Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 3.892
Filter
1.
Nat Commun ; 15(1): 3978, 2024 May 10.
Article in English | MEDLINE | ID: mdl-38729926

ABSTRACT

A key mechanism employed by plants to adapt to salinity stress involves maintaining ion homeostasis via the actions of ion transporters. While the function of cation transporters in maintaining ion homeostasis in plants has been extensively studied, little is known about the roles of their anion counterparts in this process. Here, we describe a mechanism of salt adaptation in plants. We characterized the chloride channel (CLC) gene AtCLCf, whose expression is regulated by WRKY transcription factor under salt stress in Arabidopsis thaliana. Loss-of-function atclcf seedlings show increased sensitivity to salt, whereas AtCLCf overexpression confers enhanced resistance to salt stress. Salt stress induces the translocation of GFP-AtCLCf fusion protein to the plasma membrane (PM). Blocking AtCLCf translocation using the exocytosis inhibitor brefeldin-A or mutating the small GTPase gene AtRABA1b/BEX5 (RAS GENES FROM RAT BRAINA1b homolog) increases salt sensitivity in plants. Electrophysiology and liposome-based assays confirm the Cl-/H+ antiport function of AtCLCf. Therefore, we have uncovered a mechanism of plant adaptation to salt stress involving the NaCl-induced translocation of AtCLCf to the PM, thus facilitating Cl- removal at the roots, and increasing the plant's salinity tolerance.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Cell Membrane , Chloride Channels , Golgi Apparatus , Salt Stress , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/physiology , Arabidopsis/drug effects , Cell Membrane/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Golgi Apparatus/metabolism , Chloride Channels/metabolism , Chloride Channels/genetics , Gene Expression Regulation, Plant , Protein Transport/drug effects , Salt Tolerance/genetics , Sodium Chloride/pharmacology , Plants, Genetically Modified
2.
PLoS Pathog ; 20(5): e1012245, 2024 May.
Article in English | MEDLINE | ID: mdl-38768235

ABSTRACT

Albendazole (a benzimidazole) and ivermectin (a macrocyclic lactone) are the two most commonly co-administered anthelmintic drugs in mass-drug administration programs worldwide. Despite emerging resistance, we do not fully understand the mechanisms of resistance to these drugs nor the consequences of delivering them in combination. Albendazole resistance has primarily been attributed to variation in the drug target, a beta-tubulin gene. Ivermectin targets glutamate-gated chloride channels (GluCls), but it is unknown whether GluCl genes are involved in ivermectin resistance in nature. Using Caenorhabditis elegans, we defined the fitness costs associated with loss of the drug target genes singly or in combinations of the genes that encode GluCl subunits. We quantified the loss-of-function effects on three traits: (i) multi-generational competitive fitness, (ii) fecundity, and (iii) development. In competitive fitness and development assays, we found that a deletion of the beta-tubulin gene ben-1 conferred albendazole resistance, but ivermectin resistance required the loss of two GluCl genes (avr-14 and avr-15). The fecundity assays revealed that loss of ben-1 did not provide any fitness benefit in albendazole conditions and that no GluCl deletion mutants were resistant to ivermectin. Next, we searched for evidence of multi-drug resistance across the three traits. Loss of ben-1 did not confer resistance to ivermectin, nor did loss of any single GluCl subunit or combination confer resistance to albendazole. Finally, we assessed the development of 124 C. elegans wild strains across six benzimidazoles and seven macrocyclic lactones to identify evidence of multi-drug resistance between the two drug classes and found a strong phenotypic correlation within a drug class but not across drug classes. Because each gene affects various aspects of nematode physiology, these results suggest that it is necessary to assess multiple fitness traits to evaluate how each gene contributes to anthelmintic resistance.


