been fully elucidated. It is well known that the Cl - channel controls cell volume and activates several signaling pathways for cell differentiation. The aim of the present study was to investigate the role of the Cl - channel on EMT in the OSC 20 cell line, which is an OSCC line. OSC-20 cells were cultured with low serum medium containing a Cl - channel blocker NPPB. Morphological changes, gene expression, immunoreactivity, cell volume, and signaling pathway of the NPPB-treated OSC-20 cells were evaluated. The NPPB-treated OSC-20 cells showed typical morphology of mesenchymal cells. The expression levels of the epithelial marker E-cadherin in the NPPB-treated OSC-20 cells were lower than those of the untreated and TGF-β1-treated OSC-20 cells. On the other hand, mesenchymal markers such as vimentin, ZEB1, and Snail, in the NPPB-treated OSC-20 cells were higher than those in the untreated and TGF-β1-treated OSC-20 cells. Furthermore, a large number of vimentin-positive cells also appeared in the NPPB-treated OSC-20 cells. Additionally, the cell volume of these cells was significantly increased compared to that of the untreated and TGF-β1-treated cells. Interestingly, NPPB did not activate the TGF-β/smad signaling pathway, but activated the Wnt/β-catenin signaling pathway. These results suggest that Cl - channel dysfunction promoted EMT via activation of the Wnt/β-catenin signaling pathway in OSCC., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cataracts have a high incidence and prevalence rate worldwide, and they are the leading cause of blindness. Lens epithelial cell (LEC) apoptosis is often analysed in cataract research since it is the pathological basis of cataracts, except for congenital cataract. Chloride channels are present in ocular tissues, such as in trabecular cells, LECs and other cells. They serve an important role in apoptosis and participate in endoplasmic reticulum (ER) stress and oxidative stress. However, their role in the apoptosis of LECs has not been discussed. The present study examined the effects of the chloride channel blocker 5‑nitro‑2‑(3‑phenylpropylamino) benzoic acid (NPPB) in human LECs (HLECs) to elucidate the role of NPPB in HLECs and investigate the role and mechanism of chloride channels in cataract formation. HLECs were exposed to NPPB. Cell survival rate was evaluated using Cell Counting Kit‑8 assays. Oxidative stress was detected as reactive oxygen species (ROS) in cells by using a ROS assay kit. Apoptosis was examined by assessing mitochondrial membrane potential and using a JC‑1 assay kit, and western blot analysis was performed to measure the expression levels of mitochondrial‑dependent apoptosis pathway‑associated proteins. ER stress was evaluated by determining the intracellular calcium ion fluorescence intensity, and western blot analysis was performed to measure ER stress‑associated protein expression. The results revealed that NPPB treatment decreased the viability of HLECs and increased apoptosis. Additionally, NPPB increased intracellular ROS levels, as well as the number of JC‑1 monomers and the protein expression levels of B‑cell lymphoma‑2 (Bcl‑2)‑associated X and cleaved caspase‑3, and decreased Bcl‑2 protein expression. NPPB increased intracellular calcium ions, the protein expression levels of activating transcription factor 6, JNK, C/EBP homologous protein and caspase‑12, and the phosphorylation of protein kinase R‑like endoplasmic reticulum kinase. N‑acetylcysteine and 4‑phenylbutyric acid inhibited NPPB‑induced oxidative stress, ER stress and apoptosis. Therefore, NPPB treatment decreased cell viability and promoted apoptosis of HLECs via the promotion of oxidative and ER stress.

Objectives: Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line., Methods: Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays., Results: VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane., Conclusions: We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 (IC 50 = 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., Competing Interests: Competing interest statement: A.K.K., J.D.B., and M.M. have filed for a patent “Compositions and Methods to Modulate Chloride Ion Channel Activity,” USSN 16/449,021 from the U.S. Patent & Trademark Office, January 16, 2020.

Purpose: In the present study, we attempted to explore the role of chloride channel 3 (CLC-3) in colorectal cancer (CRC) and its related mechanism., Methods: First, the expression level of CLC-3 in CRC tumor tissues and cell lines were measured by RT-qPCR, immunohistochemistry or western blot analysis. CLC-3 expression knockdown in CRC cells was achieved by siRNA transfection. The effect of CLC-3 silence on cell viability, cell cycle, invasion and migration of CRC was estimated by CCK8, flow cytometry based cell cycle assay, and transwell assay, respectively. In order to investigate whether Wnt/β-catenin signaling was perturbed by CLC-3 knockdown, CLC-3 knockdown cells were treated with pathway activator LiCl, followed by the measurement of the expressions of pathway related genes, cell viability, cell cycle, metastasis ability., Results: The expression of CLC-3 was gradually increased from normal adjacent tissues to CRC tumor tissues, and the increase in tumor tissues was related to TNM stages. CLC-3 was overexpressed in four CRC cell lines (HCT116, SW480, LoVo and SW620), compared with NCM460 cells. CLC-3 knockdown significantly reduced cell proliferation, invasion and migration ability, reflected by declined cell viability, arrested G0/G1 cell cycle, decreased invasion and migration ability. In contrast, the declined cell proliferation, invasion and migration of LoVo and SW620 cells induced by CLC-3 knockdown were reversed by the addition of Wnt/β-catenin activator LiCl., Conclusion: CLC-3 contributed to the CRC development and metastasis through Wnt/β-catenin signaling pathway. CLC-3 could be proposed as the candidate target for CRC treatment., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The use of synthetic ion transporters for alteration of the concentration of ions across cell membranes has drawn attention from scientists over the last two decades. This ion transport property has been sensibly used to reduce the viability of cancer cells mainly due to the disruption of their ion homeostasis, leading to the perturbation of their abnormal pH gradient. The use of the proanionophore strategy has been recently adopted to increase cellular deliverability and reduce unwanted cytotoxicity towards normal cells. Meanwhile, various anionophores exhibiting non-toxic behavior in epithelial cells have shown a great propensity to be used as a putative treatment for channelopathies like cystic fibrosis. Anionophores with Cl - ion transport mediated antibacterial activities have also been successfully used against clinically relevant bacterial strains, many of which are tolerant towards commercial antibiotics. Recent developments in the chloride ion transport mediated biological activities of anionophores have been mainly discussed in this article. It also highlights other aspects, such as design criteria, targeted delivery options, and others, which can be useful for the further development of selective anionophores for specific biological applications.

