One of the main pathological features of airway inflammatory diseases is hypersecretion of airway mucus, which is manifested by goblet cell hyperplasia and mucociliary clearance dysfunction. In recent years, it has been found that the molecular structure of calcium activated chloride ion channels, transmenbrane protein 16A（TMEM16A）, is closely related to airway mucus hypersecretion.TMEM16A not only mediates ion transepithelial transport and hydration, but also participates in the regulation of mucin secretion. TMEM16A is highly expressed in airway epithelium of a variety of inflammatory diseases of upper and lower airway, such as asthma, cystic fibrosis, allergic rhinitis, chronic sinusitis and so on. Understanding the expression level and regulation mechanism of TMEM16A in different airway diseases and revealing its physiological function and pathological mechanism is critical for targeted disease treatment. This paper summarizes the research status of the discovery process, structural characteristics and regulatory mechanism of TMEM16A, and then summarizes the expression level of TMEM16A in asthma, cystic fibrosis, allergic rhinitis and chronic sinusitis ant related pathological mechanisms, clarifies the potential value of TMEM16A as a therapeutic target for the above four diseases, in order to guide treatment of airway inflammatory diseases.
Humans ; Asthma/metabolism* ; Anoctamin-1 ; Cystic Fibrosis/metabolism* ; Neoplasm Proteins/metabolism* ; Sinusitis/metabolism* ; Chloride Channels/metabolism* ; Rhinitis, Allergic/metabolism* ; Inflammation

Sleep, an essential and evolutionarily conserved behavior, is regulated by numerous neurotransmitter systems. In mammals, glutamate serves as the wake-promoting signaling agent, whereas in Drosophila, it functions as the sleep-promoting signal. However, the precise molecular and cellular mechanisms through which glutamate promotes sleep remain elusive. Our study reveals that disruption of glutamate signaling significantly diminishes nocturnal sleep, and a neural cell-specific knockdown of the glutamate-gated chloride channel (GluClα) markedly reduces nocturnal sleep. We identified two pairs of neurons in the ventral nerve cord (VNC) that receive glutamate signaling input, and the GluClα derived from these neurons is crucial for sleep promotion. Furthermore, we demonstrated that GluClα mediates the glutamate-gated inhibitory input to these VNC neurons, thereby promoting sleep. Our findings elucidate that GluClα enhances nocturnal sleep by mediating the glutamate-gated inhibitory input to two pairs of VNC neurons, providing insights into the mechanism of sleep promotion in Drosophila.
Animals ; Sleep/physiology* ; Neurons/metabolism* ; Chloride Channels/genetics* ; Drosophila Proteins/genetics* ; Drosophila ; Glutamic Acid/metabolism* ; Animals, Genetically Modified

BACKGROUND: Tetrabromobisphenol A (TBBPA), one of the most widely used brominated flame-retardants, is a representative persistent organic pollutants group. Studies on TBBPA toxicity have been conducted using various target cells; however, few studies have investigated TBBPA toxicity in bone cells. Therefore, this study investigated the in vitro effects of TBBPA on osteoclasts, a cell type involved in bone metabolism. METHODS: RAW264.7 cells were cultured in medium containing 50 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL) and varying concentrations of TBBPA. To evaluate the effects of TBBPA on the differentiation and function of osteoclasts, osteoclast-specific gene expression, tartrate-resistant acid phosphatase (TRAP) activity, bone resorbing activity, mitochondrial membrane potential (MMP) and mitochondrial superoxide were measured. RESULTS: The presence of 20 μM TBBPA significantly increased TRAP activity in RANKL-stimulated RAW264.7 cells, the bone resorbing activity of osteoclasts, and the gene expression of Akt2, nuclear factor of activated T-cells cytoplasmic 1, and chloride channel voltage-sensitive 7. However, TBBPA treatment caused no change in the expression of carbonic anhydrase II, cathepsin K, osteopetrosis-associated transmembrane protein 1, Src, extracellular signal-related kinase, GAB2, c-Fos, or matrix metalloproteinase 9. Furthermore, 20 μM TBBPA caused a significant decrease in MMP and a significant increase in mitochondrial superoxide production. CONCLUSION: This study suggests that TBBPA promotes osteoclast differentiation and activity. The mechanism of TBBPA-stimulated osteoclastogenesis might include increased expression of several genes involved in osteoclast differentiation and reactive oxygen species production.
Acid Phosphatase ; Carbonic Anhydrase II ; Cathepsin K ; Chloride Channels ; Cytoplasm ; Gene Expression ; In Vitro Techniques ; Matrix Metalloproteinase 9 ; Membrane Potential, Mitochondrial ; Metabolism ; Osteoclasts ; Phosphotransferases ; RANK Ligand ; Reactive Oxygen Species ; Receptor Activator of Nuclear Factor-kappa B ; Superoxides ; T-Lymphocytes