Subject(s)
Anthelmintics , Caenorhabditis elegans , Drug Resistance , Ivermectin , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/drug effects , Anthelmintics/pharmacology , Drug Resistance/genetics , Ivermectin/pharmacology , Alleles , Genetic Fitness/drug effects , Albendazole/pharmacology , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Chloride Channels/genetics , Chloride Channels/metabolism , Tubulin/genetics , Tubulin/metabolism , Selection, Genetic
3.
Head Neck Pathol ; 18(1): 40, 2024 May 10.
Article in English | MEDLINE | ID: mdl-38727794

ABSTRACT

BACKGROUND: Odontogenic lesions constitute a heterogeneous group of lesions. CLIC4 protein regulates different cellular processes, including epithelial-mesenchymal transition and fibroblast-myofibroblast transdifferentiation. This study analyzed CLIC4, E-cadherin, Vimentin, and α-SMA immunoexpression in epithelial odontogenic lesions that exhibit different biological behavior. METHODS: It analyzed the immunoexpression of CLIC4, E-cadherin, and Vimentin in the epithelial cells, as well as CLIC4 and α-SMA in the mesenchymal cells, of ameloblastoma (AM) (n = 16), odontogenic keratocyst (OKC) (n = 20), and adenomatoid odontogenic tumor (AOT) (n = 8). Immunoexpressions were categorized as score 0 (0% positive cells), 1 (< 25%), 2 (≥ 25% - < 50%), 3 (≥ 50% - < 75%), or 4 (≥ 75%). RESULTS: Cytoplasmic CLIC4 immunoexpression was higher in AM and AOT (p < 0.001) epithelial cells. Nuclear-cytoplasmic CLIC4 was higher in OKC's epithelial lining (p < 0.001). Membrane (p = 0.012) and membrane-cytoplasmic (p < 0.001) E-cadherin immunoexpression were higher in OKC, while cytoplasmic E-cadherin expression was higher in AM and AOT (p < 0.001). Vimentin immunoexpression was higher in AM and AOT (p < 0.001). Stromal CLIC4 was higher in AM and OKC (p = 0.008). Similarly, α-SMA immunoexpression was higher in AM and OKC (p = 0.037). Correlations in these proteins' immunoexpression were observed in AM and OKC (p < 0.05). CONCLUSIONS: CLIC4 seems to regulate the epithelial-mesenchymal transition, modifying E-cadherin and Vimentin expression. In mesenchymal cells, CLIC4 may play a role in fibroblast-myofibroblast transdifferentiation. CLIC4 may be associated with epithelial odontogenic lesions with aggressive biological behavior.


Subject(s)
Ameloblastoma , Cadherins , Chloride Channels , Epithelial-Mesenchymal Transition , Odontogenic Tumors , Vimentin , Humans , Epithelial-Mesenchymal Transition/physiology , Chloride Channels/metabolism , Chloride Channels/analysis , Cadherins/metabolism , Odontogenic Tumors/pathology , Odontogenic Tumors/metabolism , Ameloblastoma/pathology , Ameloblastoma/metabolism , Vimentin/metabolism , Adult , Female , Odontogenic Cysts/pathology , Odontogenic Cysts/metabolism , Male , Actins/metabolism , Young Adult , Middle Aged , Antigens, CD/metabolism , Adolescent
4.
Genes (Basel) ; 15(5)2024 Apr 26.
Article in English | MEDLINE | ID: mdl-38790184

ABSTRACT

The ionic toxicity induced by salinization has adverse effects on the growth and development of crops. However, researches on ionic toxicity and salt tolerance in plants have focused primarily on cations such as sodium ions (Na+), with very limited studies on chloride ions (Cl-). Here, we cloned the homologous genes of Arabidopsis thaliana AtCLCc, GhCLCc-1A/D, from upland cotton (Gossypium hirsutum), which were significantly induced by NaCl or KCl treatments. Subcellular localization showed that GhCLCc-1A/D were both localized to the tonoplast. Complementation of Arabidopsis atclcc mutant with GhCLCc-1 rescued its salt-sensitive phenotype. In addition, the silencing of the GhCLCc-1 gene led to an increased accumulation of Cl- in the roots, stems, and leaves of cotton seedlings under salt treatments, resulting in compromised salt tolerance. And ectopic expression of the GhCLCc-1 gene in Arabidopsis reduced the accumulation of Cl- in transgenic lines under salt treatments, thereby enhancing salt tolerance. These findings elucidate that GhCLCc-1 positively regulates salt tolerance by modulating Cl- accumulation and could be a potential target gene for improving salt tolerance in plants.