Excitatory α7 neuronal nicotinic receptors (nAChR) are widely expressed in the central and peripheral nervous and immune systems and are important for learning, memory, and immune response regulation. Specific α7 nAChR ligands, including positive allosteric modulators are promising to treat cognitive disorders, inflammatory processes, and pain. One of them, PNU-120596, highly increased the neuron response to α7 agonists and retarded desensitization, showing selectivity for α7 as compared to heteromeric nAChRs, but was not examined at the inhibitory ligand-gated channels. We studied PNU-120596 action on anion-conducting channels using voltage-clamp techniques: it slightly potentiated the response of human glycine receptors expressed in PC12 cells, of rat GABA A receptors in cerebellar Purkinje cells and mouse GABA A Rs heterologously expressed in Xenopus oocytes. On the contrary, PNU-120596 exerted an inhibitory effect on the receptors mediating anion currents in Lymnaea stagnalis neurons: two nAChR subtypes, GABA and glutamate receptors. Acceleration of the current decay, contrary to slowing down desensitization in mammalian α7 nAChR, was observed in L. stagnalis neurons predominantly expressing one of the two nAChR subtypes. Thus, PNU-120596 effect on these anion-selective nAChRs was just opposite to the action on the mammalian cation-selective α7 nAChRs. A comparison of PNU-120596 molecule docked to the models of transmembrane domains of the human α7 AChR and two subunits of L. stagnalis nAChR demonstrated some differences in contacts with the amino acid residues important for PNU-120596 action on the α7 nAChR. Thus, our results show that PNU-120596 action depends on a particular subtype of these Cys-loop receptors., (© 2020 International Society for Neurochemistry.)

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 Ca 2+ 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 Ca 2+ elevation/depolarization without significant preference among these vasoconstrictors. Besides, chrysin inhibited both intracellular Ca 2+ release-dependent and extracellular Ca 2+ 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 Ca 2+ availability reduction and activation of cAMP/PKA and NO/cGMP signaling pathways, contributes to chrysin-induced RCA relaxation., (Copyright © 2020. Published by Elsevier Masson SAS.)

SCO-101 (Endovion) was discontinued 20 years ago as a new drug under development against sickle cell anaemia. Data from the phase 1 studies remained unpublished. New data indicate that SCO-101 might be efficacious as add-on therapy in cancer. Thus, we report the results from the four phase 1 trials performed between 2001 and 2002. Adult volunteers received SCO-101 or placebo in four independent trials. Adverse events were recorded, and SCO-101 was determined for pharmacokinetic analysis. Ninety-two volunteers completed the trials. The most remarkable adverse effect was a transient and dose-dependent increase in unconjugated bilirubin. Plasma SCO-101 elimination was approximately log linear, with apparent oral clearances of between 315 and 2103 mL/h for single doses, and between 121 and 2433 mL/h at steady state following oral administration. There was a marked decrease in clearance with increasing dose, and for repeated dose versus single dose. T max was greater, and C max and AUC ∞ were lower in the fed state compared to the fasted state. Exposure was equivalent in males and females and for African Americans and Caucasians. In conclusion, SCO-101 appears to be a safe drug with a predictable PK profile. Its efficacy as add-on to standard anticancer drugs has yet to be defined., (© 2020 The Authors. Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Macrophage foam cell formation and inflammation are a pathological hallmark of atherosclerosis. ClC-2 has been implicated in various pathological processes, including inflammation and lipid metabolic disorder. However, the functional role of ClC-2 in macrophage foam cell formation and inflammation is unclear. Here, we found that ClC-2 was dominantly expressed in macrophages of atherosclerotic plaque and increased in atherogenesis. Knockdown of ClC-2 inhibited ox-LDL -induced lipid uptake and deposition in macrophages. The increase in CD36 expression and the decrease in ABCA1 expression induced by ox-LDL were alleviated by ClC-2 downregulation. Further, ClC-2 lacking limited the ox-LDL-induced secretion of inflammatory cytokines and chemokine, and suppressed Nlrp3 inflammasome activation. Restoration of Nlrp3 expression reversed the effect of ClC-2 downregulation on macrophage lipid accumulation and inflammation. Collectively, our study demonstrates that ClC-2 knockdown ameliorates ox-LDL-induced macrophage foam cell formation and inflammation by inhibiting Nlrp3 inflammasome activation.

Using the patch-clamp method in the whole cell configuration, it was shown that new conjugates of 2-aminothiophene-3-carboxylic acid with adamantane derivatives exhibit the ability to modulate CaCC activity in the single Purkinje neurons of rat cerebellum. It was noted that, depending on the nature of the substitution in the thiophene fragment, the nature of the effect on CaCC varies from inhibition to potentiation of CaCC currents. The described compounds are also blockers of the NMDA receptor ifenprodile site, which may have an additional neuroprotective contribution to the spectrum of biological activity of these compounds.