OBJECTIVETo study the impact of the chloride channel dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) on the cytoskeleton of Sertoli cells in the mouse.

METHODSTM4 Sertoli cells were cultured and treated with CFTR(inh)-172 at the concentrations of 1, 5, 10 and 20 μmol/L for 48 hours. Then the cytotoxicity of CFT(inh)-172 was assessed by CCK-8 assay, the expressions of F-actin and Ac-tub in the TM4 Sertoli cells detected by immunofluorescence assay, and those of N-cadherin, vimentin and vinculin determined by qPCR.

RESULTSCFTR(inh)-172 produced cytotoxicity to the TM4 Sertoli cells at the concentration of 20 μmol/L. The expressions of F-actin and Ac-tub were decreased gradually in the TM4 Sertoli cells with the prolonging of treatment time and increasing concentration of CFTR(inh)-172 (P < 0.05). The results of qPCR showed that different concentrations of CFTR(inh)-172 worked no significant influence on the mRNA expressions of N-cadherin, vimentin and vinculin in the Sertoli cells.

CONCLUSIONThe CFTR chloride channel plays an important role in maintaining the normal cytoskeleton of Sertoli cells. The reduced function and expression of the CFTR chloride channel may affect the function of Sertoli cells and consequently spermatogenesis of the testis.

Actins ; metabolism ; Animals ; Benzoates ; pharmacology ; Chloride Channels ; physiology ; Cystic Fibrosis Transmembrane Conductance Regulator ; antagonists & inhibitors ; Cytoskeleton ; drug effects ; Male ; Mice ; Sertoli Cells ; drug effects ; metabolism ; Spermatogenesis ; Thiazolidines ; pharmacology ; Time Factors

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism

Microglia play a pivotal role in clearance of Aβ by degrading them in lysosomes, countering amyloid plaque pathogenesis in Alzheimer's disease (AD). Recent evidence suggests that lysosomal dysfunction leads to insufficient elimination of toxic protein aggregates. We tested whether enhancing lysosomal function with transcription factor EB (TFEB), an essential regulator modulating lysosomal pathways, would promote Aβ clearance in microglia. Here we show that microglial expression of TFEB facilitates fibrillar Aβ (fAβ) degradation and reduces deposited amyloid plaques, which are further enhanced by deacetylation of TFEB. Using mass spectrometry analysis, we firstly confirmed acetylation as a previously unreported modification of TFEB and found that SIRT1 directly interacted with and deacetylated TFEB at lysine residue 116. Subsequently, SIRT1 overexpression enhanced lysosomal function and fAβ degradation by upregulating transcriptional levels of TFEB downstream targets, which could be inhibited when TFEB was knocked down. Furthermore, overexpression of deacetylated TFEB at K116R mutant in microglia accelerated intracellular fAβ degradation by stimulating lysosomal biogenesis and greatly reduced the deposited amyloid plaques in the brain slices of APP/PS1 transgenic mice. Our findings reveal that deacetylation of TFEB could regulate lysosomal biogenesis and fAβ degradation, making microglial activation of TFEB a possible strategy for attenuating amyloid plaque deposition in AD.
Alzheimer Disease ; metabolism ; pathology ; Amyloid beta-Peptides ; metabolism ; Amyloid beta-Protein Precursor ; genetics ; metabolism ; Animals ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; chemistry ; genetics ; metabolism ; Brain ; metabolism ; Cells, Cultured ; Chloride Channels ; genetics ; metabolism ; Disease Models, Animal ; HEK293 Cells ; Humans ; Lysosomes ; genetics ; metabolism ; Mice ; Mice, Transgenic ; Microglia ; cytology ; metabolism ; Mutagenesis, Site-Directed ; Peptides ; analysis ; chemistry ; Protein Binding ; RNA Interference ; Sirtuin 1 ; antagonists & inhibitors ; genetics ; metabolism