Subject(s)
Arabidopsis , Chloride Channels , Chlorides , Gene Expression Regulation, Plant , Gossypium , Plant Proteins , Plants, Genetically Modified , Salt Tolerance , Gossypium/genetics , Gossypium/metabolism , Gossypium/growth & development , Salt Tolerance/genetics , Chloride Channels/genetics , Chloride Channels/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Chlorides/metabolism , Plants, Genetically Modified/genetics , Sodium Chloride/metabolism
5.
Int J Mol Sci ; 25(10)2024 May 07.
Article in English | MEDLINE | ID: mdl-38791133

ABSTRACT

Identifying alterations caused by aging could be an important tool for improving the diagnosis of cardiovascular diseases. Changes in vascular tone regulation involve various mechanisms, like NO synthase activity, activity of the sympathetic nervous system, production of prostaglandin, endothelium-dependent relaxing, and contracting factors, etc. Surprisingly, Ca2+-dependent Cl- channels (CaCCs) are involved in all alterations of the vascular tone regulation mentioned above. Furthermore, we discuss these mechanisms in the context of ontogenetic development and aging. The molecular and electrophysiological mechanisms of CaCCs activation on the cell membrane of the vascular smooth muscle cells (VSMC) and endothelium are explained, as well as the age-dependent changes that imply the activation or inhibition of CaCCs. In conclusion, due to the diverse intracellular concentration of chloride in VSMC and endothelial cells, the activation of CaCCs depends, in part, on intracellular Ca2+ concentration, and, in part, on voltage, leading to fine adjustments of vascular tone. The activation of CaCCs declines during ontogenetic development and aging. This decline in the activation of CaCCs involves a decrease in protein level, the impairment of Ca2+ influx, and probably other alterations in vascular tone regulation.


Subject(s)
Aging , Calcium , Chloride Channels , Muscle, Smooth, Vascular , Humans , Aging/metabolism , Aging/physiology , Animals , Calcium/metabolism , Muscle, Smooth, Vascular/metabolism , Chloride Channels/metabolism , Endothelium, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism
6.
Cell Mol Biol (Noisy-le-grand) ; 70(5): 76-81, 2024 May 27.
Article in English | MEDLINE | ID: mdl-38814232

ABSTRACT

Bladder tumors occur more frequently in men than in women and are the fourth most common malignancy after prostate, lung, and colon cancers. In this study, we examined the expression of chlorine ion channel 1 and chlorine ion channel 3 in localized bladder tumors according to their stage. We conducted a retrospective analysis of a prospective cohort study spanning from May 2018 to January 2020. This study involved a group of 55 patients who had been diagnosed with primary bladder cancer and underwent transurethral resection of bladder tumor under either general or spinal anesthesia. In addition, 30 patients who underwent cystoscopy due to etiology of hematuria and biopsies were taken from suspicious areas and whose results were normal were included as the control group. The collected samples were evaluated using real-time polymerase chain reaction in a medical genetics laboratory. In our study, it was observed that chlorine ion channel 3 gene expression increased significantly (P<0.001) in all cancer tissues compared to the control group, whereas no significant increase was found in chlorine ion channel 1 gene expression compared to the control group. The data obtained, especially for chlorine ion channel 3, are promising in terms of their use in the treatment of bladder tumors in humans.


Subject(s)
Urinary Bladder Neoplasms , Humans , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/metabolism , Female , Male , Middle Aged , Chloride Channels/genetics , Chloride Channels/metabolism , Aged , Gene Expression Regulation, Neoplastic/drug effects , Retrospective Studies , Prospective Studies
7.
Exp Neurol ; 377: 114810, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38714284

