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Sample records for stretch-sensitive chloride channel

  1. Characterization of the Circumlimbal Suture Model of Chronic IOP Elevation in Mice and Assessment of Changes in Gene Expression of Stretch Sensitive Channels

    PubMed Central

    Zhao, Da; Nguyen, Christine T. O.; Wong, Vickie H. Y.; Lim, Jeremiah K. H.; He, Zheng; Jobling, Andrew I.; Fletcher, Erica L.; Chinnery, Holly R.; Vingrys, Algis J.; Bui, Bang V.

    2017-01-01

    To consider whether a circumlimbal suture can be used to chronically elevate intraocular pressure (IOP) in mice and to assess its effect on retinal structure, function and gene expression of stretch sensitive channels. Anesthetized adult C57BL6/J mice had a circumlimbal suture (10/0) applied around the equator of one eye. In treated eyes (n = 23) the suture was left in place for 12 weeks whilst in sham control eyes the suture was removed at day two (n = 17). Contralateral eyes served as untreated controls. IOP was measured after surgery and once a week thereafter. After 12 weeks, electroretinography (ERG) was performed to assess photoreceptor, bipolar cell and retinal ganglion cell (RGC) function. Retinal structure was evaluated using optical coherence tomography. Retinae were processed for counts of ganglion cell density or for quantitative RT-PCR to quantify purinergic (P2x7, Adora3, Entpd1) or stretch sensitive channel (Panx1, Trpv4) gene expression. Immediately after suture application, IOP spiked to 33 ± 3 mmHg. After 1 day, IOP had recovered to 27 ± 3 mmHg. Between weeks 2 and 12, IOP remained elevated above baseline (control 14 ± 1 mmHg, ocular hypertensive 19 ± 1 mmHg). Suture removal at day 2 (Sham) restored IOP to baseline levels, where it remained through to week 12. ERG analysis showed that 12 weeks of IOP elevation reduced photoreceptor (−15 ± 4%), bipolar cell (−15 ± 4%) and ganglion cell responses (−19 ± 6%) compared to sham controls and respective contralateral eyes (untreated). The retinal nerve fiber layer was thinned in the presence of normal total retinal thickness. Ganglion cell density was reduced across all quadrants (superior −12 ± 5%; temporal, −7% ± 2%; inferior −9 ± 4%; nasal −8 ± 5%). Quantitative RT-PCR revealed a significant increase in Entpd1 gene expression (+11 ± 4%), whilst other genes were not significantly altered (P2x7, Adora3, Trpv4, Panx1). Our results show that circumlimbal ligation produces mild

  2. Chloride channels as drug targets

    PubMed Central

    Verkman, Alan S.; Galietta, Luis J. V.

    2013-01-01

    Chloride channels represent a relatively under-explored target class for drug discovery as elucidation of their identity and physiological roles has lagged behind that of many other drug targets. Chloride channels are involved in a wide range of biological functions, including epithelial fluid secretion, cell-volume regulation, neuroexcitation, smooth-muscle contraction and acidification of intracellular organelles. Mutations in several chloride channels cause human diseases, including cystic fibrosis, macular degeneration, myotonia, kidney stones, renal salt wasting and hyperekplexia. Chloride-channel modulators have potential applications in the treatment of some of these disorders, as well as in secretory diarrhoeas, polycystic kidney disease, osteoporosis and hypertension. Modulators of GABAA (γ-aminobutyric acid A) receptor chloride channels are in clinical use and several small-molecule chloride-channel modulators are in preclinical development and clinical trials. Here, we discuss the broad opportunities that remain in chloride-channel-based drug discovery. PMID:19153558

  3. Lubiprostone: a chloride channel activator.

    PubMed

    Lacy, Brian E; Levy, L Campbell

    2007-04-01

    In January 2006 the Food and Drug Administration approved lubiprostone for the treatment of chronic constipation in men and women aged 18 and over. Lubiprostone is categorized as a prostone, a bicyclic fatty acid metabolite of prostaglandin E1. Lubiprostone activates a specific chloride channel (ClC-2) in the gastrointestinal (GI) tract to enhance intestinal fluid secretion, which increases GI transit and improves symptoms of constipation. This article reviews the role of chloride channels in the GI tract, describes the structure, function, and pharmacokinetics of lubiprostone, and discusses clinically important data on this new medication.

  4. Studies on Chloride Channels and their Modulators.

    PubMed

    Patil, Vaishali M; Gupta, Satya P

    2016-01-01

    The prime roles of mutations in the genes, encoding chloride ion channels, in various human diseases of muscle, kidney, bone and brain, such as congenital myotonia, myotonic dystrophy, cystic fibrosis, osteopetrosis, epilepsy, glioma, etc., have been well established. Chloride ion channels are also responsible for glioma progression in brain and malaria parasite in red blood cells. The present article thus emphasises on the various diseases associated with chloride channel regulation and their modulators. Studies on various chloride channels and their modulators have been discussed in detail.

  5. Regulated trafficking of the CFTR chloride channel.

    PubMed

    Kleizen, B; Braakman, I; de Jonge, H R

    2000-08-01

    The cystic fibrosis transmembrane conductance regulator (CFTR), the ABC transporter encoded by the cystic fibrosis gene, is localized in the apical membrane of epithelial cells where it functions as a cyclic AMP-regulated chloride channel and as a regulator of other ion channels and transporters. Whereas a key role of cAMP-dependent phosphorylation in CFTR-channel gating has been firmly established, more recent studies have provided clear evidence for the existence of a second level of cAMP regulation, i.e. the exocytotic recruitment of CFFR to the plasma membrane and its endocytotic retrieval. Regulated trafficking of the CFTR Cl- channel has sofar been demonstrated only in a subset of CFTR-expressing cell types. However, with the introduction of more sensitive methods to measure CFTR cycling and submembrane localization, it might turn out to be a more general phenomenon that could contribute importantly to both the regulation of CFTR-mediated chloride transport itself and to the regulation of other transporters and CFTR-modulated cellular functions. This review aims to summarize the present state of knowledge regarding polarized and regulated CFTR trafficking and endosomal recycling in epithelial cells, to discuss present gaps in our understanding of these processes at the cellular and molecular level, and to consider its possible implications for cystic fibrosis.

  6. An Apical-Membrane Chloride Channel in Human Tracheal Epithelium

    NASA Astrophysics Data System (ADS)

    Welsh, Michael J.

    1986-06-01

    The mechanism of chloride transport by airway epithelia has been of substantial interest because airway and sweat gland-duct epithelia are chloride-impermeable in cystic fibrosis. The decreased chloride permeability prevents normal secretion by the airway epithelium, thereby interfering with mucociliary clearance and contributing to the morbidity and mortality of the disease. Because chloride secretion depends on and is regulated by chloride conductance in the apical cell membrane, the patch-clamp technique was used to directly examine single-channel currents in primary cultures of human tracheal epithelium. The cells contained an anion-selective channel that was not strongly voltage-gated or regulated by calcium in cell-free patches. The channel was also blocked by analogs of carboxylic acid that decrease apical chloride conductance in intact epithelia. When attached to the cell, the channel was activated by isoproterenol, although the channel was also observed to open spontaneously. However, in some cases, the channel was only observed after the patch was excised from the cell. These results suggest that this channel is responsible for the apical chloride conductance in airway epithelia.

  7. Chloride dependence of hyperpolarization-activated chloride channel gates

    PubMed Central

    Pusch, Michael; Jordt, Sven-Eric; Stein, Valentin; Jentsch, Thomas J

    1999-01-01

    ClC proteins are a class of voltage-dependent Cl− channels with several members mutated in human diseases. The prototype ClC-0 Torpedo channel is a dimeric protein; each subunit forms a pore that can gate independently from the other one. A common slower gating mechanism acts on both pores simultaneously; slow gating activates ClC-0 at hyperpolarized voltages. The ClC-2 Cl− channel is also activated by hyperpolarization, as are some ClC-1 mutants (e.g. D136G) and wild-type (WT) ClC-1 at certain pH values.We studied the dependence on internal Cl− ([Cl−]i) of the hyperpolarization-activated gates of several ClC channels (WT ClC-0, ClC-0 mutant P522G, ClC-1 mutant D136G and an N-terminal deletion mutant of ClC-2), by patch clamping channels expressed in Xenopus oocytes.With all these channels, reducing [Cl−]i shifted activation to more negative voltages and reduced the maximal activation at most negative voltages.We also investigated the external halide dependence of WT ClC-2 using two-electrode voltage-clamp recording. Reducing external Cl− ([Cl−]o) activated ClC-2 currents. Replacing [Cl−]o by the less permeant Br− reduced channel activity and accelerated deactivation.Gating of the ClC-2 mutant K566Q in normal [Cl−]o resembled that of WT ClC-2 in low [Cl−]o, i.e. channels had a considerable open probability (Po) at resting membrane potential. Substituting external Cl− by Br− or I− led to a decrease in Po.The [Cl−]i dependence of the hyperpolarization-activated gates of various ClC channels suggests a similar gating mechanism, and raises the possibility that the gating charge for the hyperpolarization-activated gate is provided by Cl−.The external halide dependence of hyperpolarization-activated gating of ClC-2 suggests that it is mediated or modulated by anions as in other ClC channels. In contrast to the depolarization-activated fast gates of ClC-0 and ClC-1, the absence of Cl− favours channel opening. Lysine 556 may be important

  8. Chloride Channels: Often enigmatic, rarely predictable

    PubMed Central

    Duran, Charity; Thompson, Christopher H.; Xiao, Qinghuan; Hartzell, Criss

    2010-01-01

    Until recently, anion (Cl−) channels have received considerably less attention than cation channels. One reason for this may be that many Cl− channels perform functions that might be considered cell biological, like fluid secretion and cell volume regulation, whereas cation channels have historically been associated with cellular excitability that typically happens more rapidly. In this review, we discuss the recent explosion of interest in Cl− channels with special emphasis on new and often surprising developments over the last 5 years. This is exemplified by the findings that more than half of the ClC family members are antiporters, and not channels as was previously thought, and that bestrophins, previously prime candidates for Ca2+-activated Cl− channels, have been supplanted by the newly discovered anoctamins and now hold a tenuous position in the Cl− channel world. PMID:19827947

  9. Inhibition of nitrite-induced toxicity in channel catfish by calcium chloride and sodium chloride

    USGS Publications Warehouse

    Tommasso J.R., Wright; Simco, B.A.; Davis, K.B.

    1980-01-01

    Environmental chloride has been shown to inhibit methemoglobin formation in fish, thereby offering a protective effect against nitrite toxicity. Channel catfish (Ictalurus punctatus) were simultaneously exposed to various environmental nitrite and chloride levels (as either CaCl2 or NaCl) in dechlorinated tap water (40 mg/L total hardness, 47 mg/L alkalinity, 4 mg/L chloride, pH = 6.9-7.1, and temperature 21-24°C). Methemoglobin levels in fish simultaneously exposed to 2.5 mg/L nitrite and up to 30 mg/L chloride as either CaCl2 or NaCl were similar but significantly lower than in unprotected fish. Exposure to 10 mg/L nitrite and 60 mg/L chloride resulted in methemoglobin levels similar to those of the controls; most unprotected fish died. Fish exposed to 10 mg/L nitrite had significantly lower methemoglobin levels when protected with 15.0 mg/L chloride as CaCl2 than with NaCl. Fish exposed to nitrite in the presence of 60 mg/L chloride (as either CaCl2 or NaCl) had similar 24-h LC50 values that were significantly elevated above those obtained in the absence of chloride. Calcium had little effect on tolerance to nitrite toxicity in channel catfish in contrast to its large effect reported in steelhead trout (Salmo gairdneri).