The aim of the present study was to investigate whether the physiological features of Ano1 were affected by enhanced green fluorescent protein (EGFP) fusing at Ano1 C-terminal. The eukaryotic expression vectors of Ano1 and EGFP-Ano1 were constructed, and these plasmids were transfected into Fischer rat thyroid follicular epithelial (FRT) cells using liposome. The expression and location of Ano1 were examined by using inverted fluorescence microscope. The ability of Ano1 to transport iodide was detected by kinetics experiment of fluorescence quenching. The results showed that both Ano1 and EGFP-Ano1 were expressed on FRT cell membrane and could be activated by Ca(2+). There was no significant difference of the ability to transport iodide between Ano1 and EGFP-Ano1. These results suggest Ano1 and EGFP-Ano1 have similar physiological feature.
Animals ; Anoctamin-1 ; Cell Membrane ; physiology ; Chloride Channels ; metabolism ; Epithelial Cells ; physiology ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; Microscopy, Fluorescence ; Plasmids ; Rats ; Recombinant Fusion Proteins ; metabolism ; Thyroid Gland ; cytology ; Transfection

Chloride channels belong to a superfamily of ion channels that permit passive passage of anions, mainly chloride, across cell membrane. They play a variety of important physiological roles in regulation of cytosolic pH, cell volume homeostasis, organic solute transport, cell migration, cell proliferation, and differentiation. However, little is known about the functional regulation of these channels. In this study, we generated an integrated transgenic worm strain expressing green fluorescence protein (GFP) fused CLC-type chloride channel 1 (CLH-1::GFP), a voltage-gated chloride channel in Caenorhabditis elegans (C. elegans). CLH-1::GFP was expressed in some unidentified head neurons and posterior intestinal cells of C. elegans. Interacting proteins of CLH-1::GFP were purified by GFP-Trap, a novel system for efficient isolation of GFP fusion proteins and their interacting factors. Mass spectrometry (MS) analysis revealed that a total of 27 high probability interacting proteins were co-trapped with CLHp-1::GFP. Biochemical evidence showed that eukaryotic translation elongation factor 1 (EEF-1), one of these co-trapped proteins identified by MS, physically interacted with CLH-1, in consistent with GFP-Trap experiments. Further immunostaining data revealed that the protein level of CLH-1 was significantly increased upon co-expression with EEF-1. These results suggest that the combination of GFP-Trap purification with MS is an excellent tool to identify novel interacting proteins of voltage-gated chloride channels in C. elegans. Our data also show that EEF-1 is a regulator of voltage-gated chloride channel CLH-1.
Animals ; Animals, Genetically Modified ; Caenorhabditis elegans ; genetics ; metabolism ; Caenorhabditis elegans Proteins ; metabolism ; Chloride Channels ; metabolism ; Green Fluorescent Proteins ; chemistry ; Mass Spectrometry ; Peptide Elongation Factor 1 ; metabolism

OBJECTIVETo detect the effects of ANO1 overexpression on the biological behaviors of human laryngeal squamous cell carcinoma Hep-2 cells.