ABSTRACT

Most projection neurons, including retinal ganglion cells (RGCs), undergo cell death after axotomy proximal to the cell body. Specific RGC subtypes, such as ON-OFF direction selective RGCs (ooDSGCs) are particularly vulnerable, whereas intrinsically photosensitive RGCs (ipRGCs) exhibit resilience to axonal injury. Through the application of RNA sequencing and fluorescent in situ hybridization, we show that the expression of chloride intracellular channel protein 1 and 4 (Clic1 and Clic4) are highly increased in the ooDSGCs after axonal injury. Toward determining a gene's role in RGCs, we optimized the utility and efficacy of adenovirus associated virus (AAV)-retro expressing short hairpin RNA (shRNA). Injection of AAV2-retro into the superior colliculus results in efficient shRNA expression in RGCs. Incorporating histone H2B gene fused with mGreenLantern results in bright nuclear reporter expression, thereby enhancing single RGC identification and cell quantitation in live retinas. Lastly, we demonstrate that AAV2-retro mediated knockdown of both Clic1 and Clic4 promotes RGC survival after injury. Our findings establish an integrated use of AAV2-retro-shRNA and real-time fundus imaging and reveal CLICs' contribution to RGC death.


Subject(s)
Cell Death , Chloride Channels , Dependovirus , Retinal Ganglion Cells , Animals , Retinal Ganglion Cells/metabolism , Dependovirus/genetics , Chloride Channels/genetics , Chloride Channels/metabolism , Cell Death/physiology , Mice , Mice, Inbred C57BL , Male , RNA, Small Interfering/genetics
8.
Elife ; 122024 Apr 09.
Article in English | MEDLINE | ID: mdl-38593125

ABSTRACT

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier, coupled with microbial dysbiosis, has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability, and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7-dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.


Subject(s)
Colitis , Goblet Cells , Animals , Humans , Mice , Chloride Channels/genetics , Chloride Channels/metabolism , Colitis/chemically induced , Colitis/metabolism , Colon/metabolism , Disease Models, Animal , Goblet Cells/metabolism , Homeostasis , Inflammation/metabolism , Intestinal Mucosa/metabolism , Mice, Inbred C57BL
9.
Brain Res ; 1834: 148915, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38582414

ABSTRACT

Bestrophin-1 and anoctamin-1 are members of the calcium-activated chloride channels (CaCCs) family and are involved in inflammatory and neuropathic pain. However, their role in pain hypersensitivity induced by REM sleep deprivation (REMSD) has not been studied. This study aimed to determine if anoctamin-1 and bestrophin-1 are involved in the pain hypersensitivity induced by REMSD. We used the multiple-platform method to induce REMSD. REM sleep deprivation for 48 h induced tactile allodynia and a transient increase in corticosterone concentration at the beginning of the protocol (12 h) in female and male rats. REMSD enhanced c-Fos and α2δ-1 protein expression but did not change activating transcription factor 3 (ATF3) and KCC2 expression in dorsal root ganglia and dorsal spinal cord. Intrathecal injection of CaCCinh-A01, a non-selective bestrophin-1 blocker, and T16Ainh-A01, a specific anoctamin-1 blocker, reverted REMSD-induced tactile allodynia. However, T16Ainh-A01 had a higher antiallodynic effect in male than female rats. In addition, REMSD increased bestrophin-1 protein expression in DRG but not in DSC in male and female rats. In marked contrast, REMSD decreased anoctamin-1 protein expression in DSC but not in DRG, only in female rats. Bestrophin-1 and anoctamin-1 promote pain and maintain tactile allodynia induced by REM sleep deprivation in both male and female rats, but their expression patterns differ between the sexes.


Subject(s)
Bestrophins , Ganglia, Spinal , Hyperalgesia , Sleep Deprivation , Spinal Cord , Animals , Sleep Deprivation/metabolism , Sleep Deprivation/complications , Hyperalgesia/metabolism , Male , Female , Rats , Ganglia, Spinal/metabolism , Spinal Cord/metabolism , Bestrophins/metabolism , Chloride Channels/metabolism , Sleep, REM/physiology , Rats, Wistar , Anoctamin-1 , Calcium Channels, L-Type
10.
Life Sci Alliance ; 7(7)2024 Jul.
Article in English | MEDLINE | ID: mdl-38670633

ABSTRACT

Mutations in Cl-/H+ antiporter ClC-5 cause Dent's disease type 1 (DD1), a rare tubulopathy that progresses to renal fibrosis and kidney failure. Here, we have used DD1 human cellular models and renal tissue from DD1 mice to unravel the role of ClC-5 in renal fibrosis. Our results in cell systems have shown that ClC-5 deletion causes an increase in collagen I (Col I) and IV (Col IV) intracellular levels by promoting their transcription through the ß-catenin pathway and impairing their lysosomal-mediated degradation. Increased production of Col I/IV in ClC-5-depleted cells ends up in higher release to the extracellular medium, which may lead to renal fibrosis. Furthermore, our data have revealed that 3-mo-old mice lacking ClC-5 (Clcn5 +/- and Clcn5 -/- ) present higher renal collagen deposition and fibrosis than WT mice. Altogether, we describe a new regulatory mechanism for collagens' production and release by ClC-5, which is altered in DD1 and provides a better understanding of disease progression to renal fibrosis.