  10. Functional architecture of the CFTR chloride channel.

    PubMed

    Linsdell, Paul

    2014-02-01

    Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) family of membrane transport proteins. CFTR is unique among ABC proteins in that it functions not as an active transporter but as an ATP-gated Cl(-) channel. As an ion channel, the function of the CFTR transmembrane channel pore that mediates Cl(-) movement has been studied in great detail. On the other hand, only low resolution structural data is available on the transmembrane parts of the protein. The structure of the channel pore has, however, been modeled on the known structure of active transporter ABC proteins. Currently, significant barriers exist to building a unified view of CFTR pore structure and function. Reconciling functional data on the channel with indirect structural data based on other proteins with very different transport functions and substrates has proven problematic. This review summarizes current structural and functional models of the CFTR Cl(-) channel pore, including a comprehensive review of previous electrophysiological investigations of channel structure and function. In addition, functional data on the three-dimensional arrangement of pore-lining helices, as well as contemporary hypotheses concerning conformational changes in the pore that occur during channel opening and closing, are discussed. Important similarities and differences between different models of the pore highlight current gaps in our knowledge of CFTR structure and function. In order to fill these gaps, structural and functional models of the membrane-spanning pore need to become better integrated.

  11. Epithelial Sodium and Chloride Channels and Asthma

    PubMed Central

    Wang, Wen; Ji, Hong-Long

    2015-01-01

    Objective: To focus on the asthmatic pathogenesis and clinical manifestations related to epithelial sodium channel (ENaC)/chlorine ion channel. Data Sources: The data analyzed in this review were the English articles from 1980 to 2015 from journal databases, primarily PubMed and Google Scholar. The terms used in the literature search were: (1) ENaCs; cystic fibrosis (CF) transmembrane conductance regulator (CFTR); asthma/asthmatic, (2) ENaC/sodium salt; CF; asthma/asthmatic, (3) CFTR/chlorine ion channels; asthma/asthmatic, (4) ENaC/sodium channel/scnn1a/scnn1b/scnn1g/scnn1d/amiloride-sensitive/amiloride-inhibtable sodium channels/sodium salt; asthma/asthmatic, lung/pulmonary/respiratory/tracheal/alveolar, and (5) CFTR; CF; asthma/asthmatic (ti). Study Selection: These studies included randomized controlled trials or studies covering asthma pathogenesis and clinical manifestations related to ENaC/chlorine ion channels within the last 25 years (from 1990 to 2015). The data involving chronic obstructive pulmonary disease and CF obtained from individual studies were also reviewed by the authors. Results: Airway surface liquid dehydration can cause airway inflammation and obstruction. ENaC and CFTR are closely related to the airway mucociliary clearance. Ion transporters may play a critical role in pathogenesis of asthmatic exacerbations. Conclusions: Ion channels have been the center of many studies aiming to understand asthmatic pathophysiological mechanisms or to identify therapeutic targets for better control of the disease. PMID:26265620

  12. Phosphatase inhibitors activate normal and defective CFTR chloride channels.

    PubMed Central

    Becq, F; Jensen, T J; Chang, X B; Savoia, A; Rommens, J M; Tsui, L C; Buchwald, M; Riordan, J R; Hanrahan, J W

    1994-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epithelial cells. We report that the alkaline phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophylline, and vanadate slow the rundown of CFTR channel activity in excised membrane patches and reduce dephosphorylation of CFTR protein in isolated membranes. It was also found that in unstimulated cells, CFTR channels can be activated by exposure to phosphatase inhibitors alone. Most importantly, exposure of mammalian cells to phosphatase inhibitors alone activates CFTR channels that have disease-causing mutations, provided the mutant channels are present in the plasma membrane (R117H, G551D, and delta F508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membrane-associated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis. Images PMID:7522329

  13. Monoclonal Antibodies to the Apical Chloride Channel in Necturus Gallbladder Inhibit the Chloride Conductance

    NASA Astrophysics Data System (ADS)

    Finn, Arthur L.; Tsai, Lih-Min; Falk, Ronald J.

    1989-10-01

    Monoclonal antibodies raised by injecting Necturus gallbladder cells into mice were tested for their ability to inhibit the apical chloride conductance induced by elevation of cellular cAMP. Five of these monoclonal antibodies bound to the apical cells, as shown by indirect immunofluorescence microscopy, and inhibited the chloride conductance; one antibody that bound only to subepithelial smooth muscle, by indirect immunofluorescence microscopy, showed no inhibition of chloride transport. The channel or a closely related molecule is present in the membrane whether or not the pathway is open, since, in addition to inhibiting the conductance of the open channel, the antibody also bound to the membrane in the resting state and prevented subsequent opening of the channel. The antibody was shown to recognize, by ELISA, epitopes from the Necturus gallbladder and small intestine. Finally, by Western blot analysis of Necturus gallbladder homogenates, the antibody was shown to recognize two protein bands of Mr 219,000 and Mr 69,000. This antibody should permit isolation and characterization of this important ion channel.

  14. Lack of conventional ATPase properties in CFTR chloride channel gating.

    PubMed

    Schultz, B D; Bridges, R J; Frizzell, R A

    1996-05-01

    CFTR shares structural homology with the ABC transporter superfamily of proteins which hydrolyze ATP to effect the transport of compounds across cell membranes. Some superfamily members are characterized as P-type ATPases because ATP-dependent transport is sensitive to the presence of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrolytic activity in channel gating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride channel activity under conditions known to inhibit the activity of ATPases; i.e., in the absence of divalent cations and in the presence of a variety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, reduced both the opening and closing rates of CFTR suggesting that Mg2+ plays a modulatory role in channel gating. However, channels continued to both open and close showing that Mg2+ is not an absolute requirement for channel activity. The nonselective P-type ATPase inhibitor, vanadate, did not alter the gating of CFTR when used at concentrations which completely inhibit the activity of other ABC transporters (1 mM). Higher concentrations of vanadate (10 mM) blocked the closing of CFTR, but did not affect the opening of the channel. As expected, more selective P-type (Sch28080, ouabain), V-type (bafilomycin A1, SCN-) and F-type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics. Vanadate inhibition of channel closure might suggest that a hydrolytic step is involved although the requirement for a high concentration raises the possibility of previously uncharacterized effects of this compound. Most conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for

  15. Swell activated chloride channel function in human neutrophils

    SciTech Connect

    Salmon, Michael D.; Ahluwalia, Jatinder

    2009-04-17

    Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I{sub e} has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I{sub e} must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.

  16. Mitochondrial chloride channels: electrophysiological characterization and pH induction of channel pore dilation.

    PubMed

    Misak, Anton; Grman, Marian; Malekova, Lubica; Novotova, Marta; Markova, Jana; Krizanova, Olga; Ondrias, Karol; Tomaskova, Zuzana

    2013-09-01

    Physiological and pathological functions of mitochondria are highly dependent on the properties and regulation of mitochondrial ion channels. There is still no clear understanding of the molecular identity, regulation, and properties of anion mitochondrial channels. The inner membrane anion channel (IMAC) was assumed to be equivalent to mitochondrial centum picosiemens (mCS). However, the different properties of IMAC and mCS channels challenges this opinion. In our study, we characterized the single-channel anion selectivity and pH regulation of chloride channels from purified cardiac mitochondria. We observed that channel conductance decreased in the order: Cl⁻ > Br⁻ > I⁻ > chlorate ≈ formate > acetate, and that gluconate did not permeate under control conditions. The selectivity sequence was Br⁻ ≥ chlorate ≥ I⁻ ≥ Cl⁻ ≥ formate ≈ acetate. Measurement of the concentration dependence of chloride conductance revealed altered channel gating kinetics, which was demonstrated by prolonged mean open time value with increasing chloride concentration. The observed mitochondrial chloride channels were in many respects similar to those of mCS, but not those of IMAC. Surprisingly, we observed that acidic pH increased channel conductance and that an increase of pH from 7.4 to 8.5 reduced it. The gluconate current appeared and gradually increased when pH decreased from pH 7.0 to 5.6. Our results indicate that pH regulates the channel pore diameter in such a way that dilation increases with more acidic pH. We assume this newly observed pH-dependent anion channel property may be involved in pH regulation of anion distribution in different mitochondrial compartments.

  17. Gating the Selectivity Filter in ClC Chloride Channels

    NASA Astrophysics Data System (ADS)

    Dutzler, Raimund; Campbell, Ernest B.; MacKinnon, Roderick

    2003-04-01

    ClC channels conduct chloride (Cl-) ions across cell membranes and thereby govern the electrical activity of muscle cells and certain neurons, the transport of fluid and electrolytes across epithelia, and the acidification of intracellular vesicles. The structural basis of ClC channel gating was studied. Crystal structures of wild-type and mutant Escherichia coli ClC channels bound to a monoclonal Fab fragment reveal three Cl- binding sites within the 15-angstrom neck of an hourglass-shaped pore. The Cl- binding site nearest the extracellular solution can be occupied either by a Cl- ion or by a glutamate carboxyl group. Mutations of this glutamate residue in Torpedo ray ClC channels alter gating in electrophysiological assays. These findings reveal a form of gating in which the glutamate carboxyl group closes the pore by mimicking a Cl- ion.

  18. A synthetic chloride channel restores chloride conductance in human cystic fibrosis epithelial cells.

    PubMed

    Shen, Bing; Li, Xiang; Wang, Fei; Yao, Xiaoqiang; Yang, Dan

    2012-01-01

    Mutations in the gene-encoding cystic fibrosis transmembrane conductance regulator (CFTR) cause defective transepithelial transport of chloride (Cl(-)) ions and fluid, thereby becoming responsible for the onset of cystic fibrosis (CF). One strategy to reduce the pathophysiology associated with CF is to increase Cl(-) transport through alternative pathways. In this paper, we demonstrate that a small synthetic molecule which forms Cl(-) channels to mediate Cl(-) transport across lipid bilayer membranes is capable of restoring Cl(-) permeability in human CF epithelial cells; as a result, it has the potential to become a lead compound for the treatment of human diseases associated with Cl(-) channel dysfunction.

  19. A Synthetic Chloride Channel Restores Chloride Conductance in Human Cystic Fibrosis Epithelial Cells

    PubMed Central

    Wang, Fei; Yao, Xiaoqiang; Yang, Dan

    2012-01-01

    Mutations in the gene-encoding cystic fibrosis transmembrane conductance regulator (CFTR) cause defective transepithelial transport of chloride (Cl−) ions and fluid, thereby becoming responsible for the onset of cystic fibrosis (CF). One strategy to reduce the pathophysiology associated with CF is to increase Cl− transport through alternative pathways. In this paper, we demonstrate that a small synthetic molecule which forms Cl− channels to mediate Cl− transport across lipid bilayer membranes is capable of restoring Cl− permeability in human CF epithelial cells; as a result, it has the potential to become a lead compound for the treatment of human diseases associated with Cl− channel dysfunction. PMID:22514656

  20. Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator

    PubMed Central

    Schwiebert, Erik M.; Morales, Marcelo M.; Devidas, Sreenivas; Egan, Marie E.; Guggino, William B.

    1998-01-01

    CFTR is a cyclic AMP (cAMP)-activated chloride (Cl−) channel and a regulator of outwardly rectifying Cl− channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl− channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3–1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36Cl− efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl− channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted α-helices 5 and 6, forms an essential part of the Cl− channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl− channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved. PMID:9482946

  1. Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst

    SciTech Connect

    Ahluwalia, Jatinder

    2008-10-31

    Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I{sub e}. In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 {mu}M), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.