Chloride intracellular channel protein 4 (CLIC4) has been implicated in different types of cancers, but the role of CLIC4 in the development of gastric cancer (GC) remains unknown. We analyzed the expression of CLIC4 in 102 pairs of gastric adenocarcinomas by western blot and real-time PCR. Our data revealed that the expression of CLIC4 is reduced in GC tumor tissues compared with adjacent normal tissues. The expression levels of CLIC4 correlate inversely with the clinical stage of GC. CLIC4 expression is lowest in MKN45 cells, which have the highest tumorigenic potential and express the highest levels of cancer stem cell markers CD44 and OCT4, compared with N87 and AGS cells. Exogenous overexpression of CLIC4 downregulated the expression of CD44 and OCT4, and inhibited migration, invasion and epithelial-mesenchymal transition (EMT). Moreover, anchorage-independent growth of GC cells was decreased and the cells became more sensitive to 5-fluorouracil and etoposide treatment when CLIC4 was overexpressed. The ability of N87 cells to form tumors in nude mice was enhanced when CLIC4 was silenced. We, for the first time, demonstrate that CLIC4 suppresses tumor growth by inhibiting cancer cell stemness and EMT., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Adenosine plays an important role in various aspects of kidney physiology, but the specific targets and mechanisms of actions are not completely understood. The collecting duct has the highest expression of adenosine receptors, particularly adenosine A 1 receptors (A 1 Rs). Interstitial adenosine levels are greatly increased up to a micromolar range in response to dietary salt loading. We have previously shown that the basolateral membrane of principal cells has primarily K + conductance mediated by K ir 4.1/5.1 channels to mediate K + recycling and to set up a favorable driving force for Na + /K + exchange (47). Intercalated cells express the Cl - ClC-K2/b channel mediating transcellular Cl - reabsorption. Using patch-clamp electrophysiology in freshly isolated mouse collecting ducts, we found that acute application of adenosine reversely inhibits ClC-K2/b open probability from 0.31 ± 0.04 to 0.17 ± 0.06 and to 0.10 ± 0.05 for 1 and 10 µM, respectively. In contrast, adenosine (10 µM) had no measureable effect on K ir 4.1/5.1 channel activity in principal cells. The inhibitory effect of adenosine on ClC-K2/b was abolished in the presence of the A 1 R blocker 8-cyclopentyl-1,3-dipropylxanthine (10 µM). Consistently, application of the A 1 R agonist N 6 -cyclohexyladenosine (1 µM) recapitulated the inhibitory action of adenosine on ClC-K2/b open probability. The effects of adenosine signaling in the collecting duct were independent from its purinergic counterpartner, ATP, having no measurable actions on ClC-K2/b and K ir 4.1/5.1. Overall, we demonstrated that adenosine selectively inhibits ClC-K2/b activity in intercalated cells by targeting A 1 Rs. We propose that inhibition of transcellular Cl - reabsorption in the collecting duct by adenosine would aid in augmenting NaCl excretion during high salt intake.

5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) is a non-specific chloride channel blocker. Peritoneal adhesion is an inevitable complication of abdominal surgery and remains an important clinical problem, leading to chronic pain, intestinal obstruction, and female infertility. The aim of this study is to observe the effects of NPPB on peritoneal adhesions and uncover the underlying mechanism. The formation of postoperative peritoneal adhesions was induced by mechanical injury to the peritoneum of rats. MTT assay and wound-healing assay were used to evaluate proliferation and migration of primary cultured adhesion fibroblasts (AFB) respectively. Whole-cell chloride currents were measured using a fully automated patch-clamp workstation. Cell volume changes were monitored by light microscopy and video imaging. Our results demonstrated that NPPB could significantly prevent the formation of peritoneal adhesion in rats and inhibit the proliferation of AFB in a concentration-dependent manner. NPPB also reduced the migration of AFB cells with an IC 50 of 53.09 μM. A 47% hypotonic solution successfully activated the I Cl,vol in AFB cells. The current could be blocked by extracellular treatment with NPPB. Moreover, 100 μM NPPB almost completely eliminated the capacity of regulatory volume decrease (RVD) in these cells. These data indicate that NPPB could prevent the formation of postoperative peritoneal adhesions. The possible mechanism may be through the inhibition of the proliferation and migration of AFB cells by modulating I Cl,vol and cell volume. These results suggest a potential clinical use of NPPB for preventing the formation of peritoneal adhesions.

Ca 2+ -activated Cl - channels (CaCCs) wildly exist in many tissues which play an important role in ion transport and excitation conduction, especially fluid secretion and smooth muscle contraction in epithelial tissues. TMEM16A as a classic CaCC expresses in the intestine, and has become a potential target of intestinal physiological and pathological researches and therapeutic drug screening. In this study, we identified trans-δ-viniferin (TVN), a resveratrol dimmer, could inhibit TMEM16A activity in TMEM16A expressed FRT cells with IC 50 of 19.7 μM, it also prevented Ca 2+ -activated Cl - current in HT-29 cells with IC 50 of 4.65 μM and in colonic mucosa. In the mechanism studies, TVN showed no significant inhibition on CFTR and basal Na + /K + -ATPase in both intestinal epithelial cells and colonic tissues, except for inhibition of calcium concentration and Ca 2+ -activated K + channel to some degree. In anti-diarrheal studies, TVN could effectively prevent diarrhea caused by rotavirus infection and reduce the pellet number in IBS-D mice. These physiological effects are at least partially attributed to the inhibitory effect of TVN on CaCC-mediated intestinal fluid secretion and the reduction of smooth muscle contraction force by inhibiting TMEM16A. Collectively, the present study identified a new pharmacological target of TVN which provided the theoretical basis for the application of TVN in the treatment of rotavirus-infected diarrhea and IBS-D., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The mechanisms by which common risk variants of small effect interact to contribute to complex genetic disorders are unclear. Here, we apply a genetic approach, using isogenic human induced pluripotent stem cells, to evaluate the effects of schizophrenia (SZ)-associated common variants predicted to function as SZ expression quantitative trait loci (eQTLs). By integrating CRISPR-mediated gene editing, activation and repression technologies to study one putative SZ eQTL (FURIN rs4702) and four top-ranked SZ eQTL genes (FURIN, SNAP91, TSNARE1 and CLCN3), our platform resolves pre- and postsynaptic neuronal deficits, recapitulates genotype-dependent gene expression differences and identifies convergence downstream of SZ eQTL gene perturbations. Our observations highlight the cell-type-specific effects of common variants and demonstrate a synergistic effect between SZ eQTL genes that converges on synaptic function. We propose that the links between rare and common variants implicated in psychiatric disease risk constitute a potentially generalizable phenomenon occurring more widely in complex genetic disorders.