Subject(s)
Chloride Channels , Fibrosis , Lysosomes , Mice, Knockout , beta Catenin , Animals , Chloride Channels/metabolism , Chloride Channels/genetics , Lysosomes/metabolism , Humans , Mice , beta Catenin/metabolism , Fibrosis/metabolism , Kidney/metabolism , Kidney/pathology , Collagen Type I/metabolism , Dent Disease/metabolism , Dent Disease/genetics , Proteolysis , Signal Transduction
11.
Methods Enzymol ; 696: 109-154, 2024.
Article in English | MEDLINE | ID: mdl-38658077

ABSTRACT

The use of molecular dynamics (MD) simulations to study biomolecular systems has proven reliable in elucidating atomic-level details of structure and function. In this chapter, MD simulations were used to uncover new insights into two phylogenetically unrelated bacterial fluoride (F-) exporters: the CLCF F-/H+ antiporter and the Fluc F- channel. The CLCF antiporter, a member of the broader CLC family, has previously revealed unique stoichiometry, anion-coordinating residues, and the absence of an internal glutamate crucial for proton import in the CLCs. Through MD simulations enhanced with umbrella sampling, we provide insights into the energetics and mechanism of the CLCF transport process, including its selectivity for F- over HF. In contrast, the Fluc F- channel presents a novel architecture as a dual topology dimer, featuring two pores for F- export and a central non-transported sodium ion. Using computational electrophysiology, we simulate the electrochemical gradient necessary for F- export in Fluc and reveal details about the coordination and hydration of both F- and the central sodium ion. The procedures described here delineate the specifics of these advanced techniques and can also be adapted to investigate other membrane protein systems.


Subject(s)
Biochemistry , Computational Biology , Fluorides , Molecular Dynamics Simulation , Fluorides/metabolism , Membrane Transport Proteins/metabolism , Ion Transport/physiology , Chloride Channels/chemistry , Chloride Channels/metabolism , Electrophysiology , Biochemistry/methods , Computational Biology/methods , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Biological Transport, Active/physiology
12.
Hum Genet ; 143(5): 667-681, 2024 May.
Article in English | MEDLINE | ID: mdl-38578438

ABSTRACT

CLCN4-related disorder is a rare X-linked neurodevelopmental condition with a pathogenic mechanism yet to be elucidated. CLCN4 encodes the vesicular 2Cl-/H+ exchanger ClC-4, and CLCN4 pathogenic variants frequently result in altered ClC-4 transport activity. The precise cellular and molecular function of ClC-4 remains unknown; however, together with ClC-3, ClC-4 is thought to have a role in the ion homeostasis of endosomes and intracellular trafficking. We reviewed our research database for patients with CLCN4 variants and epilepsy, and performed thorough phenotyping. We examined the functional properties of the variants in mammalian cells using patch-clamp electrophysiology, protein biochemistry, and confocal fluorescence microscopy. Three male patients with developmental and epileptic encephalopathy were identified, with differing phenotypes. Patients #1 and #2 had normal growth parameters and normal-appearing brains on MRI, while patient #3 had microcephaly, microsomia, complete agenesis of the corpus callosum and cerebellar and brainstem hypoplasia. The p.(Gly342Arg) variant of patient #1 significantly impaired ClC-4's heterodimerization capability with ClC-3 and suppressed anion currents. The p.(Ile549Leu) variant of patient #2 and p.(Asp89Asn) variant of patient #3 both shift the voltage dependency of transport activation by 20 mV to more hyperpolarizing potentials, relative to the wild-type, with p.(Asp89Asn) favouring higher transport activity. We concluded that p.(Gly342Arg) carried by patient #1 and the p.(Ile549Leu) expressed by patient #2 impair ClC-4 transport function, while the p.(Asp89Asn) variant results in a gain-of-transport function; all three variants result in epilepsy and global developmental impairment, but with differences in epilepsy presentation, growth parameters, and presence or absence of brain malformations.


