Estimation of Chloride Channel Residual Function and Assessment of Targeted Drugs Efficiency in the Presence of a Complex Allele [L467F;F508del] in the CFTR Gene.

Complex alleles of the CFTR gene complicate the diagnosis of cystic fibrosis (CF), the classification of its pathogenic variants, affect the clinical picture of the disease and can affect the efficiency of targeted drugs. The total frequency of complex allele [L467F;F508del] in the Russian population of patients with CF is 0.74%, and in patients with the F508del/F508del genotype, its frequency reaches 8%. This article presents multi-faceted study of the complex allele [L467F;F508del] in a cohort of patients with genotypes [L467F;F508del]/class I (c.3532_3535dup, c.1766+2T>C, W1310X, 712-1G>T), and data for a unique patient with the genotype [L467F;F508del]/[L467F;F508del]. Using the intestinal current measurement method, it was demonstrated the absence of CFTR function for [L467F;F508del]/class I and [L467F;F508del]/[L467F;F508del] genotypes. In intestinal organoids, it was shown that [L467F;F508del] in combination with class I variants and in the homozygotes abolishes the efficacy of both two-component (ivacaftor+lumacaftor; ivacaftor+tezacaftor) and three-component (ivacaftor+tezacaftor+elexacaftor) targeted drugs. When prescribing ivacaftor+tezacaftor+elexacaftor to three patients, they did not have a clinical effect after 6-12 months.

Identification of an ionic mechanism for ERα-mediated rapid excitation in neurons.

The major female ovarian hormone, 17ß-estradiol (E2), can alter neuronal excitability within milliseconds to regulate a variety of physiological processes. Estrogen receptor-α (ERα), classically known as a nuclear receptor, exists as a membrane-bound receptor to mediate this rapid action of E2, but the ionic mechanisms remain unclear. Here, we show that a membrane channel protein, chloride intracellular channel protein-1 (Clic1), can physically interact with ERα with a preference to the membrane-bound ERα. Clic1-mediated currents can be enhanced by E2 and reduced by its depletion. In addition, Clic1 currents are required to mediate the E2-induced rapid excitations in multiple brain ERα populations. Further, genetic disruption of Clic1 in hypothalamic ERα neurons blunts the regulations of E2 on female body weight balance. In conclusion, we identified the Clic1 chloride channel as a key mediator for E2-induced rapid neuronal excitation, which may have a broad impact on multiple neurobiological processes regulated by E2.

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

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

In Silico Analysis: Molecular Characterization and Evolutionary Study of CLCN Gene Family in Buffalo.

Chloride channels (ClCs) have received global interest due to their significant role in the regulation of ion homeostasis, fluid transport, and electrical excitability of tissues and organs in different mammals and contributing to various functions, such as neuronal signaling, muscle contraction, and regulating the electrolytes' balance in kidneys and other organs. In order to define the chloride voltage-gated channel (CLCN) gene family in buffalo, this study used in silico analyses to examine physicochemical properties, evolutionary patterns, and genome-wide identification. We identified eight CLCN genes in buffalo. The ProtParam tool analysis identified a number of important physicochemical properties of these proteins, including hydrophilicity, thermostability, in vitro instability, and basic nature. Based on their evolutionary relationships, a phylogenetic analysis divided the eight discovered genes into three subfamilies. Furthermore, a gene structure analysis, motif patterns, and conserved domains using TBtool demonstrated the significant conservation of this gene family among selected species over the course of evolution. A comparative amino acid analysis using ClustalW revealed similarities and differences between buffalo and cattle CLCN proteins. Three duplicated gene pairs were identified, all of which were segmental duplications except for CLCN4-CLCN5, which was a tandem duplication in buffalo. For each gene pair, the Ka/Ks test ratio findings showed that none of the ratios was more than one, indicating that these proteins were likely subject to positive selection. A synteny analysis confirmed a conserved pattern of genomic blocks between buffalo and cattle. Transcriptional control in cells relies on the binding of transcription factors to specific sites in the genome. The number of transcription factor binding sites (TFBSs) was higher in cattle compared to buffalo. Five main recombination breakpoints were identified at various places in the recombination analysis. The outcomes of our study provide new knowledge about the CLCN gene family in buffalo and open the door for further research on candidate genes in vertebrates through genome-wide studies.

A Focus on the Proximal Tubule Dysfunction in Dent Disease Type 1.