The swelling-activated chloride current ( I Cl,swell ) is induced when a cell swells and plays a central role in maintaining cell volume in response to osmotic stress. The major contributor of I Cl,swell is the volume-regulated anion channel (VRAC). Leucine-rich repeat containing 8A (LRRC8A; SWELL1) was recently identified as an essential component of VRAC, but the mechanisms of VRAC activation are still largely unknown; moreover, other Cl - channels, such as anoctamin 1 (ANO1), were also suggested to contribute to I Cl,swell . In this present study, we investigated the roles of LRRC8A and ANO1 in activation of I Cl,swell ; we also explored the role of intracellular Ca 2+ in I Cl,swell activation. We used a CRISPR/Cas9 gene editing approach, electrophysiology, live fluorescent imaging, selective pharmacology, and other approaches to show that both LRRC8A and ANO1 can be activated by cell swelling in HEK293 cells. Yet, both channels contribute biophysically and pharmacologically distinct components to I Cl,swell , with LRRC8A being the major component. Cell swelling induced oscillatory Ca 2+ transients, and these Ca 2+ signals were required to activate both the LRRC8A- and ANO1-dependent components of I Cl,swell . Both I Cl,swell components required localized rather than global Ca 2+ for activation. Interestingly, while intracellular Ca 2+ was necessary and sufficient to activate ANO1, it was necessary but not sufficient to activate LRRC8A-mediated currents. Finally, Ca 2+ transients linked to the I Cl,swell activation were mediated by the G protein-coupled receptor-independent PLC isoforms.

Background: Although ClC-2 channels are important in colonic Cl - secretion, it is unclear about their roles in small intestinal anion secretion. Therefore, we sought to examine whether ClC-2 channels play important roles in anion secretion, particularly duodenal bicarbonate secretion (DBS)., Methods: Duodenal mucosae from mice were stripped of seromuscular layers and mounted in Ussing chambers. Both duodenal short-circuit current (I sc ) and HCO 3 - secretion in vitro were simultaneously recorded. DBS in vivo was measured by a CO 2 -sensitive electrode., Results: Lubiprostone, a selective ClC-2 activator, concentration-dependently increased both duodenal I sc and DBS only when applied basolaterally, but not when applied apically. Removal of extracellular Cl - abolished lubiprostone-induced duodenal I sc , but did not alter HCO 3 - secretion even in the presence of DIDS, a Cl - /HCO 3 - exchanger inhibitor. However, further addition of glibenclamide, a CFTR channel blocker, abolished lubiprostone-evoked HCO 3 - secretion. Moreover, lubiprostone-induced HCO 3 - secretion was impaired in CFTR -/- mice compared to wild-type littermates. Luminal perfusion of duodenal lumen with lubiprostone did not alter basal DBS in vivo, but lubiprostone (i.p.) was able to induce DBS, which was also significantly inhibited by Cd 2+ , a ClC-2 channel blocker. [Ca 2+ ] cyt level, Ca 2+ -activated K + channel- and cAMP-mediated duodenal I sc , and HCO 3 - secretion were unchanged by lubiprostone., Conclusions: We have provided the first evidence for the novel functional role of basolateral ClC-2 channels in the regulation of duodenal anion secretion.

The aim of the present study was to establish a cell model of volume-regulated anion channel subunit LRRC8A and investigate the physiological characteristics of LRRC8A. The eukaryotic expression vectors of LRRC8A and YFP-H148Q/I152L were constructed and transfected into Fischer rat thyroid (FRT) cells by Lipofectamine 2000. The FRT cell lines co-expressing LRRC8A and YFP-H148Q/I152L were obtained by antibiotic screening. The expression of LRRC8A and YFP-H148Q/I152L in FRT cells was detected by the inverted fluorescence microscope. The fluorescence quenching kinetic experiment was done to verify the function and effectiveness of the cell model. Then the cell model was utilized to study the physiological characteristics of LRRC8A, such as the characteristics of anion transport, the opening of LRRC8A by osmotic pressure, the effect of anion transport velocity, and the effect of chloride channel inhibitors on LRRC8A anion channel. The results of the inverted fluorescence microscope showed that LRRC8A was expressed on the cell membrane and YFP-H148Q/I152L was expressed in the cytoplasm. The results of fluorescence quenching kinetic test showed that under the condition of low osmotic state, LRRC8A could transport some kinds of anions, such as iodine and chloride ions. Osmotic pressure played a key role in the regulation of LRRC8A volume-regulated anion channel opening. Chloride channel inhibitors inhibited ion transport of LRRC8A channel in a dose-dependent manner. It is suggested that LRRC8A has the characteristics of classic volume-regulated anion channels by using the cell model of FRT cells co-expressing LRRC8A and YFP-H148Q/I152L.