Subject(s)
Chloride Channels , Epilepsy , Genetic Association Studies , Humans , Chloride Channels/genetics , Chloride Channels/metabolism , Male , Epilepsy/genetics , Child, Preschool , Child , Phenotype , Infant , Mutation
13.
Biosci Rep ; 44(5)2024 May 29.
Article in English | MEDLINE | ID: mdl-38573803

ABSTRACT

Chloride is a key anion involved in cellular physiology by regulating its homeostasis and rheostatic processes. Changes in cellular Cl- concentration result in differential regulation of cellular functions such as transcription and translation, post-translation modifications, cell cycle and proliferation, cell volume, and pH levels. In intracellular compartments, Cl- modulates the function of lysosomes, mitochondria, endosomes, phagosomes, the nucleus, and the endoplasmic reticulum. In extracellular fluid (ECF), Cl- is present in blood/plasma and interstitial fluid compartments. A reduction in Cl- levels in ECF can result in cell volume contraction. Cl- is the key physiological anion and is a principal compensatory ion for the movement of the major cations such as Na+, K+, and Ca2+. Over the past 25 years, we have increased our understanding of cellular signaling mediated by Cl-, which has helped in understanding the molecular and metabolic changes observed in pathologies with altered Cl- levels. Here, we review the concentration of Cl- in various organs and cellular compartments, ion channels responsible for its transportation, and recent information on its physiological roles.


Subject(s)
Chlorides , Humans , Chlorides/metabolism , Animals , Homeostasis , Chloride Channels/metabolism , Chloride Channels/genetics , Signal Transduction , Extracellular Fluid/metabolism , Ion Transport
14.
J Extracell Vesicles ; 13(4): e12430, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38602325

ABSTRACT

Chloride channel accessory 2 (CLCA2) is a transmembrane protein, which promotes adhesion of keratinocytes and their survival in response to hyperosmotic stress. Here we show that CLCA2 is transported to the nucleus of keratinocytes via extracellular vesicles. The nuclear localization is functionally relevant, since wild-type CLCA2, but not a mutant lacking the nuclear localization signal, suppressed migration of keratinocytes and protected them from hyperosmotic stress-induced cell death. In the nucleus, CLCA2 bound to and activated ß-catenin, resulting in enhanced expression of Wnt target genes. Mass-spectrometry-based interaction screening and functional rescue studies identified RNA binding protein 3 as a key effector of nuclear CLCA2. This is of likely relevance in vivo because both proteins co-localize in the human epidermis. Together, these results identify an unexpected nuclear function of CLCA2 in keratinocytes under homeostatic and stress conditions and suggest a role of extracellular vesicles and their nuclear transport in the control of key cellular activities.


Subject(s)
Extracellular Vesicles , Humans , Extracellular Vesicles/metabolism , Keratinocytes/metabolism , Cell Death , Chloride Channels/genetics , Chloride Channels/metabolism
15.
Exp Lung Res ; 50(1): 85-95, 2024.
Article in English | MEDLINE | ID: mdl-38597420

ABSTRACT

Recent research has revealed that airway epithelial calcium-activated chloride channel-1 (CLCA1) is implicated in the inflammation of multiple human respiratory diseases, but the specific role in acute respiratory distress syndrome (ARDS) remains unknown. To investigate the role of CLCA1 in ARDS, 80 participants, including 26 ARDS patients, 26 patients with community-acquired pneumonia (CAP) and 28 control subjects, were enrolled in this study. As the result shows, the level of CLCA1 was significantly increased in ARDS patients and positively correlated with neutrophil infiltration and the poor prognosis of ARDS. Then, the level of CLCA1 also elevated in the LPS-induced ARDS mouse model, and the administration of CLCA1 significantly regulated the phenotypes of ARDS in mice, such as lung injury score, BALF protein concentration, neutrophils infiltration and the secretions of inflammatory factors. Furthermore, administration of CLCA1 substantially altered the phosphorylation of p38 in the ARDS mouse model, whereas repressing the expression of CLCA1 or inhibiting the activation of p38 both alleviated the inflammatory response of ARDS. In summary, CLCA1 was notably correlated with ARDS and exacerbated the ARDS phenotypes through the p38 MAPK pathway.