Dent disease type 1 is a rare X-linked recessive inherited renal disorder affecting mainly young males, generally leading to end-stage renal failure and for which there is no cure. It is caused by inactivating mutations in the gene encoding ClC-5, a 2Cl-/H+ exchanger found on endosomes in the renal proximal tubule. This transporter participates in reabsorbing all filtered plasma proteins, which justifies why proteinuria is commonly observed when ClC-5 is defective. In the context of Dent disease type 1, a proximal tubule dedifferentiation was shown to be accompanied by a dysfunctional cell metabolism. However, the exact mechanisms linking such alterations to chronic kidney disease are still unclear. In this review, we gather knowledge from several Dent disease type 1 models to summarize the current hypotheses generated to understand the progression of this disorder. We also highlight some urinary biomarkers for Dent disease type 1 suggested in different studies.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The Ca2+-activated Cl- channel TMEM16B shapes the response time course of olfactory sensory neurons.

Mammalian olfactory sensory neurons (OSNs) generate an odorant-induced response by sequentially activating two ion channels, which are in their ciliary membranes. First, a cationic, Ca2+-permeable cyclic nucleotide-gated channel is opened following odorant stimulation via a G protein-coupled transduction cascade and an ensuing rise in cAMP. Second, the increase in ciliary Ca2+ opens the excitatory Ca2+-activated Cl- channel TMEM16B, which carries most of the odorant-induced receptor current. While the role of TMEM16B in amplifying the response has been well established, it is less understood how this secondary ion channel contributes to response kinetics and action potential generation during single as well as repeated stimulation and, on the other hand, which response properties the cyclic nucleotide-gated (CNG) channel determines. We first demonstrate that basic membrane properties such as input resistance, resting potential and voltage-gated currents remained unchanged in OSNs that lack TMEM16B. The CNG channel predominantly determines the response delay and adaptation during odorant exposure, while the absence of the Cl- channels shortens both the time the response requires to reach its maximum and the time to terminate after odorant stimulation. This faster response termination in Tmem16b knockout OSNs allows them, somewhat counterintuitively despite the large reduction in receptor current, to fire action potentials more reliably when stimulated repeatedly in rapid succession, a phenomenon that occurs both in isolated OSNs and in OSNs within epithelial slices. Thus, while the two olfactory ion channels act in concert to generate the overall response, each one controls specific aspects of the odorant-induced response. KEY POINTS: Mammalian olfactory sensory neurons (OSNs) generate odorant-induced responses by activating two ion channels sequentially in their ciliary membranes: a Na+, Ca2⁺-permeable cyclic nucleotide-gated (CNG) channel and the Ca2⁺-activated Cl⁻ channel TMEM16B. The CNG channel controls response delay and adaptation during odorant exposure, while TMEM16B amplifies the response and influences the time required for the response to reach its peak and terminate. OSNs lacking TMEM16B display faster response termination, allowing them to fire action potentials more reliably during rapid repeated stimulation. The CNG and TMEM16B channels have distinct and complementary roles in shaping the kinetics and reliability of odorant-induced responses in OSNs.

BK channels promote action potential repolarization in skeletal muscle but contribute little to myotonia.

Patients with myotonia congenita suffer from slowed relaxation of muscle (myotonia), due to hyperexcitability caused by loss-of-function mutations in the ClC-1 chloride channel. A recent study suggested that block of large-conductance voltage- and Ca2+- activated K+ channels (BK) may be effective as therapy. The mechanism underlying efficacy was suggested to be lessening of the depolarizing effect of build-up of K+ in t-tubules of muscle during repetitive firing. BK channels are widely expressed in the nervous system and have been shown to play a central role in regulation of excitability, but their contribution to muscle excitability has not been determined. We performed intracellular recordings as well as force measurements in both wild type and BK-/- mouse extensor digitorum longus muscles. Action potential width was increased in BK-/- muscle due to slowing of repolarization, consistent with the possibility K+ build-up in t-tubules is lessened by block of BK channels in myotonic muscle. However, there was no difference in the severity of myotonia triggered by block of muscle Cl- channels with 9-anthracenecarboxylic acid (9AC) in wild type and BK-/- muscle fibers. Further study revealed no difference in the interspike membrane potential during repetitive firing suggesting there was no reduction in K+ build-up in t-tubules of BK-/- muscle. Force recordings following block of muscle Cl- channels demonstrated little reduction in myotonia in BK-/- muscle. In contrast, the current standard of care, mexiletine, significantly reduced myotonia. Our data suggest BK channels regulate muscle excitability, but are not an attractive target for therapy of myotonia.

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

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

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

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