Background and Purpose: Anion-selective Cys-loop receptors (GABA and glycine receptors) provide the main inhibitory drive in the CNS. Both types of receptor operate via chloride-selective ion channels, though with different kinetics, pharmacological profiles, and localization. Disequilibrium in their function leads to a variety of disorders, which are often treated with allosteric modulators. The few available GABA and glycine receptor channel blockers effectively suppress inhibitory currents in neurons, but their systemic administration is highly toxic. With the aim of developing an efficient light-controllable modulator of GABA receptors, we constructed azobenzene-nitrazepam (Azo-NZ1), which is composed of a nitrazepam moiety merged to an azobenzene photoisomerizable group., Experimental Approach: The experiments were carried out on cultured cells expressing Cys-loop receptors of known subunit composition and in brain slices using patch-clamp. Site-directed mutagenesis and molecular modelling approaches were applied to evaluate the mechanism of action of Azo-NZ1., Key Results: At visible light, being in trans-configuration, Azo-NZ1 blocked heteromeric α1/β2/γ2 GABA A receptors, ρ2 GABA A (GABA C ), and α2 glycine receptors, whereas switching the compound into cis-state by UV illumination restored the activity. Azo-NZ1 successfully photomodulated GABAergic currents recorded from dentate gyrus neurons. We demonstrated that in trans-configuration, Azo-NZ1 blocks the Cl-selective ion pore of GABA receptors interacting mainly with the 2' level of the TM2 region., Conclusions and Implications: Azo-NZ1 is a soluble light-driven Cl-channel blocker, which allows photo-modulation of the activity induced by anion-selective Cys-loop receptors. Azo-NZ1 is able to control GABAergic postsynaptic currents and provides new opportunities to study inhibitory neurotransmission using patterned illumination., (© 2019 Institut de Neurosciences des Systémes. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Chloride intracellular channel 1 (CLIC1) is a promising therapeutic target in cancer due to its intrinsic characteristics; it is overexpressed in specific tumor types and its localization changes from cytosolic to surface membrane depending on activities and cell cycle progression. Ca 2+ and reactive oxygen species (ROS) are critical signaling molecules that modulate diverse cellular functions, including cell death. In this study, we investigated the function of CLIC1 in Ca 2+ and ROS signaling in A549 human lung cancer cells. Depletion of CLIC1 via shRNAs in A549 cells increased DNA double-strand breaks both under control conditions and under treatment with the putative anticancer agent chelerythrine, accompanied by a concomitant increase in the p-JNK level. CLIC1 knockdown greatly increased basal ROS levels, an effect prevented by BAPTA-AM, an intracellular calcium chelator. Intracellular Ca 2+ measurements clearly showed that CLIC1 knockdown significantly increased chelerythrine-induced Ca 2+ signaling as well as the basal Ca 2+ level in A549 cells compared to these levels in control cells. Suppression of extracellular Ca 2+ restored the basal Ca 2+ level in CLIC1-knockdown A549 cells relative to that in control cells, implying that CLIC1 regulates [Ca 2+ ] i through Ca 2+ entry across the plasma membrane. Consistent with this finding, the L-type Ca 2+ channel (LTCC) blocker nifedipine reduced the basal Ca 2+ level in CLIC1 knockdown cells to that in control cells. Taken together, our results demonstrate that CLIC1 knockdown induces an increase in the intracellular Ca 2+ level via LTCC, which then triggers excessive ROS production and consequent JNK activation. Thus, CLIC1 is a key regulator of Ca 2+ signaling in the control of cancer cell survival.

Thromboxane A 2 (TXA 2 ) has been implicated in the pathogenesis of vascular complications, but the underlying mechanism remains unclear. The contraction of renal arterial rings in mice was measured by a Multi Myograph System. The intracellular calcium concentration ([Ca 2+ ] i ) in vascular smooth muscle cells (VSMCs) was obtained by using a fluo-4/AM dye and a confocal laser scanning microscopy. The results show that the U46619-induced vasoconstriction of renal artery was completely blocked by a TXA 2 receptor antagonist GR32191, significantly inhibited by a selective phospholipase C (PI-PLC) inhibitor U73122 at 10 μmol/L and partially inhibited by a Phosphatidylcholine - specific phospholipase C (PC-PLC) inhibitor D609 at 50 μmol/L. Moreover, the U46619-induced vasoconstriction was inhibited by a general protein kinase C (PKC) inhibitor chelerythrine at 10 μmol/L, and a selective PKCδ inhibitor rottlerin at 10 μmol/L. In addition, the PKC-induced vasoconstriction was partially inhibited by a Rho-kinase inhibitor Y-27632 at 10 μmol/L and was further completely inhibited together with a putative IP 3 receptor antagonist and store-operated Ca 2+ (SOC) entry inhibitor 2-APB at 100 μmol/L. On the other hand, U46619-induced vasoconstriction was partially inhibited by L-type calcium channel (Cav1.2) inhibitor nifedipine at 1 μmol/L and 2-APB at 50 and 100 μmol/L. Last, U46619-induced vasoconstriction was partially inhibited by a cell membrane Ca 2+ activated C1 - channel blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) at 50 and 100 μmol/L. Our results suggest that the U46619-induced contraction of mouse intrarenal arteries is mediated by Cav1.2 and SOC channel, through the activation of thromboxane-prostanoid receptors and its downstream signaling pathway., (© 2019 John Wiley & Sons Australia, Ltd.)

Secretory diarrhea is one of the most common causes of death world-wide especially in children under 5 years old. Isoliquiritigenin (ISLQ), a plant-derived chalcone, has previously been shown to exert anti-secretory action in vitro and in vivo by inhibiting CFTR Cl - channels. However, its CFTR inhibition potency is considerably low (IC 50  > 10 μM) with unknown mechanism of action. This study aimed to identify novel chalcone derivatives with improved potency and explore their mechanism of action. Screening of 27 chalcone derivatives identified CHAL-025 as the most potent chalcone analog that reversibly inhibited CFTR-mediated Cl - secretion in T84 cells with an IC 50 of ∼1.5 μM. As analyzed by electrophysiological and biochemical analyses, the mechanism of CFTR inhibition by CHAL-025 is through AMP-activated protein kinase (AMPK), a negative regulator of CFTR activity. Furthermore, Western blot analyses and molecular dynamics (MD) results suggest that CHAL-025 activates AMPK by binding at the allosteric site of an upstream kinase calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, CHAL-025 inhibited both cholera toxin (CT) and bile acid-induced Cl - secretion in T84 cells and prevented CT-induced intestinal fluid secretion in mice. Therefore, CHAL-025 represents a promising anti-diarrheal agent that inhibits CFTR Cl - channel activity via CaMKKβ-AMPK pathways., (Copyright © 2019 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Neonatal seizures are different from adult seizures, and many antiepileptic drugs that are effective in adults often fail to treat neonates. Here, we report that gluconate inhibits neonatal seizure by inhibiting CLC-3 chloride channels. We detect a voltage-dependent outward rectifying Cl - current mediated by CLC-3 Cl - channels in early developing brains but not adult mouse brains. Blocking CLC-3 Cl - channels by gluconate inhibits seizure activity both in neonatal brain slices and in neonatal animals with in vivo EEG recordings. Consistently, neonatal neurons of CLC-3 knockout mice lack the outward rectifying Cl - current and show reduced epileptiform activity upon stimulation. Mechanistically, we demonstrate that activation of CLC-3 Cl - channels alters intracellular Cl - homeostasis and enhances GABA excitatory activity. Our studies suggest that gluconate can suppress neonatal seizure activities through inhibiting CLC-3 Cl - channels in developing brains.