Subject(s)
Pneumonia , Respiratory Distress Syndrome , Animals , Mice , Chloride Channels/metabolism , Lipopolysaccharides , Lung/metabolism , p38 Mitogen-Activated Protein Kinases , Pneumonia/metabolism , Respiratory Distress Syndrome/genetics , Humans
16.
Cell Physiol Biochem ; 58(2): 172-181, 2024 Apr 20.
Article in English | MEDLINE | ID: mdl-38643508

ABSTRACT

BACKGROUND/AIMS: Extracellular acidic conditions impair cellular activities; however, some cancer cells drive cellular signaling to adapt to the acidic environment. It remains unclear how ovarian cancer cells sense changes in extracellular pH. This study was aimed at characterizing acid-inducible currents in an ovarian cancer cell line and evaluating the involvement of these currents in cell viability. METHODS: The biophysical and pharmacological properties of membrane currents in OV2944, a mouse ovarian cancer cell line, were studied using the whole-cell configuration of the patch-clamp technique. Viability of this cell type in acidic medium was evaluated using the MTT assay. RESULTS: OV2944 had significant acid-sensitive outwardly rectifying (ASOR) Cl- currents at a pH50 of 5.3. The ASOR current was blocked by pregnenolone sulfate (PS), a steroid ion channel modulator that blocks the ASOR channel as one of its targets. The viability of the cells was reduced after exposure to an acidic medium (pH 5.3) but was slightly restored upon PS administration. CONCLUSION: These results offer first evidence for the presence of ASOR Cl- channel in ovarian cancer cells and indicate its involvement in cell viability under acidic environment.


Subject(s)
Cell Survival , Ovarian Neoplasms , Pregnenolone , Animals , Female , Mice , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Cell Line, Tumor , Pregnenolone/pharmacology , Hydrogen-Ion Concentration , Cell Survival/drug effects , Chloride Channels/metabolism , Chloride Channels/antagonists & inhibitors , Patch-Clamp Techniques , Membrane Potentials/drug effects
17.
J Phys Chem B ; 128(11): 2697-2706, 2024 Mar 21.
Article in English | MEDLINE | ID: mdl-38447081

ABSTRACT

CLCF fluoride/proton antiporters move fluoride ions out of bacterial cells, leading to fluoride resistance in these bacteria. However, many details about their operating mechanisms remain unclear. Here, we report a combined quantum-mechanical/molecular-mechanical (QM/MM) study of a CLCF homologue from Enterococci casseliflavus (Eca), in accord with the previously proposed windmill mechanism. Our multiscale modeling sheds light on two critical steps in the transport cycle: (i) the external gating residue E118 pushing a fluoride in the external binding site into the extracellular vestibule and (ii) an incoming fluoride reconquering the external binding site by forcing out E118. Both steps feature competitions for the external binding site between the negatively charged carboxylate of E118 and the fluoride. Remarkably, the displaced E118 by fluoride accepts a proton from the nearby R117, initiating the next transport cycle. We also demonstrate the importance of accurate quantum descriptions of fluoride solvation. Our results provide clues to the mysterious E318 residue near the central binding site, suggesting that the transport activities are unlikely to be disrupted by the glutamate interacting with a well-solvated fluoride at the central binding site. This differs significantly from the structurally similar CLC chloride/proton antiporters, where a fluoride trapped deep in the hydrophobic pore causes the transporter to be locked down. A free-energy barrier of 10-15 kcal/mol was estimated via umbrella sampling for a fluoride ion traveling through the pore to repopulate the external binding site.