  2. ClC-3 chloride channel functions as a mechanically sensitive channel in osteoblasts.

    PubMed

    Wang, Huan; Wang, Rong; Wang, Zhe; Liu, Qian; Mao, Yong; Duan, Xiaohong

    2015-12-01

    Mechanical stimulation usually causes the volume changes of osteoblasts. Whether these volume changes could be sensed by the ClC-3 chloride channel, a volume-sensitive ion channel, and further promote the osteodifferentiation in osteoblasts has not been determined. In this study, we applied persistent static compression on MC3T3-E1 cells to detect the expression changes of ClC-3, osteogenic markers, as well as some molecules related with signaling transduction pathway. We tested the key role of ClC-3 in transferring the mechanical signal to osteoinduction by ClC-3 overexpressing and siRNA technique. We found that ClC-3 level was up-regulated by mechanical stimulation in MC3T3-E1 cells. Mechanical force also up-regulated the mRNA level of osteogenic markers such as alkaline phosphatase (Alp), bone sialoprotein (Bsp), and osteocalcin (Oc), which could be blocked or strengthened by Clcn3 siRNA or overexpressing, and Alp expression was more sensitive to the changes of ClC-3 level. We also found that runt-related transcription factor 2 (Runx2), transforming growth factor beta 1 (TGF-β1), and Wnt pathway might be involved in ClC-3 mediated mechanical transduction in osteoblasts. The data from the current study suggest that the ClC-3 chloride channel acts as a mechanically sensitive channel to regulate osteodifferentiation in osteoblasts.

  3. Regulation of Fast-Spiking Basket Cell Synapses by the Chloride Channel ClC–2

    PubMed Central

    Földy, Csaba; Lee, Sang-Hun; Morgan, Robert J.; Soltesz, Ivan

    2010-01-01

    Parvalbumin-expressing, fast-spiking basket cells play key roles in the generation of synchronous, rhythmic population activities in the hippocampus. Here we show that GABAA receptor-mediated synaptic inputs from murine parvalbumin-expressing basket cells are selectively modulated by the membrane voltage- and intracellular chloride-dependent chloride channel ClC–2. These data demonstrate a novel cell type-specific regulation of intracellular chloride homeostasis in the perisomatic region of hippocampal pyramidal neurons. PMID:20676104

  4. Insecticide sensitivity of native chloride and sodium channels in a mosquito cell line.

    PubMed

    Jenson, Lacey J; Anderson, Troy D; Bloomquist, Jeffrey R

    2016-06-01

    The aim of this study was to investigate the utility of cultured Anopheles gambiae Sua1B cells for insecticide screening applications without genetic engineering or other treatments. Sua1B cells were exposed to the known insecticidal compounds lindane and DIDS, which inhibited cell growth at micromolar concentrations. In patch clamp studies, DIDS produced partial inhibition (69%) of chloride current amplitudes, and an IC50 of 5.1μM was determined for Sua1B cells. A sub-set of chloride currents showed no response to DIDS; however, inhibition (64%) of these currents was achieved using a low chloride saline solution, confirming their identity as chloride channels. In contrast, lindane increased chloride current amplitude (EC50=116nM), which was reversed when cells were bathed in calcium-free extracellular solution. Voltage-sensitive chloride channels were also inhibited by the presence of fenvalerate, a type 2 pyrethroid, but not significantly blocked by type 1 allethrin, an effect not previously shown in insects. Although no evidence of fast inward currents typical of sodium channels was observed, studies with fenvalerate in combination with veratridine, a sodium channel activator, revealed complete inhibition of cell growth that was best fit by a two-site binding model. The high potency effect was completely inhibited in the presence of tetrodotoxin, a specific sodium channel blocker, suggesting the presence of some type of sodium channel. Thus, Sua1B cells express native insect ion channels with potential utility for insecticide screening.

  5. Chloride channels involve in hydrogen peroxide-induced apoptosis of PC12 cells.

    PubMed

    Zuo, Wanhong; Zhu, Linyan; Bai, Zhiquan; Zhang, Haifeng; Mao, Jianwen; Chen, Lixin; Wang, Liwei

    2009-10-02

    Chloride channel activity is one of the critical factors responsible for cell apoptotic volume decrease (AVD). However, the roles of chloride channels in apoptosis have not been fully understood. In the current study, we assessed the role of chloride channels in hydrogen peroxide (H(2)O(2))-induced apoptosis of pheochromocytoma cells (PC12). Extracellular application of H(2)O(2) activated a chloride current and induced cell volume decrease in a few minutes. Incubation of cells with H(2)O(2) elevated significantly the membrane permeability to the DNA dye Hoechst 33258 in 1h and induced apoptosis of most PC12 cells tested in 24h. The chloride channel blocker NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) prevented appearance of H(2)O(2)-induced high membrane permeability and cell shrinkage, suppressed H(2)O(2)-activated chloride currents and protected PC12 cells from apoptosis induced by H(2)O(2). The results suggest that chloride channels may contribute to H(2)O(2)-induced apoptosis by ways of elevation of membrane permeability and AVD in PC12 cells.

  6. Characterization of the Chloride Channel-Like, AtCLCg, Involved in Chloride Tolerance in Arabidopsis thaliana.

    PubMed

    Nguyen, Chi Tam; Agorio, Astrid; Jossier, Mathieu; Depré, Sylvain; Thomine, Sébastien; Filleur, Sophie

    2016-04-01

    In plant cells, anion channels and transporters are essential for key functions such as nutrition, ion homeostasis and resistance to biotic or abiotic stresses. We characterized AtCLCg, a member of the chloride channel (CLC) family in Arabidopsis localized in the vacuolar membrane. When grown on NaCl or KCl, atclcg knock-out mutants showed a decrease in biomass. In the presence of NaCl, these mutants overaccumulate chloride in shoots. No difference in growth was detected in response to osmotic stress by mannitol. These results suggest a physiological function of AtCLCg in the chloride homeostasis during NaCl stress. AtCLCg shares a high degree of identity (62%) with AtCLCc, another vacuolar CLC essential for NaCl tolerance. However, the atclcc atclccg double mutant is not more sensitive to NaCl than single mutants. As the effects of both mutations are not additive, gene expression analyses were performed and revealed that: (i)AtCLCg is expressed in mesophyll cells, hydathodes and phloem while AtCLCc is expressed in stomata; and (ii)AtCLCg is repressed in the atclcc mutant background, and vice versa. Altogether these results demonstrate that both AtCLCc and AtCLCg are important for tolerance to excess chloride but not redundant, and form part of a regulatory network controlling chloride sensitivity.

  7. Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex.

    PubMed

    Zhang, Weiping; Schmelzeisen, Steffen; Parthier, Daniel; Frings, Stephan; Möhrlen, Frank

    2015-01-01

    Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum.

  8. Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

    PubMed Central

    Parthier, Daniel; Frings, Stephan; Möhrlen, Frank

    2015-01-01

    Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum. PMID:26558388

  9. Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride.

    PubMed

    Ratner, Martha A; Decker, Sarah E; Aller, Stephen G; Weber, Gerhard; Forrest, John N

    2006-03-01

    In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action.

  10. Mechanism of HERG potassium channel inhibition by tetra-n-octylammonium bromide and benzethonium chloride

    SciTech Connect

    Long, Yan; Lin, Zuoxian; Xia, Menghang; Zheng, Wei; Li, Zhiyuan

    2013-03-01

    Tetra-n-octylammonium bromide and benzethonium chloride are synthetic quaternary ammonium salts that are widely used in hospitals and industries for the disinfection and surface treatment and as the preservative agent. Recently, the activities of HERG channel inhibition by these compounds have been found to have potential risks to induce the long QT syndrome and cardiac arrhythmia, although the mechanism of action is still elusive. This study was conducted to investigate the mechanism of HERG channel inhibition by these compounds by using whole-cell patch clamp experiments in a CHO cell line stably expressing HERG channels. Tetra-n-octylammonium bromide and benzethonium chloride exhibited concentration-dependent inhibitions of HERG channel currents with IC{sub 50} values of 4 nM and 17 nM, respectively, which were also voltage-dependent and use-dependent. Both compounds shifted the channel activation I–V curves in a hyperpolarized direction for 10–15 mV and accelerated channel activation and inactivation processes by 2-fold. In addition, tetra-n-octylammonium bromide shifted the inactivation I–V curve in a hyperpolarized direction for 24.4 mV and slowed the rate of channel deactivation by 2-fold, whereas benzethonium chloride did not. The results indicate that tetra-n-octylammonium bromide and benzethonium chloride are open-channel blockers that inhibit HERG channels in the voltage-dependent, use-dependent and state-dependent manners. - Highlights: ► Tetra-n-octylammonium and benzethonium are potent HERG channel inhibitors. ► Channel activation and inactivation processes are accelerated by the two compounds. ► Both compounds are the open-channel blockers to HERG channels. ► HERG channel inhibition by both compounds is use-, voltage- and state dependent. ► The in vivo risk of QT prolongation needs to be studied for the two compounds.

  11. Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation.

    PubMed

    Untiet, Verena; Kovermann, Peter; Gerkau, Niklas J; Gensch, Thomas; Rose, Christine R; Fahlke, Christoph

    2017-02-01

    Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride-sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl(-) ]int to be controlled by two opposing transport processes: chloride is actively accumulated by the Na(+) -K(+) -2Cl(-) cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl(-) ]int to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion-selective channels during glutamate transport, and thus represent a class of ligand-gated anion channels. Age-dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT-1 increased [Cl(-) ]int in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl(-) ]int . Other tested chloride channels or chloride transporters do not contribute to [Cl(-) ]int under our experimental conditions. Neither genetic removal of ClC-2 nor pharmacological block of K(+) -Cl(-) cotransporter change resting Bergmann glial [Cl(-) ]int in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388-400.

  12. Cytoprotection of kidney epithelial cells by compounds that target amino acid gated chloride channels.

    PubMed

    Venkatachalam, M A; Weinberg, J M; Patel, Y; Saikumar, P; Dong, Z

    1996-02-01

    Glycine, strychnine and certain chloride channel blockers were reported to protect cells against lethal cell injury. These effects have been attributed to interactions with membrane proteins related to CNS glycine gated chloride channel receptors. We have investigated the pharmacology of these actions. Madin-Darby canine kidney (MDCK) epithelial cells were depleted of adenosine triphosphate (ATP) by incubation in glucose free medium containing a mitochondrial uncoupler. Medium Ca2+ was adjusted to 100 nM in the presence of an ionophore such that intracellular Ca2+ did not increase, and Ca(2+)-related injury mechanisms were inhibited. This permitted more sensitive quantitation of protection against cell injury attributable to glycine or other agents whose actions might be related to those of the amino acid. Two classes of compounds showed cytoprotective activity in this system: (1) ligands at chloride channel receptors, such as glycine, strychnine and avermectin B1a; (2) chloride channel blockers, including cyanotriphenylboron and niflumic acid, both of which are known to bind to channel domains of CNS glycine receptors. Morphological and functional studies showed that the compounds preserved plasma membrane integrity, but permitted cell swelling. Substitution of medium chloride by gluconate, or chloride salts by sucrose, did not substantially modify lethal damage or its prevention by glycine or other drugs. The compounds did not modify ATP declines. At least for some compounds, cytoprotection appeared to be specific to structural features on the molecules. These observations are consistent with the hypothesis that a plasma membrane protein related to glycine-gated chloride channel receptors plays a significant role in cell injury, but indicate that the mechanisms of injury and protection by compounds active in this system are not related to chloride fluxes.

  13. Chloride channels in cancer: Focus on chloride intracellular channel 1 and 4 (CLIC1 AND CLIC4) proteins in tumor development and as novel therapeutic targets.