Multiple types of Cl - channels regulate smooth muscle excitability and contractility in vascular, gastrointestinal, and airway smooth muscle cells. However, little is known about Cl - channels in detrusor smooth muscle (DSM) cells. Here, we used inside-out single channel and whole cell patch-clamp recordings for detailed biophysical and pharmacological characterizations of Cl - channels in freshly isolated guinea pig DSM cells. The recorded single Cl - channels displayed unique gating with multiple subconductive states, a fully opened single-channel conductance of 164 pS, and a reversal potential of -41.5 mV, which is close to the E Cl of -65 mV, confirming preferential permeability to Cl - . The Cl - channel demonstrated strong voltage dependence of activation (half-maximum of mean open probability, V 0.5 , ~-20 mV) and robust prolonged openings at depolarizing voltages. The channel displayed similar gating when exposed intracellularly to solutions containing Ca 2+ -free or 1 mM Ca 2+ . In whole cell patch-clamp recordings, macroscopic current demonstrated outward rectification, inhibitions by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid, and insensitivity to chlorotoxin. The outward current was reversibly reduced by 94% replacement of extracellular Cl - with I - , Br - , or methanesulfonate (MsO - ), resulting in anionic permeability sequence: Cl - >Br - >I - >MsO - . While intracellular Ca 2+ levels (0, 300 nM, and 1 mM) did not affect the amplitude of Cl - current and outward rectification, high Ca 2+ slowed voltage-step current activation at depolarizing voltages. In conclusion, our data reveal for the first time the presence of a Ca 2+ -independent DIDS and niflumic acid-sensitive, voltage-dependent Cl - channel in the plasma membrane of DSM cells. This channel may be a key regulator of DSM excitability.

Immune cells of myeloid origin show robust expression of ATP-gated P2X7 receptors, two-transmembrane ion channels permeable to Na + , K + , and Ca 2+ Receptor activation promotes inflammasome activation and release of the proinflammatory cytokines IL-1β and IL-18. In this study, we show that ATP generates facilitating cationic currents in monocyte-derived human macrophages and permeabilizes the plasma membrane to polyatomic cationic dyes. We find that antagonists of PLA 2 and Cl - channels abolish P2X7 receptor-mediated current facilitation, membrane permeabilization, blebbing, phospholipid scrambling, inflammasome activation, and IL-1β release. Our data demonstrate significant differences in the actions of ATP in murine and human macrophages and suggest that PLA 2 and Cl - channels mediate innate immunity downstream of P2X7 receptors in human macrophages., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Glutamate-gated chloride channel receptors (GluClRs) mediate inhibitory neurotransmission at invertebrate synapses and are primary targets of parasites that impact drastically on agriculture and human health. Ivermectin (IVM) is a broad-spectrum pesticide that binds and potentiates GluClR activity. Resistance to IVM is a major economic and health concern, but the molecular and synaptic mechanisms of resistance are ill-defined. Here we focus on GluClRs of the agricultural endoparasite, Haemonchus contortus. We demonstrate that IVM potentiates inhibitory input by inducing a tonic current that plateaus over 15 minutes and by enhancing post-synaptic current peak amplitude and decay times. We further demonstrate that IVM greatly enhances the active durations of single receptors. These effects are greatly attenuated when endogenous IVM-insensitive subunits are incorporated into GluClRs, suggesting a mechanism of IVM resistance that does not affect glutamate sensitivity. We discovered functional groups of IVM that contribute to tuning its potency at different isoforms and show that the dominant mode of access of IVM is via the cell membrane to the receptor., Competing Interests: The authors have declared that no competing interests exist.

Glutamate-gated chloride channels (GluCls) are found only in invertebrates and mediate fast inhibitory neurotransmission. The structural and functional diversity of GluCls are produced through assembly of multiple subunits and via posttranscriptional alternations. Alternative splicing is the most common way to achieve this in insect GluCls and splicing occurs primarily at exons 3 and 9. As expression pattern and pharmacological properties of exon 9 alternative splices in invertebrate GluCls remain poorly understood, the cDNAs encoding three alternative splice variants (9a, 9b and 9c) of the PxGluCl gene from the diamondback moth Plutella xylostella were constructed and their pharmacological characterizations were examined using electrophysiological studies. Alternative splicing of exon 9 had little to no impact on PxGluCl sensitivity towards the agonist glutamate when subunits were singly or co-expressed in Xenopus oocytes. In contrast, the allosteric modulator abamectin and the chloride channel blocker fipronil had differing effects on PxGluCl splice variants. PxGluCl9c channels were more resistant to abamectin and PxGluCl9b channels were more sensitive to fipronil than other homomeric channels. In addition, heteromeric channels containing different splice variants showed similar sensitivity to abamectin (except for 9c) and reduced sensitivity to fipronil than homomeric channels. These findings suggest that functionally indistinguishable but pharmacologically distinct GluCls could be formed in P. xylostella and that the upregulated constitutive expression of the specific variants may contribute to the evolution of insecticide resistance in P. xylostella and other arthropods., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chloride channels represent a group of potential drug targets; their blockers showed significant protecting effect on impaired cells by modulating apoptosis, autophagy, and other cell signals. However, clinical drugs with chloride channel inhibitory properties have not yet been developed. Natural product extract becomes an underlying candidate satisfied the clinical requirements for its low toxicity, low cost, and abundant sources. Here, a fluorescence-based EYFP-H148Q/I153L-HeLa cell line model was constructed by molecular cloning and verified by real-time polymerase chain reaction and Western blotting assay. By using this chloride channel blocker screening model, seven hit compounds chosen from 6988 natural compounds showed the channel blocking activity. Then the hit compounds were further validated by electrophysiological patch-clamp analysis. Our study preliminarily identified PC-4 as a new chloride channel inhibitor and demonstrated the reliability and sensitivity of fluorescence-based high throughput screening technologies for discovery of biologically active compounds from natural herbal compounds.