METHODSA Hep-2 cell line stably overexpressing ANO1 were examined with flow cytometry, soft agar assay, wound healing assay, siRNA experiments, and chloride channel block with DIDS to observe the effect of ANO1 overexpression on the growth, migration and invasion of the cells.

RESULTSFlow cytometry revealed a comparable cell percentage in G0/G1 phase between ANO1-overexpressing cells and the control cells (P>0.05). The two cells showed no significant difference in soft agar assay (P>0.05), but in wound healing experiments, ANO1-overexpressing cells showed significantly accelerated migration (P<0.05), whereas siRNA-mediated silencing of ANO1 significantly inhibited the cell migration (P<0.05). Treatment with DIDS resulted in an effective block of the ANO1 chloride channel activity and obviously decreased the migration speed of Hep-2 cells.

CONCLUSIONANO1 overexpression does not significantly affect the proliferation of cancer cells, but can enhance the migration ability of head and neck squamous cell carcinoma, suggesting the value of ANO1 as a new gene therapy target for head and neck squamous cell carcinoma.

Anoctamin-1 ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Chloride Channels ; metabolism ; Gene Silencing ; Head and Neck Neoplasms ; metabolism ; pathology ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Neoplasm Proteins ; metabolism ; RNA, Small Interfering

Spontaneous, rhythmical contractions, or vasomotion, can be recorded from cerebral vessels under both normal physiological and pathophysiological conditions. We investigated the cellular mechanisms underlying vasomotion in the cerebral basilar artery (BA) of Wistar rats. Pressure myograph video microscopy was used to study the changes in cerebral artery vessel diameter. The main results of this study were as follows: (1) The diameters of BA and middle cerebral artery (MCA) were 314.5±15.7 μm (n=15) and 233.3±10.1 μm (n=12) at 10 mmHg working pressure (P<0.05), respectively. Pressure-induced vasomotion occurred in BA (22/28, 78.6%), but not in MCA (4/31, 12.9%) from 0 to 70 mmHg working pressure. As is typical for vasomotion, the contractile phase of the response was more rapid than the relaxation phase; (2) The frequency of vasomotion response and the diameter were gradually increased in BA from 0 to 70 mmHg working pressure. The amplitude of the rhythmic contractions was relatively constant once stable conditions were achieved. The frequency of contractions was variable and the highest value was 16.7±4.7 (n=13) per 10 min at 60 mmHg working pressure; (3) The pressure-induced vasomotion of the isolated BA was attenuated by nifedipine, NFA, 18β-GA, TEA or in Ca(2+)-free medium. Nifedipine, NFA, 18β-GA or Ca(2+)-free medium not only dampened vasomotion, but also kept BA in relaxation state. In contrasts, TEA kept BA in contraction state. These results suggest that the pressure-induced vasomotion of the isolated BA results from an interaction between Ca(2+)-activated Cl(-) channels (CaCCs) currents and K(Ca) currents. We hypothesize that vasomotion of BA depends on the depolarizing of the vascular smooth muscle cells (VSMCs) to activate CaCCs. Depolarization in turn activates voltage-dependent Ca(2+) channels, synchronizing contractions of adjacent cells through influx of extracellular calcium and the flow of calcium through gap junctions. Subsequent calcium-induced calcium release from ryanodine-sensitive stores activates K(Ca) channels and hyperpolarizes VSMCs, which provides a negative feedback loop for regenerating the contractile cycle.
Animals ; Basilar Artery ; cytology ; metabolism ; physiology ; Chloride Channels ; metabolism ; Female ; Male ; Membrane Potentials ; physiology ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Potassium Channels, Calcium-Activated ; metabolism ; Rats ; Rats, Wistar ; Vasoconstriction ; physiology ; Vasodilation ; physiology