    PubMed

    Peretti, Marta; Angelini, Marina; Savalli, Nicoletta; Florio, Tullio; Yuspa, Stuart H; Mazzanti, Michele

    2015-10-01

    In recent decades, growing scientific evidence supports the role of ion channels in the development of different cancers. Both potassium selective pores and chloride permeabilities are considered the most active channels during tumorigenesis. High rate of proliferation, active migration, and invasiveness into non-neoplastic tissues are specific properties of neoplastic transformation. All these actions require partial or total involvement of chloride channel activity. In this context, this class of membrane proteins could represent valuable therapeutic targets for the treatment of resistant tumors. However, this encouraging premise has not so far produced any valid new channel-targeted antitumoral molecule for cancer treatment. Problematic for drug design targeting ion channels is their vital role in normal cells for essential physiological functions. By targeting these membrane proteins involved in pathological conditions, it is inevitable to cause relevant side effects in healthy organs. In light of this, a new protein family, the chloride intracellular channels (CLICs), could be a promising class of therapeutic targets for its intrinsic individualities: CLIC1 and CLIC4, in particular, not only are overexpressed in specific tumor types or their corresponding stroma but also change localization and function from hydrophilic cytosolic to integral transmembrane proteins as active ionic channels or signal transducers during cell cycle progression in certain cases. These changes in intracellular localization, tissue compartments, and channel function, uniquely associated with malignant transformation, may offer a unique target for cancer therapy, likely able to spare normal cells. This article is part of a special issue itled "Membrane Channels and Transporters in Cancers."

  14. Spatial Distribution of Calcium-Gated Chloride Channels in Olfactory Cilia

    PubMed Central

    French, Donald A.; Badamdorj, Dorjsuren; Kleene, Steven J.

    2010-01-01

    Background In vertebrate olfactory receptor neurons, sensory cilia transduce odor stimuli into changes in neuronal membrane potential. The voltage changes are primarily caused by the sequential openings of two types of channel: a cyclic-nucleotide-gated (CNG) cationic channel and a calcium-gated chloride channel. In frog, the cilia are 25 to 200 µm in length, so the spatial distributions of the channels may be an important determinant of odor sensitivity. Principal Findings To determine the spatial distribution of the chloride channels, we recorded from single cilia as calcium was allowed to diffuse down the length of the cilium and activate the channels. A computational model of this experiment allowed an estimate of the spatial distribution of the chloride channels. On average, the channels were concentrated in a narrow band centered at a distance of 29% of the ciliary length, measured from the base of the cilium. This matches the location of the CNG channels determined previously. This non-uniform distribution of transduction proteins is consistent with similar findings in other cilia. Conclusions On average, the two types of olfactory transduction channel are concentrated in the same region of the cilium. This may contribute to the efficient detection of weak stimuli. PMID:21209888

  15. The ClC-0 chloride channel is a 'broken' Cl-/H+ antiporter.

    PubMed

    Lísal, Jirí; Maduke, Merritt

    2008-08-01

    Ion channels have historically been viewed as distinct from secondary active transporters. However, the recent discovery that the CLC 'chloride channel' family is made up of both channels and active transporters has led to the hypothesis that the ion-transport mechanisms of these two types of membrane proteins may be similar. Here we use single-channel analysis to demonstrate that ClC-0 channel gating (opening and closing) involves the transmembrane movement of protons. This result indicates that ClC-0 is a 'broken' Cl(-)/H(+) antiporter in which one of the conformational states has become leaky for chloride ions. This finding clarifies the evolutionary relationship between the channels and transporters and conveys that similar mechanisms and analogous protein movements are used by both.

  16. Projection structure of a ClC-type chloride channel at 6.5Å resolution

    NASA Astrophysics Data System (ADS)

    Mindell, Joseph A.; Maduke, Merritt; Miller, Christopher; Grigorieff, Nikolaus

    2001-01-01

    Virtually all cells in all eukaryotic organisms express ion channels of the ClC type, the only known molecular family of chloride-ion-selective channels. The diversity of ClC channels highlights the multitude and range of functions served by gated chloride-ion conduction in biological membranes, such as controlling electrical excitability in skeletal muscle, maintaining systemic blood pressure, acidifying endosomal compartments, and regulating electrical responses of GABA (γ-aminobutyric acid)-containing interneurons in the central nervous system. Previously, we expressed and purified a prokaryotic ClC channel homologue. Here we report the formation of two-dimensional crystals of this ClC channel protein reconstituted into phospholipid bilayer membranes. Cryo-electron microscopic analysis of these crystals yields a projection structure at 6.5Å resolution, which shows off-axis water-filled pores within the dimeric channel complex.

  17. Stimulation effect of wide type CFTR chloride channel by the naturally occurring flavonoid tangeretin.

    PubMed

    Jiang, Yu; Yu, Bo; Wang, Xue; Sui, Yujie; Zhang, Yaofang; Yang, Shuang; Yang, Hong; Ma, Tonghui

    2014-12-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed in the apical membrane of serous epithelial cells. Both deficiency and overactivation of CFTR may cause fluid and salt secretion related diseases. In the present study, we identified tangeretin from Pericarpium Citri Reticulatae Viride as a CFTR activator using high-throughput screening based on FRT cell-based fluorescence assay. The activation effect of tangeretin on CFTR chloride channel and the possible underlying mechanisms were investigated. Fluorescence quenching tests showed that tangeretin dose- and time-dependently activated CFTR chloride channel, the activity had rapid and reversible characteristics and the activation effect could be completely reversed by the CFTR specific blocker CFTRinh-172. Primary mechanism studies indicated that the activation effect of tangeretin on CFTR chloride channel was FSK dependent as well as had additional effect with FSK and IBMX suggesting that tangeretin activates CFTR by direct interacting with the protein. Ex-vivo tests revealed that tangeretin could accelerate the speed of the submucosal gland fluid secretion. Short-circuit current measurement demonstrated that tangeretin activated rat colonic mucosa chloride current. Thus, CFTR Cl(-) channel is a molecular target of natural compound tangeretin. Tangeretin may have potential use for the treatment of CFTR-related diseases like cystic fibrosis, bronchiectasis and habitual constipation.

  18. Effect of trimethyllead chloride on slowly activating (SV) channels in red beet (Beta vulgaris L.) taproots.

    PubMed

    Trela, Zenon; Burdach, Zbigniew; Przestalski, Stanisław; Karcz, Waldemar

    2012-12-01

    The patch-clamp technique was used to examine the effect of trimethyllead chloride (Met(3)PbCl) on SV channel activity in red beet (Beta vulgaris L.) taproot vacuoles. It was found that in the control bath the macroscopic currents showed the typical slow activation and a strong outward rectification of the steady-state currents. An addition of Met(3)PbCl to the bath solution blocked, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant τ increased several times in the presence of 100 μM trimethyllead chloride at all voltages tested. When single channel properties were analyzed, only little channel activity could be recorded in the presence of 100 μM Met(3)PbCl. Trimethyllead chloride decreased significantly (by about one order of magnitude) the open probability of single channels. The recordings of single channel activity obtained in the presence and absence of Met(3)PbCl showed that organolead only slightly (by ca. 10%) decreased the unitary conductance of single channels. It was also found that Met(3)PbCl diminished significantly the number of SV channel openings, whereas it did not change the opening times of the channels. Taken together, these results suggest that Met(3)PbCl binding site is located outside the channel selectivity filter.

  19. Chloride and potassium channels in cystic fibrosis airway epithelia

    NASA Astrophysics Data System (ADS)

    Welsh, Michael J.; Liedtke, Carole M.

    1986-07-01

    Cystic fibrosis, the most common lethal genetic disease in Caucasians, is characterized by a decreased permeability in sweat gland duct and airway epithelia. In sweat duct epithelium, a decreased Cl- permeability accounts for the abnormally increased salt content of sweat1. In airway epithelia a decreased Cl- permeability, and possibly increased sodium absorption, may account for the abnormal respiratory tract fluid2,3. The Cl- impermeability has been localized to the apical membrane of cystic fibrosis airway epithelial cells4. The finding that hormonally regulated Cl- channels make the apical membrane Cl- permeable in normal airway epithelial cells5 suggested abnormal Cl- channel function in cystic fibrosis. Here we report that excised, cell-free patches of membrane from cystic fibrosis epithelial cells contain Cl- channels that have the same conductive properties as Cl- channels from normal cells. However, Cl- channels from cystic fibrosis cells did not open when they were attached to the cell. These findings suggest defective regulation of Cl- channels in cystic fibrosis epithelia; to begin to address this issue, we performed two studies. First, we found that isoprenaline, which stimulates Cl- secretion, increases cellular levels of cyclic AMP in a similar manner in cystic fibrosis and non-cystic fibrosis epithelial cells. Second, we show that adrenergic agonists open calcium-activated potassium channels, indirectly suggesting that calcium-dependent stimulus-response coupling is intact in cystic fibrosis. These data suggest defective regulation of Cl- channels at a site distal to cAMP accumulation.

  20. Evidence for a role of GABA- and glutamate-gated chloride channels in olfactory memory.

    PubMed

    Boumghar, Katia; Couret-Fauvel, Thomas; Garcia, Mikael; Armengaud, Catherine

    2012-11-01

    In the honeybee, we investigated the role of transmissions mediated by GABA-gated chloride channels and glutamate-gated chloride channels (GluCls) of the mushroom bodies (MBs) on olfactory learning using a single-trial olfactory conditioning paradigm. The GABAergic antagonist picrotoxin (PTX) or the GluCl antagonist L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) was injected alone or in combination into the α-lobes of MBs. PTX impaired early long-term olfactory memory when injected before conditioning or before testing. L-trans-PDC alone induced no significant effect on learning and memory but induced a less specific response to the conditioned odor. When injected before PTX, L-trans-PDC was able to modulate PTX effects. These results emphasize the role of MB GABA-gated chloride channels in consolidation processes and strongly support that GluCls are involved in the perception of the conditioned stimulus.

  1. Hormonally controlled chloride movement across Drosophila tubules is via ion channels in stellate cells.

    PubMed

    O'Donnell, M J; Rheault, M R; Davies, S A; Rosay, P; Harvey, B J; Maddrell, S H; Kaiser, K; Dow, J A

    1998-04-01

    Anion conductance across the Drosophila melanogaster Malpighian (renal) tubule was investigated by a combination of physiological and transgenic techniques. Patch-clamp recordings identified clusters of 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive "maxi-chloride" channels in a small domain of the apical membrane. Fluid secretion assays demonstrated sensitivity to the chloride channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid, diphenylamine-2-carboxylate, anthracene-9-carboxylic acid, and niflumic acid. Electrophysiological analysis showed that the calcium-mediated increase in anion conductance was blocked by the same agents. Vibrating probe analysis revealed a small number of current density hot spots, coincident with "stellate" cells, that were abolished by low-chloride saline or the same chloride channel blockers. GAL-4-targeted expression of an aequorin transgene revealed that the neurohormone leucokinin elicits a rapid increase in intracellular calcium levels in stellate cells that precedes the fastest demonstrable physiological effect. Taken together, these data show that leucokinins act on stellate cells through intracellular calcium to increase transcellular chloride conductance through channels. As electrogenic cation conductance is confined to principal cells, the two pathways are spatially segregated in this tissue.

  2. The AQP-3 water channel is a pivotal modulator of glycerol-induced chloride channel activation in nasopharyngeal carcinoma cells.