The aim of this study was to develop a method to silence a very specific set of cells in a spatially and temporally refined manner. Here, an approach is presented that combines the use of a transgenic mouse line, expressing cre recombinase under a nestin promoter, with lentiviral delivery of a floxed, ivermectin (IVM)-gated chloride channel construct to the dentate gyrus. This approach was used to express an IVM-sensitive chloride channel in newly born granule cells in adult mouse brains, and its ability to silence neuronal activity was tested by analyzing the effect on immediate early gene expression in vitro in cre-transgenic primary neuronal cultures. IVM treatment of cells expressing the chloride channel prevented gabazine-induced expression of the immediate early gene product EGR1, while cells expressing a control inactive channel or no channel retained their EGR1 response. Thus, a genetic strategy is presented for targeting a specific neurogenic niche for transgene expression in the adult mouse brain, and proof of principle is shown that it can be used in vitro as a method for silencing neuronal activity.

Background: Although pharmacological treatment has increased the average life expectancy of patients with cystic fibrosis, the median survival of females is shorter than that of males. In vitro and in vivo studies have shown that estrogens play a relevant role in the disease progression. The aim of this study was to investigate the effects of 17β-estradiol and tamoxifen citrate (TMX) on calcium-activated chloride channel (CaCC) currents in human bronchial epithelial cells carrying the ΔPhe508-CFTR mutation both in homozygosis and in heterozygosis., Methods: Perforated patch clamp experiments were performed on single cells of the immortalized cell lines CFBE and IB3-1. Gramicidin (10 or 20 μM) was added to the electrode solution to reach the whole cell configuration. The electrical stimulation protocol consisted of square voltages ranging from - 80 to + 80 mV, in steps of 20 mV and with a duration of 800 msec., Results: The presence of 17β-estradiol significantly reduced the CaCC currents, both in basal conditions and in the presence of ATP (100 μM). The addition of TMX (10 μM) completely restored the currents abolished by 17β-estradiol, in basal conditions and after stimulation with ATP in both CFBE and IB3-1 cells. TMX had a strong, direct action on membrane current density, which significantly increased more than 4-fold in both cases. The membrane current stimulation produced by TMX was further enhanced by the addition of ATP. CFBE cells incubated for 24 h with 3 μM VX-809 (a CFTR corrector) and then acutely stimulated with VX-770 (a CFTR potentiator) in the presence of forskolin, showed an increase of chloride currents which were abolished by Inh-172. The chloride current density induced by TMX + ATP was, on average, greater than that obtained with VX-809 + VX-770 + forskolin. The currents elicited by TMX + ATP were abolished by the addition of NPPB, a CaCC inhibitor. The combined administration of TMX/ATP and VXs/FSK had an additional effect on chloride currents., Conclusions: Our results show that TMX restores CaCC currents inhibited by 17ß-estradiol and directly activates the transmembrane chloride currents potentiated by ATP, an effect which is mutation independent. The combined effect of TMX with current used treatments for cystic fibrosis could be of benefit to patients.

Fluxametamide is a novel wide-spectrum insecticide that was discovered and synthesized by Nissan Chemical Industries, Ltd. To identify the mode of action of fluxametamide, we first performed [ 3 H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) binding assays. Fluxametamide potently inhibited the specific binding of [ 3 H]EBOB to housefly-head membranes, suggesting that fluxametamide affects insect γ-aminobutyric acid (GABA)-gated chloride channels (GABACls). Next, the antagonism of housefly GABACls and glutamate-gated chloride channels (GluCls) was examined using the two-electrode voltage clamp (TEVC) method. Fluxametamide inhibited agonist responses in both ion channels expressed in Xenopus oocytes in the nanomolar range, indicating that this insecticide is a ligand-gated chloride channel (LGCC) antagonist. The insecticidal and LGCC antagonist potencies of fluxametamide against fipronil-susceptible and fipronil-resistant strains of small brown planthoppers and two-spotted spider mites, which are insensitive to fipronil, were evaluated. Fluxametamide exhibited similar levels of both activities in these fipronil-susceptible and fipronil-resistant arthropod pests. These data indicate that fluxametamide exerts distinctive antagonism of arthropod GABACls by binding to a site different from those for existing antagonists. In contrast to its profound actions on the arthropod LGCCs, the antagonistic activity of fluxametamide against rat GABACls and human glycine-gated chloride channels was nearly insignificant, suggesting that fluxametamide has high target-site selectivity for arthropods over mammals. Overall, fluxametamide is a new type of LGCC antagonist insecticide with excellent safety for mammals at the target-site level., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Phagocytes destroy ingested microbes by producing hypochlorous acid (HOCl) from chloride ions (Cl - ) and hydrogen peroxide within phagolysosomes, using the enzyme myeloperoxidase. HOCl, the active ingredient in bleach, has antibacterial/antiviral properties. As myeloperoxidase is needed for HOCl production, non-myeloid cells are considered incapable of producing HOCl. Here, we show that epithelial, fibroblast and hepatic cells have enhanced antiviral activity in the presence of increasing concentrations of sodium chloride (NaCl). Replication of enveloped/non-enveloped, DNA (herpes simplex virus-1, murine gammaherpesvirus 68) and RNA (respiratory syncytial virus, influenza A virus, human coronavirus 229E, coxsackievirus B3) viruses are inhibited in a dose-dependent manner. Whilst treatment with sodium channel inhibitors did not prevent NaCl-mediated virus inhibition, a chloride channel inhibitor reversed inhibition by NaCl, suggesting intracellular chloride is required for antiviral activity. Inhibition is also reversed in the presence of 4-aminobenzoic hydrazide, a myeloperoxidase inhibitor, suggesting epithelial cells have a peroxidase to convert Cl - to HOCl. A significant increase in intracellular HOCl production is seen early in infection. These data suggest that non-myeloid cells possess an innate antiviral mechanism dependent on the availability of Cl - to produce HOCl. Antiviral activity against a broad range of viral infections can be augmented by increasing availability of NaCl.