Subject(s)
Antiporters , Protons , Antiporters/chemistry , Antiporters/metabolism , Fluorides/chemistry , Models, Molecular , Membrane Transport Proteins/metabolism , Chlorides/chemistry , Chloride Channels/chemistry , Chloride Channels/metabolism , Ion Transport
18.
J Ovarian Res ; 17(1): 67, 2024 Mar 25.
Article in English | MEDLINE | ID: mdl-38528613

ABSTRACT

BACKGROUND: Premature ovarian insufficiency (POI) is a severe disorder leading to female infertility. Genetic mutations are important factors causing POI. TP63-truncating mutation has been reported to cause POI by increasing germ cell apoptosis, however what factors mediate this apoptosis remains unclear. METHODS: Ninety-three patients with POI were recruited from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Whole-exome sequencing (WES) was performed for each patient. Sanger sequencing was used to confirm potential causative genetic variants. A minigene assay was performed to determine splicing effects of TP63 variants. A TP63-truncating plasmid was constructed. Real-time quantitative PCR, western blot analyses, dual luciferase reporter assays, immunofluorescence staining, and cell apoptosis assays were used to study the underlying mechanism of a TP63-truncating mutation causing POI. RESULTS: By WES of 93 sporadic patients with POI, we found a 14-bp deletion covering the splice site in the TP63 gene. A minigene assay demonstrated that the 14-bp deletion variant led to exon 13 skipping during TP63 mRNA splicing, resulting in the generation of a truncated TP63 protein (TP63-mut). Overexpression of TP63-mut accelerated cell apoptosis. Mechanistically, the TP63-mut protein could bind to the promoter region of CLCA2 and activate the transcription of CLCA2 several times compared to that of the TP63 wild-type protein. Silencing CLCA2 using a specific small interfering RNA (siRNA) or inhibiting the Ataxia Telangiectasia Mutated (ATM) pathway using the KU55933 inhibitor attenuated cell apoptosis caused by TP63-mut protein expression. CONCLUSION: Our findings revealed a crucial role for CLCA2 in mediating apoptosis in POI pathogenesis, and suggested that CLCA2 is a potential therapeutic target for POI.


Subject(s)
Menopause, Premature , Primary Ovarian Insufficiency , Transcription Factors , Tumor Suppressor Proteins , Female , Humans , Chloride Channels/genetics , Chloride Channels/metabolism , Exons , Menopause, Premature/genetics , Mutation , Primary Ovarian Insufficiency/genetics , Primary Ovarian Insufficiency/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation , Tumor Suppressor Proteins/genetics
19.
Nat Commun ; 15(1): 2085, 2024 Mar 07.
Article in English | MEDLINE | ID: mdl-38453905

ABSTRACT

Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs' function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose the function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs' transition to a membrane-associated conformation, and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal extension in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.


Subject(s)
Caenorhabditis elegans , Chlorides , Animals , Chlorides/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Chloride Channels/metabolism , Liposomes
20.
Biochim Biophys Acta Mol Basis Dis ; 1870(4): 167059, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38336104

ABSTRACT

Oviductal smooth muscle exhibits spontaneous rhythmic contraction (SRC) and controls the passage of the ova at the exact time, but its mechanistic regulation remains to be determined. In this study, female mice with Ano1SMKO (smooth muscle-specific deletion of Ano1) had reduced fertility. Deficiency of Ano1 in mice resulted in impaired oviductal SRC function and reduced calcium signaling in individual smooth muscle cells in the oviduct. The Ano1 antagonist T16Ainh-A01 dose-dependently inhibited SRCs and [Ca2+]i in the oviducts of humans and mice. A similar inhibitory effect of SRCs and [Ca2+]i was observed after treatment with nifedipine. In our study, ANO1 acted primarily as an activator or amplifier in [Ca2+]i and contraction of tubal smooth muscle cells. We found that tubal SRC was markedly attenuated in patients with ectopic pregnancy. Then, our study was designed to determine whether chloride channel Ano1-mediated smooth muscle motility is associated with tubal SRC. Our findings reveal a new mechanism for the regulation of tubal motility that may be associated with abnormal pregnancies such as ectopic pregnancies.


Subject(s)
Calcium , Muscle, Smooth , Animals , Female , Humans , Mice , Pregnancy , Calcium/metabolism , Chloride Channels/genetics , Chloride Channels/metabolism , Muscle, Smooth/metabolism , Myocytes, Smooth Muscle/metabolism , Oviducts/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...