    PubMed

    Zhang, Haifeng; Deng, Zhiqin; Yang, Lili; Luo, Hai; Liu, Shanwen; Li, Yuan; Wei, Yan; Peng, Shuang; Zhu, Linyan; Wang, Liwei; Chen, Lixin

    2016-03-01

    Aquaporin (AQP) and chloride channels are ubiquitous in virtually all living cells, playing pivotal roles in cell proliferation, migration and apoptosis. We previously reported that AQP-3 aquaglyceroporin and ClC-3 chloride channels could form complexes to regulate cell volume in nasopharyngeal carcinoma cells. In this study, the roles of AQP-3 in their hetero-complexes were further investigated. Glycerol entered the cells via AQP-3 and induced two different Cl(-) currents through cell swelling-dependent or -independent pathways. The swelling-dependent Cl(-) current was significantly inhibited by pretreatment with CuCl2 and AQP-3-siRNA. After siRNA-induced AQP-3 knock-down, the 140 mM glycerol isoosmotic solution swelled cells by 22% (45% in AQP-3-intact cells) and induced a smaller Cl(-) current; this current was smaller than that activated by 8% cell volume swelling, which induced by the 140 mM glycerol hyperosmotic solution in AQP-3-intact cells. This suggests that the interaction between AQP-3 and ClC-3 plays an important role in cell volume regulation and that AQP-3 may be a modulator that opens volume-regulated chloride channels. The swelling-independent Cl(-) current, which was activated by extracellular glycerol, was reduced by CuCl2 and AQP-3-siRNA pretreatment. Dialyzing glycerol into cells via the pipette directly induced the swelling-independent Cl(-) current; however this current was blocked by AQP-3 down-regulation, suggesting AQP-3 is essential for the opening of chloride channels. In conclusion, AQP-3 is the pathway for water, glycerol and other small solutes to enter cells, and it may be an essential modulator for the gating of chloride channels.

  3. Conformational changes opening and closing the CFTR chloride channel: insights from cysteine scanning mutagenesis.

    PubMed

    El Hiani, Yassine; Linsdell, Paul

    2014-12-01

    Cystic fibrosis, the most common lethal genetic disease affecting young people in North America, is caused by failure of the chloride ion channel known as CFTR (cystic fibrosis transmembrane conductance regulator). CFTR belongs to the large family of ATP-binding cassette (ABC) membrane transporters. In CFTR, ATP-driven events at the nucleotide-binding domains (NBDs) open and close a gate that controls chloride permeation. However, the conformational changes concomitant with opening and closing of the CFTR gate are unknown. Diverse techniques including substituted cysteine accessibility method, disulfide cross-linking, and patch-clamp recording have been used to explore CFTR channel structure. Here, we consider the architecture of both the open and the closed CFTR channel. We review how CFTR channel structure changes between the closed and the open channel conformations and portray the relative function of both cytoplasmic and vestigial gates during the gating cycle. Understanding how the CFTR channel gates chloride permeation is central for understanding how CFTR defects lead to CF. Such knowledge opens the door for novel ways to maximize CFTR channel activity in a CF setting.

  4. Anion conductance selectivity mechanism of the CFTR chloride channel.

    PubMed

    Linsdell, Paul

    2016-04-01

    All ion channels are able to discriminate between substrate ions to some extent, a process that involves specific interactions between permeant anions and the so-called selectivity filter within the channel pore. In the cystic fibrosis transmembrane conductance regulator (CFTR) anion-selective channel, both anion relative permeability and anion relative conductance are dependent on anion free energy of hydration--anions that are relatively easily dehydrated tend to show both high permeability and low conductance. In the present work, patch clamp recording was used to investigate the relative conductance of different anions in CFTR, and the effect of mutations within the channel pore. In constitutively-active E1371Q-CFTR channels, the anion conductance sequence was Cl(-) > NO3(-) > Br(-) > formate > SCN(-) > I(-). A mutation that disrupts anion binding in the inner vestibule of the pore (K95Q) disrupted anion conductance selectivity, such that anions with different permeabilities showed almost indistinguishable conductances. Conversely, a mutation at the putative narrowest pore region that is known to disrupt anion permeability selectivity (F337A) had minimal effects on anion relative conductance. Ion competition experiments confirmed that relatively tight binding of permeant anions resulted in relatively low conductance. These results suggest that the relative affinity of ion binding in the inner vestibule of the pore controls the relative conductance of different permeant anions in CFTR, and that the pore has two physically distinct anion selectivity filters that act in series to control anion conductance selectivity and anion permeability selectivity respectively.

  5. Regulation of CFTR chloride channel macroscopic conductance by extracellular bicarbonate.

    PubMed

    Li, Man-Song; Holstead, Ryan G; Wang, Wuyang; Linsdell, Paul

    2011-01-01

    The CFTR contributes to Cl⁻ and HCO₃⁻ transport across epithelial cell apical membranes. The extracellular face of CFTR is exposed to varying concentrations of Cl⁻ and HCO₃⁻ in epithelial tissues, and there is evidence that CFTR is sensitive to changes in extracellular anion concentrations. Here we present functional evidence that extracellular Cl⁻ and HCO₃⁻ regulate anion conduction in open CFTR channels. Using cell-attached and inside-out patch-clamp recordings from constitutively active mutant E1371Q-CFTR channels, we show that voltage-dependent inhibition of CFTR currents in intact cells is significantly stronger when the extracellular solution contains HCO₃⁻ than when it contains Cl⁻. This difference appears to reflect differences in the ability of extracellular HCO₃⁻ and Cl⁻ to interact with and repel intracellular blocking anions from the pore. Strong block by endogenous cytosolic anions leading to reduced CFTR channel currents in intact cells occurs at physiologically relevant HCO₃⁻ concentrations and membrane potentials and can result in up to ∼50% inhibition of current amplitude. We propose that channel block by cytosolic anions is a previously unrecognized, physiologically relevant mechanism of channel regulation that confers on CFTR channels sensitivity to different anions in the extracellular fluid. We further suggest that this anion sensitivity represents a feedback mechanism by which CFTR-dependent anion secretion could be regulated by the composition of the secretions themselves. Implications for the mechanism and regulation of CFTR-dependent secretion in epithelial tissues are discussed.

  6. Myotonia caused by mutations in the muscle chloride channel gene CLCN1.

    PubMed

    Pusch, Michael

    2002-04-01

    Pure non-syndromic, non-dystrophic myotonia in humans is caused by mutations in the genes coding for the skeletal muscle sodium channel (SCN5A) or the skeletal muscle chloride channel (CLCN1) with similar phenotypes. Chloride-channel myotonia can be dominant (Thomsen-type myotonia) or recessive (Becker-type myotonia). More than 60 myotonia-causing mutations in the CLCN1 gene have been identified, with only a few of them being dominant. A common phenotype of dominant mutations is a dominant negative effect of mutant subunits in mutant-WT heterodimers, causing a large shift of the steady-state open probability voltage-dependence towards more positive, unphysiological voltages. The study of the properties of disease causing mutations has helped in understanding the functional properties of the CLC-1 channel that is part of a nine-member gene family of chloride channels. The large body of knowledge obtained for CLC-1 may also help to better understand the other CLC channels, three of which are also involved in genetic diseases.

  7. Permeation and Block of the Skeletal Muscle Chloride Channel, ClC-1, by Foreign Anions

    PubMed Central

    Rychkov, G.Y.; Pusch, M.; Roberts, M.L.; Jentsch, T.J.; Bretag, A.H.

    1998-01-01

    A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl− as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO3−, BrO3−); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl− at the regulatory binding site but impair Cl− passage through the channel pore (Br−, NO3−, ClO3−, I−, ClO4−, SCN−). The permeability sequence for rClC-1, SCN− ∼ ClO4− > Cl− > Br− > NO3− ∼ ClO3− > I− >> BrO3− > HCO3− >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the Popen curve, SCN− ∼ ClO4− > I− > NO3− ∼ ClO3− ∼ methanesulfonate > Br− > cyclamate > BrO3− > HCO3− > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl− and SCN− or ClO4− suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may

  8. Modulation of Chloride Channel Functions by the Plant Lignan Compounds Kobusin and Eudesmin

    PubMed Central

    Jiang, Yu; Yu, Bo; Fang, Fang; Cao, Huanhuan; Ma, Tonghui; Yang, Hong

    2015-01-01

    Plant lignans are diphenolic compounds widely present in vegetables, fruits, and grains. These compounds have been demonstrated to have protective effect against cancer, hypertension and diabetes. In the present study, we showed that two lignan compounds, kobusin and eudesmin, isolated from Magnoliae Flos, could modulate intestinal chloride transport mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCCs). The compounds activated CFTR channel function in both FRT cells and in HT-29 cells. The modulating effects of kobusin and eudesmin on the activity of CaCCgie (CaCC expressed in gastrointestinal epithelial cells) were also investigated, and the result showed that both compounds could stimulate CaCCgie-mediated short-circuit currents and the stimulation was synergistic with ATP. In ex vivo studies, both compounds activated CFTR and CaCCgie chloride channel activities in mouse colonic epithelia. Remarkably, the compounds showed inhibitory effects toward ANO1/CaCC-mediated short-circuit currents in ANO1/CaCC-expressing FRT cells, with IC50 values of 100 μM for kobusin and 200 μM for eudesmin. In charcoal transit study, both compounds mildly reduced gastrointestinal motility in mice. Taken together, these results revealed a new kind of activity displayed by the lignan compounds, one that is concerned with the modulation of chloride channel function. PMID:26635857

  9. Cloning and expression of Ca2+-activated chloride channel from rat brain.

    PubMed

    Jeong, Sang Min; Park, Hye-Kyung; Yoon, In-Soo; Lee, Jun-Ho; Kim, Jong-Hoon; Jang, Choon-Gon; Lee, C Justin; Nah, Seung-Yeol

    2005-08-26

    To clone the gene product responsible for the calcium-activated chloride channel (CLCA) in rat brain cerebrum, we performed a reverse transcription-PCR (RT-PCR) with gene-specific primers of a rat EST clone. We successfully cloned a rat brain CLCA (rbCLCA). The full-length cDNA is 2895 bp long and codes for a 902 amino acid protein. The clone consists of four transmembrane domains and shows a 79.1% of significant homology with previously reported mouse smooth muscle chloride channel sequence. We also performed RT-PCR using single neuron and glia, and various tissues to determine the tissue expression of rbCLCA. We found that rbCLCA was expressed in both neuron and glia. In peripheral organs, rbCLCA showed the predominant expressions in cerebrum, cerebellum, kidney, small intestine, and stomach but not in heart, large intestine, liver, lung, and spleen. Whole-cell patch clamp studies in HEK293 cells transfected with the clone identified a niflumic acid (a CLCA channel blocker)-sensitive and voltage-dependent chloride current but we could not observe this chloride current in mock-transfected cells. The identification of genes belonging to the CLCA family from rat brain and its functional expression will help to evaluate its physiological role in brain as anion channel.

  10. Chloride Channel 3 Channels in the Activation and Migration of Human Blood Eosinophils in Allergic Asthma.

    PubMed

    Gaurav, Rohit; Bewtra, Againdra K; Agrawal, Devendra K

    2015-08-01

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is responsible for respiratory burst in immune cells. Chloride channel 3 (CLC3) has been linked to the respiratory burst in eosinophils and neutrophils. The effect of cytokines and the involvement of CLC3 in the regulation of NADPH-dependent oxidative stress and on cytokine-mediated migration of eosinophils are not known. Human peripheral blood eosinophils were isolated from healthy individuals and from individuals with asthma by negative selection. Real-time PCR was used to detect the expression of NADPH oxidases in eosinophils. Intracellular reactive oxygen species (ROS) measurement was done with flow cytometry. Superoxide generation was measured with transforming growth factor (TGF)-β, eotaxin, and CLC3 blockers. CLC3 dependence of eosinophils in TGF-β- and eotaxin-induced migration was also examined. The messenger RNA (mRNA) transcripts of NADPH oxidase (NOX) 2, dual oxidase (DUOX) 1, and DUOX2 were detected in blood eosinophils, with very low expression of NOX1, NOX3, and NOX5 and no NOX4 mRNA. The level of NOX2 mRNA transcripts increased with disease severity in the eosinophils of subjects with asthma compared with healthy nonatopic volunteers. Change in granularity and size in eosinophils, but no change in intracellular ROS, was observed with phorbol myristate acetate (PMA). PMA, TGF-β, and eotaxin used the CLC3-dependent pathway to increase superoxide radicals. TGF-β and eotaxin induced CLC3-dependent chemotaxis of eosinophils. These findings support the requirement of CLC3 in the activation and migration of human blood eosinophils and may provide a potential novel therapeutic target to regulate eosinophil hyperactivity in allergic airway inflammation in asthma.