Ca 2+ -activated Cl - channels (CaCCs) are anionic channels that regulate many important physiological functions associated with chloride and calcium flux in some somatic cells. The molecular identity of CaCCs was revealed to be TMEM16A and TMEM16B (also known as Anoctamin or ANO1 and ANO2, respectively) in all eukaryotes. A recent study suggests the presence of TMEM16A in human sperm and a relationship with the rhZP-induced acrosome reaction. However, to the best of our knowledge, little is known about the role of TMEM16A in other spermatic processes such as capacitation or motility. In this study, we evaluated the effects of two TMEM16A antagonists on capacitation, acrosome reaction, and motility in guinea pig sperm; these antagonists were T16Ainh-A01, belonging to a second generation of potent antagonists of TMEM16A, and niflumic acid (NFA), a well-known antagonist of TMEM16A (CaCCs). First of all, we confirmed that the absence of Cl - in the capacitation medium changes motility parameters, capacitation, and the progesterone-induced acrosome reaction. Using a specific antibody, TMEM16A was found as a protein band of ∼120 kDa, which localization was in the apical crest of the acrosome and the middle piece of the flagellum. Inhibition of TMEM16A by T16Ainh-A01 affected sperm physiology by reducing capacitation, blocking the progesterone-induced acrosome reaction under optimal capacitation conditions, inhibiting progressive motility, and the acquisition of hyperactivated motility, diminishing [Ca 2+ ]i, and increasing [Cl - ]i. These changes in sperm kinematic parameters provide new evidence of the important role played by TMEM16A in the production of sperm capable of fertilizing oocytes., (© 2018 Wiley Periodicals, Inc.)

Background: Mishandling of intracellular chloride (Cl - ) concentration ([Cl - ] i ) in cerebrovascular smooth muscle cells is implicated in several pathological processes, including hyperplasia and remodeling. We investigated the effects of ClC-2-mediated Cl - efflux on the proliferation of human brain vascular smooth muscle cells (HBVSMCs) induced by angiotensin II (AngII)., Methods: Cell proliferation and motility were determined using the CCK-8, bromodeoxyuridine staining, wound healing and invasion assays. ClC-2, PCNA, Ki67, survivin and cyclin D1 expression, and β-catenin and GSK-3β phosphorylation were examined using western blotting. Histological analyses were performed using hematoxylin and eosin staining and α-SMA staining., Results: Our results showed that AngII-induced HBVSMC proliferation was accompanied by a decrease in [Cl - ] i and an increase in ClC-2 expression. Inhibition of ClC-2 by siRNA prevented AngII from inducing the efflux of Cl - . AngII-induced HBVSMC proliferation, migration and invasion were significantly attenuated by ClC-2 downregulation. The inhibitory effects of ClC-2 knockout on HBVSMC proliferation and motility were associated with inactivation of the Wnt/β-catenin signaling pathway, as evidenced by inhibition of β-catenin phosphorylation and nuclear translocation, and decrease of GSK-3β phosphorylation and survivin and cyclin D1 expression. Recombinant Wnt3a treatment markedly reversed the effect of ClC-2 knockdown on HBVSMC viability. An in vivo study revealed that knockdown of ClC-2 with shRNA adenovirus ameliorated basilar artery remodeling by inhibiting Wnt/β-catenin signaling in AngII-treated mice., Conclusion: This study demonstrates that blocking ClC-2-mediated Cl - efflux inhibits AngII-induced cerebrovascular smooth muscle cell proliferation and migration by inhibiting the Wnt/β-catenin pathway. Our data indicate that downregulation of ClC-2 may be a viable strategy in the prevention of hyperplasia and remodeling of cerebrovascular smooth muscle cells., Competing Interests: All animal procedures were in accordance with the institutional guidelines of the Henan People’s Hospital and were approved by the Institutional Animal Ethics Committee.This study is approved by all authors for publication.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Morbidity and mortality associated with diarrheal diseases remain significant burdens on global health. In the developing world, the major sources of secretory diarrhea are infectious, including those caused by bacteria such as enterotoxic Escherichia coli , and viruses such as rotavirus. In many cases of secretory diarrhea, activation of pathways for cyclic nucleotides and/or Ca 2+ signaling in the apical membrane of enterocytes increases the conductance of Cl - channels at the enterocyte lumen-facing membrane. Those channels include the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca 2+ -activated Cl - channel (CaCC). Inhibition of enterocyte Cl - channels is an effective strategy for anti-secretory drug therapy. Small molecules and natural peptides with Cl - channel inhibitory activity have shown efficacy in diarrhea models. Screening of natural peptides via the patch-clamp technique provides evidence that such channel inhibition by an extract of black tea may be responsible for its anti-diarrhea benefits.