  11. Location of Release Sites and Calcium-Activated Chloride Channels Relative to Calcium Channels at the Photoreceptor Ribbon Synapse

    PubMed Central

    Mercer, A. J.; Rabl, K.; Riccardi, G. E.; Brecha, N. C.; Stella, S. L.

    2011-01-01

    Vesicle release from photoreceptor ribbon synapses is regulated by L-type Ca2+ channels, which are in turn regulated by Cl− moving through calcium-activated chloride [Cl(Ca)] channels. We assessed the proximity of Ca2+ channels to release sites and Cl(Ca) channels in synaptic terminals of salamander photoreceptors by comparing fast (BAPTA) and slow (EGTA) intracellular Ca2+ buffers. BAPTA did not fully block synaptic release, indicating some release sites are <100 nm from Ca2+ channels. Comparing Cl(Ca) currents with predicted Ca2+ diffusion profiles suggested that Cl(Ca) and Ca2+ channels average a few hundred nanometers apart, but the inability of BAPTA to block Cl(Ca) currents completely suggested some channels are much closer together. Diffuse immunolabeling of terminals with an antibody to the putative Cl(Ca) channel TMEM16A supports the idea that Cl(Ca) channels are dispersed throughout the presynaptic terminal, in contrast with clustering of Ca2+ channels near ribbons. Cl(Ca) currents evoked by intracellular calcium ion concentration ([Ca2+]i) elevation through flash photolysis of DM-nitrophen exhibited EC50 values of 556 and 377 nM with Hill slopes of 1.8 and 2.4 in rods and cones, respectively. These relationships were used to estimate average submembrane [Ca2+]i in photoreceptor terminals. Consistent with control of exocytosis by [Ca2+] nanodomains near Ca2+ channels, average submembrane [Ca2+]i remained below the vesicle release threshold (∼400 nM) over much of the physiological voltage range for cones. Positioning Ca2+ channels near release sites may improve fidelity in converting voltage changes to synaptic release. A diffuse distribution of Cl(Ca) channels may allow Ca2+ influx at one site to influence relatively distant Ca2+ channels. PMID:21084687

  12. Anion exchanger and chloride channel in cat carotid body chemotransduction.

    PubMed

    Iturriaga, R; Mokashi, A; Lahiri, S

    1998-05-28

    In order to test the hypothesis that carotid body (CB) chemoreception depends on the functions of anion channels and HCO3-/Cl- exchangers, we studied the effects of the anion channel blocker anthracene-9-carboxylic acid (9-ANC), the carbonic anhydrase inhibitor methazolamide, and the HCO3-/Cl- exchanger blocker 4,4 diisothiocyanatostilbene-2-2'disulfonic acid (DIDS) on the chemosensory discharges of cat CB, perfused-superfused in vitro at 36.5 +/- 0.5 degrees C, with a modified Tyrode solution. The chemosensory responses to hypoxia (PO2 approximately 50 Torr), hypercapnia (PCO2 approximately 60 Torr, pH = 7.10), nicotine (2-4 nmol) and NaCN (20-40 nmol) were recorded. 9-ANC (2 microM) and DIDS (10 microM) decreased the chemosensory baseline activity, and eliminated the initial peak responses to hypercapnia and hypoxia and increased the time to achieve it. Methazolamide (0.13 mM) did not alter the effect of 9-ANC. The steady state responses to hypoxia and hypercapnia were not diminished after 9-ANC but DIDS lowered the responses. Responses to NaCN effects were all diminished but those to nicotine were not affected. The results suggest that the functions of anion channels and HCO3-/Cl- exchangers are important for the resting dischargers and for the fast responses to hypoxia and hypercapnia.

  13. Simultaneous Optical Recording in Multiple Cells by Digital Holographic Microscopy of Chloride Current Associated to Activation of the Ligand-Gated Chloride Channel GABAA Receptor

    PubMed Central

    Jourdain, Pascal; Boss, Daniel; Rappaz, Benjamin; Moratal, Corinne; Hernandez, Maria-Clemencia; Depeursinge, Christian

    2012-01-01

    Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABAA mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABAA receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABAA receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM. PMID:23236427

  14. Mechanism of allosteric activation of TMEM16A/ANO1 channels by a commonly used chloride channel blocker

    PubMed Central

    Ta, Chau M; Adomaviciene, Aiste; Rorsman, Nils J G; Garnett, Hannah

    2016-01-01

    Background and Purpose Calcium‐activated chloride channels (CaCCs) play varied physiological roles and constitute potential therapeutic targets for conditions such as asthma and hypertension. TMEM16A encodes a CaCC. CaCC pharmacology is restricted to compounds with relatively low potency and poorly defined selectivity. Anthracene‐9‐carboxylic acid (A9C), an inhibitor of various chloride channel types, exhibits complex effects on native CaCCs and cloned TMEM16A channels providing both activation and inhibition. The mechanisms underlying these effects are not fully defined. Experimental Approach Patch‐clamp electrophysiology in conjunction with concentration jump experiments was employed to define the mode of interaction of A9C with TMEM16A channels. Key Results In the presence of high intracellular Ca2+, A9C inhibited TMEM16A currents in a voltage‐dependent manner by entering the channel from the outside. A9C activation, revealed in the presence of submaximal intracellular Ca2+ concentrations, was also voltage‐dependent. The electric distance of A9C inhibiting and activating binding site was ~0.6 in each case. Inhibition occurred according to an open‐channel block mechanism. Activation was due to a dramatic leftward shift in the steady‐state activation curve and slowed deactivation kinetics. Extracellular A9C competed with extracellular Cl−, suggesting that A9C binds deep in the channel's pore to exert both inhibiting and activating effects. Conclusions and Implications A9C is an open TMEM16A channel blocker and gating modifier. These effects require A9C to bind to a region within the pore that is accessible from the extracellular side of the membrane. These data will aid the future drug design of compounds that selectively activate or inhibit TMEM16A channels. PMID:26562072

  15. The ABC protein turned chloride channel whose failure causes cystic fibrosis

    NASA Astrophysics Data System (ADS)

    Gadsby, David C.; Vergani, Paola; Csanády, László

    2006-03-01

    CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.

  16. The ABC protein turned chloride channel whose failure causes cystic fibrosis.

    PubMed

    Gadsby, David C; Vergani, Paola; Csanády, László

    2006-03-23

    CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.

  17. Shikonin Inhibits Intestinal Calcium-Activated Chloride Channels and Prevents Rotaviral Diarrhea

    PubMed Central

    Jiang, Yu; Yu, Bo; Yang, Hong; Ma, Tonghui

    2016-01-01

    Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl- current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl- currents in mouse colonic epithelia but did not affect cytoplasmic Ca2+ concentration as well as the other major enterocyte chloride channel conductance regulator. Characterization study found that shikonin inhibited basolateral K+ channel activity without affecting Na+/K+-ATPase activities. In vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in vivo. Taken together, the results suggested that shikonin inhibited enterocyte calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K+ channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea. PMID:27601995

  18. Shikonin Inhibits Intestinal Calcium-Activated Chloride Channels and Prevents Rotaviral Diarrhea.

    PubMed

    Jiang, Yu; Yu, Bo; Yang, Hong; Ma, Tonghui

    2016-01-01

    Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl(-) current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl(-) currents in mouse colonic epithelia but did not affect cytoplasmic Ca(2+) concentration as well as the other major enterocyte chloride channel conductance regulator. Characterization study found that shikonin inhibited basolateral K(+) channel activity without affecting Na(+)/K(+)-ATPase activities. In vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in vivo. Taken together, the results suggested that shikonin inhibited enterocyte calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K(+) channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea.

  19. Control of volume-sensitive chloride channel inactivation by the coupled action of intracellular chloride and extracellular protons.

    PubMed

    Hernández-Carballo, Carmen Y; De Santiago-Castillo, José A; Rosales-Saavedra, Teresa; Pérez-Cornejo, Patricia; Arreola, Jorge

    2010-08-01

    The volume-sensitive chloride current (I(ClVol)) exhibit a time-dependent decay presumably due to channel inactivation. In this work, we studied the effects of chloride ions (Cl(-)) and H(+) ions on I(ClVol) decay recorded in HEK-293 and HL-60 cells using the whole-cell patch clamp technique. Under control conditions ([Cl(-)](e) = [Cl(-)](i) = 140 mM and pH(i) = pH(e) = 7.3), I(ClVol) in HEK cells shows a large decay at positive voltages but in HL-60 cells I(ClVol) remained constant independently of time. In HEK-293 cells, simultaneously raising the [Cl(-)](e) and [Cl(-)](i) from 25 to 140 mM (with pH(e) = pH(i) = 7.3) increased the fraction of inactivated channels (FIC). This effect was reproduced by elevating [Cl(-)](i) while keeping the [Cl(-)](e) constant. Furthermore, a decrease in pH(e) from 7.3 to 5.5 accelerated current decay and increased FIC when [Cl(-)] was 140 mM but not 25 mM. In HL-60 cells, a slight I(ClVol) decay was seen when the pH(e) was reduced from 7.3 to 5.5. Our data show that inactivation of I(ClVol) can be controlled by changing either the Cl(-) or H(+) concentration or both. Based on our results and previously published data, we have built a model that explains VRAC inactivation. In the model the H(+) binding site is located outside the electrical field near the extracellular entry whilst the Cl(-) binding site is intracellular. The model depicts inactivation as a pore constriction that happens by simultaneous binding of H(+) and Cl(-) ions to the channel followed by a voltage-dependent conformational change that ultimately causes inactivation.

  20. Two tonoplast MATE proteins function as turgor-regulating chloride channels in Arabidopsis.

    PubMed

    Zhang, Haiwen; Zhao, Fu-Geng; Tang, Ren-Jie; Yu, Yuexuan; Song, Jiali; Wang, Yuan; Li, Legong; Luan, Sheng

    2017-02-15

    The central vacuole in a plant cell occupies the majority of the cellular volume and plays a key role in turgor regulation. The vacuolar membrane (tonoplast) contains a large number of transporters that mediate fluxes of solutes and water, thereby adjusting cell turgor in response to developmental and environmental signals. We report that two tonoplast Detoxification efflux carrier (DTX)/Multidrug and Toxic Compound Extrusion (MATE) transporters, DTX33 and DTX35, function as chloride channels essential for turgor regulation in Arabidopsis Ectopic expression of each transporter in Nicotiana benthamiana mesophyll cells elicited a large voltage-dependent inward chloride current across the tonoplast, showing that DTX33 and DTX35 each constitute a functional channel. Both channels are highly expressed in Arabidopsis tissues, including root hairs and guard cells that experience rapid turgor changes during root-hair elongation and stomatal movements. Disruption of these two genes, either in single or double mutants, resulted in shorter root hairs and smaller stomatal aperture, with double mutants showing more severe defects, suggesting that these two channels function additively to facilitate anion influx into the vacuole during cell expansion. In addition, dtx35 single mutant showed lower fertility as a result of a defect in pollen-tube growth. Indeed, patch-clamp recording of isolated vacuoles indicated that the inward chloride channel activity across the tonoplast was impaired in the double mutant. Because MATE proteins are widely known transporters of organic compounds, finding MATE members as chloride channels expands the functional definition of this large family of transporters.

  1. Two tonoplast MATE proteins function as turgor-regulating chloride channels in Arabidopsis

    PubMed Central

    Zhang, Haiwen; Zhao, Fu-Geng; Tang, Ren-Jie; Yu, Yuexuan; Song, Jiali; Wang, Yuan; Li, Legong; Luan, Sheng

    2017-01-01

    The central vacuole in a plant cell occupies the majority of the cellular volume and plays a key role in turgor regulation. The vacuolar membrane (tonoplast) contains a large number of transporters that mediate fluxes of solutes and water, thereby adjusting cell turgor in response to developmental and environmental signals. We report that two tonoplast Detoxification efflux carrier (DTX)/Multidrug and Toxic Compound Extrusion (MATE) transporters, DTX33 and DTX35, function as chloride channels essential for turgor regulation in Arabidopsis. Ectopic expression of each transporter in Nicotiana benthamiana mesophyll cells elicited a large voltage-dependent inward chloride current across the tonoplast, showing that DTX33 and DTX35 each constitute a functional channel. Both channels are highly expressed in Arabidopsis tissues, including root hairs and guard cells that experience rapid turgor changes during root-hair elongation and stomatal movements. Disruption of these two genes, either in single or double mutants, resulted in shorter root hairs and smaller stomatal aperture, with double mutants showing more severe defects, suggesting that these two channels function additively to facilitate anion influx into the vacuole during cell expansion. In addition, dtx35 single mutant showed lower fertility as a result of a defect in pollen-tube growth. Indeed, patch-clamp recording of isolated vacuoles indicated that the inward chloride channel activity across the tonoplast was impaired in the double mutant. Because MATE proteins are widely known transporters of organic compounds, finding MATE members as chloride channels expands the functional definition of this large family of transporters. PMID:28202726

  2. Activation and inhibition of TMEM16A calcium-activated chloride channels.

    PubMed

    Ni, Yu-Li; Kuan, Ai-Seon; Chen, Tsung-Yu

    2014-01-01

    Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca(2+)-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca(2+), Sr(2+), and Ba(2+), and discovered that Mg(2+) competes with Ca(2+) in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore-as revealed by the permeability ratios of these anions-appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1.

  3. Activation and Inhibition of TMEM16A Calcium-Activated Chloride Channels

    PubMed Central

    Ni, Yu-Li; Kuan, Ai-Seon; Chen, Tsung-Yu

    2014-01-01

    Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca2+-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca2+, Sr2+, and Ba2+, and discovered that Mg2+ competes with Ca2+ in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore–as revealed by the permeability ratios of these anions–appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1. PMID:24489780

  4. Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels.

    PubMed

    Vocke, Kerstin; Dauner, Kristin; Hahn, Anne; Ulbrich, Anne; Broecker, Jana; Keller, Sandro; Frings, Stephan; Möhrlen, Frank

    2013-10-01

    Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca(2+)/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca(2+)/calmodulin, one at submicromolar Ca(2+) concentrations and one in the micromolar Ca(2+) range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca(2+)/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca(2+) signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca(2+) regulation in anoctamin Cl(-) channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.

  5. Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

    PubMed Central

    Vocke, Kerstin; Dauner, Kristin; Hahn, Anne; Ulbrich, Anne; Broecker, Jana; Keller, Sandro; Frings, Stephan

    2013-01-01

    Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca2+/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types. PMID:24081981

  6. Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons

    PubMed Central

    Dibattista, Michele; Amjad, Asma; Maurya, Devendra Kumar; Sagheddu, Claudia; Montani, Giorgia; Tirindelli, Roberto

    2012-01-01

    The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of −261 pA was measured at −50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction. PMID:22732308

  7. Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons.

    PubMed

    Dibattista, Michele; Amjad, Asma; Maurya, Devendra Kumar; Sagheddu, Claudia; Montani, Giorgia; Tirindelli, Roberto; Menini, Anna

    2012-07-01

    The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of -261 pA was measured at -50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction.

  8. Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB

    SciTech Connect

    Zheng, Kai; Chen, Maoyun; Xiang, Yangfei; Ma, Kaiqi; Jin, Fujun; Wang, Xiao; Wang, Xiaoyan; Wang, Shaoxiang; Wang, Yifei

    2014-04-18

    Highlights: • We analyze the anti-HSV potential of chloride channel inhibitors. • Tamoxifen and NPPB show anti-HSV-1 and anti-ACV-resistant HSV-1 activities. • HSV-1 infection induces intracellular chloride concentration increasing. • Tamoxifen and NPPB inhibit HSV-1 early infection. • Tamoxifen and NPPB prevent the fusion process of HSV-1. - Abstract: Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections.

  9. CFTR chloride channel is a molecular target of the natural cancer preventive agent resveratrol.

    PubMed

    Yang, Shuang; Yu, B O; Sui, Yujie; Zhang, Yaofang; Wang, Xue; Hou, Shuguang; Ma, Tonghui; Yang, Hong

    2013-09-01

    The naturally occurring polyphenol compound resveratrol (RES) has been receiving wide attention because of its variety of health benefits and favourable biological activities. Previous studies have shown that RES could induce intestinal chloride secretion in mouse jejunum and stimulate cAMP-dependent Cl- secretion in T84, primary cultured murine nasal septal and human sinonasal epithelial cells, but the precise molecular target is not clear. We therefore tested the hypothesis that RES may stimulate the activity of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Using cell-based fluorescent assays, transepithelial short-circuit current measurements and excised inside-out patch-clamp analysis; we found that RES dose-dependently potentiate CFTR Cl- channel activities, which was reversed by CFTR inhibitors CFTR(inh)-172 and GlyH101. Transepithelial Cl- secretion by CFTR-expressing FRT cells was stimulated by RES with half maximal concentration -80 microM. Intracellular cAMP content was not elevated by RES in FRT cells. Excised inside-out patch-clamp analysis indicated that RES significantly increased the chloride currents of CFTR. In ex vivo studies, RES stimulated the transmucosal chloride current of rat colon by short-circuit current assay. These data suggested that CFTR is a molecular target of RES. Our findings add a new molecular target to RES, and RES may represent a novel class of therapeutic lead compounds in treating CFTR-related diseases including CF and habitual constipation.

  10. Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone.

    PubMed

    Buckingham, Steven D; Higashino, Yoshiaki; Sattelle, David B

    2009-11-01

    The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D(f)) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D(f) motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.

  11. Chloride channels are involved in sperm motility and are downregulated in spermatozoa from patients with asthenozoospermia.

    PubMed

    Liu, Shan-Wen; Li, Yuan; Zou, Li-Li; Guan, Yu-Tao; Peng, Shuang; Zheng, Li-Xin; Deng, Shun-Mei; Zhu, Lin-Yan; Wang, Li-Wei; Chen, Li-Xin

    2016-06-03

    Human spermatozoa encounter an osmotic decrease from 330 to 290 mOsm l-1 when passing through the female reproductive tract. We aimed to evaluate the role of chloride channels in volume regulation and sperm motility from patients with asthenozoospermia. Spermatozoa were purified using Percoll density gradients. Sperm volume was measured as the forward scatter signal using flow cytometry. Sperm motility was analyzed using computer-aided sperm analysis (CASA). When transferred from an isotonic solution (330 mOsm l-1 ) to a hypotonic solution (290 mOsm l-1 ), cell volume was not changed in spermatozoa from normozoospermic men; but increased in those from asthenozoospermic samples. The addition of the chloride channel blockers, 4,4'-diisothiocyanatostilbene-2,2'- isulfonic acid (DIDS) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) to the hypotonic solution caused the normal spermatozoa to swell but did not increase the volume of those from the asthenozoospermic semen. DIDS and NPPB decreased sperm motility in both sets of semen samples. The inhibitory effect of NPPB on normal sperm motility was much stronger than on spermatozoa from the asthenozoospermic samples. Both sperm types expressed ClC-3 chloride channels, but the expression levels in the asthenozoospermic samples were much lower, especially in the neck and mid-piece areas. Spermatozoa from men with asthenozoospermia demonstrated lower volume regulating capacity, mobility, and ClC-3 expression levels (especially in the neck) than did normal spermatozoa. Thus, chloride channels play important roles in the regulation of sperm volume and motility and are downregulated in cases of asthenozoospermia.

  12. International Union of Basic and Clinical Pharmacology. LXXXV: Calcium-Activated Chloride Channels

    PubMed Central

    Huang, Fen; Wong, Xiuming

    2012-01-01

    Calcium-activated chloride channels (CaCCs) are widely expressed in various tissues and implicated in physiological processes such as sensory transduction, epithelial secretion, and smooth muscle contraction. Transmembrane proteins with unknown function 16 (TMEM16A) has recently been identified as a major component of CaCCs. Detailed molecular analysis of TMEM16A will be needed to understand its structure-function relationships. The role this channel plays in physiological systems remains to be established and is currently a subject of intense investigation. PMID:22090471

  13. Structure of a CLC chloride ion channel by cryo-electron microscopy.

    PubMed

    Park, Eunyong; Campbell, Ernest B; MacKinnon, Roderick

    2017-01-26

    CLC proteins transport chloride (Cl(-)) ions across cellular membranes to regulate muscle excitability, electrolyte movement across epithelia, and acidification of intracellular organelles. Some CLC proteins are channels that conduct Cl(-) ions passively, whereas others are secondary active transporters that exchange two Cl(-) ions for one H(+). The structural basis underlying these distinctive transport mechanisms is puzzling because CLC channels and transporters are expected to share the same architecture on the basis of sequence homology. Here we determined the structure of a bovine CLC channel (CLC-K) using cryo-electron microscopy. A conserved loop in the Cl(-) transport pathway shows a structure markedly different from that of CLC transporters. Consequently, the cytosolic constriction for Cl(-) passage is widened in CLC-K such that the kinetic barrier previously postulated for Cl(-)/H(+) transporter function would be reduced. Thus, reduction of a kinetic barrier in CLC channels enables fast flow of Cl(-) down its electrochemical gradient.

  14. Effects of chloride channel blockers on hypoxic injury in rat proximal tubules.

    PubMed

    Reeves, W B

    1997-05-01

    These studies examined the pathways and consequences of chloride uptake into proximal tubule cells during in vitro hypoxia. The chloride channel blocker diphenylamine-2-carboxylate (DPC) markedly reduced the degree of hypoxia-induced membrane damage as measured by the release of lactate dehydrogenase (LDH). DPC reduced the release of LDH from hypoxic tubules from 38 +/- 2.7% to 16 +/- 1.7% after 30 minutes of hypoxia (P < 0.001, N = 16) and also reduced 36Cl- uptake by hypoxic tubules. The reduction in LDH release was not associated with better preservation of cell ATP content or with protection against hypoxia-induced DNA damage. Other Cl- channel blockers, such as niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihyrdo-2-methyl-1-oxo-1H-in den-5-yl)oxy] acetic acid (IAA-94) provided even greater protection than DPC and were as effective as 2 mM glycine. The Cl- channel blockers appear to act late in the course of hypoxic injury since DNA damage, an early manifestation of injury, is not prevented by the blockers and since addition of the Cl- channel blocker after the hypoxic injury has begun reduces further membrane damage. These results support the conclusion that transport through Cl- channels contributes to hypoxic cell injury in proximal tubular cells.

  15. Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

    PubMed

    Al Khamici, Heba; Brown, Louise J; Hossain, Khondker R; Hudson, Amanda L; Sinclair-Burton, Alxcia A; Ng, Jane Phui Mun; Daniel, Elizabeth L; Hare, Joanna E; Cornell, Bruce A; Curmi, Paul M G; Davey, Mary W; Valenzuela, Stella M

    2015-01-01

    The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

  16. ClC-K chloride channels: emerging pathophysiology of Bartter syndrome type 3.

    PubMed

    Andrini, Olga; Keck, Mathilde; Briones, Rodolfo; Lourdel, Stéphane; Vargas-Poussou, Rosa; Teulon, Jacques

    2015-06-15

    The mutations in the CLCNKB gene encoding the ClC-Kb chloride channel are responsible for Bartter syndrome type 3, one of the four variants of Bartter syndrome in the genetically based nomenclature. All forms of Bartter syndrome are characterized by hypokalemia, metabolic alkalosis, and secondary hyperaldosteronism, but Bartter syndrome type 3 has the most heterogeneous presentation, extending from severe to very mild. A relatively large number of CLCNKB mutations have been reported, including gene deletions and nonsense or missense mutations. However, only 20 CLCNKB mutations have been functionally analyzed, due to technical difficulties regarding ClC-Kb functional expression in heterologous systems. This review provides an overview of recent progress in the functional consequences of CLCNKB mutations on ClC-Kb chloride channel activity. It has been observed that 1) all ClC-Kb mutants have an impaired expression at the membrane; and 2) a minority of the mutants combines reduced membrane expression with altered pH-dependent channel gating. Although further investigation is needed to fully characterize disease pathogenesis, Bartter syndrome type 3 probably belongs to the large family of conformational diseases, in which the mutations destabilize channel structure, inducing ClC-Kb retention in the endoplasmic reticulum and accelerated channel degradation.

  17. Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels in Drosophila melanogaster

    PubMed Central

    Remnant, Emily J.; Williams, Adam; Lumb, Chris; Yang, Ying Ting; Chan, Janice; Duchêne, Sebastian; Daborn, Phillip J.; Batterham, Philip; Perry, Trent

    2016-01-01

    Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927, and CG11340, are highly expressed in nonneuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here, we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization to examine cell type and tissue-specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including the salivary gland, ejaculatory duct, malpighian tubules, and early midgut. CG11340 is found in malpighian tubules and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific nonneuronal role for this subunit in D. melanogaster. Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets. PMID:27172217

  18. State-dependent blocker interactions with the CFTR chloride channel: implications for gating the pore.

    PubMed

    Linsdell, Paul

    2014-12-01

    Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is subject to voltage-dependent open-channel block by a diverse range of cytoplasmic anions. However, in most cases the ability of these blocking substances to influence the pore opening and closing process has not been reported. In the present work, patch clamp recording was used to investigate the state-dependent block of CFTR by cytoplasmic Pt(NO2)4(2-) ions. Two major effects of Pt(NO2)4(2-) were identified. First, this anion caused fast, voltage-dependent block of open channels, leading to an apparent decrease in single-channel current amplitude. Secondly, Pt(NO2)4(2-) also decreased channel open probability due to an increase in interburst closed times. Interestingly, mutations in the pore that weakened (K95Q) or strengthened (I344K, V345K) interactions with Pt(NO2)4(2-) altered blocker effects both on Cl(-) permeation and on channel gating, suggesting that both these effects are a consequence of Pt(NO2)4(2-) interaction with a single site within the pore. Experiments at reduced extracellular Cl(-) concentration hinted that Pt(NO2)4(2-) may have a third effect, possibly increasing channel activity by interfering with channel closure. These results suggest that Pt(NO2)4(2-) can enter from the cytoplasm into the pore inner vestibule of both open and closed CFTR channels, and that Pt(NO2)4(2-) bound in the inner vestibule blocks Cl(-) permeation as well as interfering with channel opening and, perhaps, channel closure. Implications for the location of the channel gate in the pore, and the operation of this gate, are discussed.

  19. Calmodulin regulation of TMEM16A and 16B Ca2+-activated chloride channels

    PubMed Central

    Yang, Tingting; Colecraft, Henry M

    2016-01-01

    Ca2+-activated chloride channels encoded by TMEM16A and 16B are important for regulating epithelial mucus secretion, cardiac and neuronal excitability, smooth muscle contraction, olfactory transduction, and cell proliferation. Whether and how the ubiquitous Ca2+ sensor calmodulin (CaM) regulates the activity of TMEM16A and 16B channels has been controversial and the subject of an ongoing debate. Recently, using a bioengineering approach termed ChIMP (Channel Inactivation induced by Membrane-tethering of an associated Protein) we argued that Ca2+-free CaM (apoCaM) is pre-associated with functioning TMEM16A and 16B channel complexes in live cells. Further, the pre-associated apoCaM mediates Ca2+-dependent sensitization of activation (CDSA) and Ca2+-dependent inactivation (CDI) of some TMEM16A splice variants. In this review, we discuss these findings in the context of previous and recent results relating to Ca2+-dependent regulation of TMEM16A/16B channels and the putative role of CaM. We further discuss potential future directions for these nascent ideas on apoCaM regulation of TMEM16A/16B channels, noting that such future efforts will benefit greatly from the pioneering work of Dr. David T. Yue and colleagues on CaM regulation of voltage-dependent calcium channels. PMID:26083059

  20. Glutamate-gated chloride channels inhibit juvenile hormone biosynthesis in the cockroach, Diploptera punctata.

    PubMed

    Liu, Hsin-Ping; Lin, Shu-Chen; Lin, Chi-Yen; Yeh, Shih-Rung; Chiang, Ann-Shyn

    2005-11-01

    Juvenile hormone (JH) synthesized and released from endocrine gland corpus allatum (CA) plays an important role in insect metamorphosis, vitellogenesis and reproduction. Glutamate is a major neurotransmitter in the nervous system and its activated receptors possess excitatory and inhibitory forms in muscle fibers of invertebrates. Previously, we have shown that the rise of intracellular calcium through excitatory glutamate receptors, N-methyl-d-aspartate (NMDA) and non-NMDA-type channels stimulates JH synthesis in the cockroach, Diploptera punctata. Here, we demonstrate the occurrence of inhibitory chloride permeable glutamate (GluCl) receptors on CA cell membranes. Application of the GluCl channel activators, ibotenic acid (Ibo) and ivermectin, but not gamma-aminobutyric acid caused a decline in JH synthesis in glands of either high or low activity during the gonadotrophic cycle. Also, while recording the membrane potential of the isolated whole CA glands intracellularly, Ibo induced a hyperpolarizated response. Both changes in the membrane potential and inhibition of JH synthesis could be abolished by the application of the chloride channel blocker picrotoxin. Finally, we found both excitatory and inhibitory glutamate receptors cause antagonistic effects on rates of JH synthesis. These results indicate a novel function of GluCl channels in the inhibition of JH synthesis that could be a potential pathway for developing a new generation of insecticides.

  1. Calcium-activated chloride channels anoctamin 1 and 2 promote murine uterine smooth muscle contractility

    PubMed Central

    Bernstein, Kyra; Vink, Joy Y; Fu, Xiao Wen; Wakita, Hiromi; Danielsson, Jennifer; Wapner, Ronald; Gallos, George

    2014-01-01

    Objective To determine the presence of calcium activated chloride channels anoctamin 1 and 2 in human and murine uterine smooth muscle and evaluate the physiologic role for these ion channels in murine myometrial contractility. Study Design We performed reverse transcription polymerase chain reaction to determine if anoctamin 1 and 2 are expressed in human and murine uterine tissue to validate the study of this protein in mouse models. Immunohistochemical staining of anoctamin 1 and 2 was then performed to determine protein expression in murine myometrial tissue. The function of anoctamin 1 and 2 in murine uterine tissue was evaluated using electrophysiological studies, organ bath, and calcium flux experiments. Results Anoctamin 1 and 2 are expressed in human and murine USM cells. Functional studies show that selective antagonism of these channels promotes relaxation of spontaneous murine uterine smooth muscle contractions. Blockade of anoctamin 1 and 2 inhibits both agonist-induced and spontaneous transient inward currents and abolishes G-protein coupled receptor (oxytocin) mediated elevations in intracellular calcium. Conclusion The calcium activated chloride channels ANO 1 and 2 are present in human and murine myometrial tissue and may provide novel potential therapeutic targets to achieve effective tocolysis. PMID:24928056

  2. Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2.

    PubMed

    Ponissery Saidu, Samsudeen; Stephan, Aaron B; Talaga, Anna K; Zhao, Haiqing; Reisert, Johannes

    2013-06-01

    Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca(2+)-activated Cl(-) channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5' rapid amplification of cDNA ends analysis was conducted to characterize the 5' end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5' end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca(2+) sensitivity and that the exon 4-encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.

  3. Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB.

    PubMed

    Zheng, Kai; Chen, Maoyun; Xiang, Yangfei; Ma, Kaiqi; Jin, Fujun; Wang, Xiao; Wang, Xiaoyan; Wang, Shaoxiang; Wang, Yifei

    2014-04-18

    Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections.

  4. Identification of natural coumarin compounds that rescue defective DeltaF508-CFTR chloride channel gating.

    PubMed

    Xu, Li-Na; Na, Wan-Li; Liu, Xin; Hou, Shu-Guang; Lin, Sen; Yang, Hong; Ma, Tong-Hui

    2008-08-01

    1. Deletion of phenylalanine at position 508 (DeltaF508) of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the most common mutation causing cystic fibrosis (CF). Effective pharmacological therapy of CF caused by the DeltaF508-CFTR mutation requires the rescue of both intracellular processing and channel gating defects. 2. We identified a class of natural coumarin compounds that can correct the defective DeltaF508-CFTR chloride channel gating by screening a collection of 386 single natural compounds from Chinese medicinal herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells coexpressing DeltaF508-CFTR and an iodide-sensitive fluorescent indicator (YFP-H148Q/I152L). 3. Dose-dependent potentiation of defective DeltaF508-CFTR chloride channel gating by five coumarin compounds was demonstrated by the fluorescent iodide influx assay and confirmed by an Ussing chamber short-circuit current assay. Activation was fully abolished by the specific CFTR inhibitor CFTR(inh)-172. Two potent compounds, namely imperatorin and osthole, have activation K(d) values of approximately 10 micromol/L, as determined by the short-circuit current assay. The active coumarin compounds do not elevate intracellular cAMP levels. Activation of DeltaF508-CFTR by the coumarin compounds requires cAMP agonist, suggesting direct interaction with the mutant CFTR molecule. Kinetics analysis indicated rapid activation of DeltaF508-CFTR by the coumarin compounds, with half-maximal activation of < 5 min. The activating effect was fully reversed for all five active compounds 45 min after washout. 4. In conclusion, the natural coumarin DeltaF508-CFTR activators may represent a new class of natural lead compounds for the development of pharmacological therapies for CF caused by the DeltaF508 mutation.

  5. A functional tandem between transient receptor potential canonical channels 6 and calcium-dependent chloride channels in human epithelial cells.

    PubMed

    Bertrand, Johanna; Dannhoffer, Luc; Antigny, Fabrice; Vachel, Laura; Jayle, Christophe; Vandebrouck, Clarisse; Becq, Frédéric; Norez, Caroline

    2015-10-15

    TRPC6 plays important human physiological functions, notably in artery and arterioles constriction, in regulation of vascular volume and in bronchial muscle constriction. It is implicated in pulmonary hypertension, cardiovascular disease, and focal segmental glomerulosclerosis and seems to play a role in cancer development. Previously, we identified Guanabenz, an α2-adrenergic agonist used for hypertension treatment (Wytensin®), as an activator of calcium-dependent chloride channels (CaCC) in human Cystic Fibrosis (CF) nasal epithelial cells by transiently increasing [Ca2+]i via an influx of extracellular Ca2+. In this study, using assays to measure chloride channel activity, we show that guanabenz is an activator of CaCC in freshly dissociated human bronchial epithelial cells from three CF patients with various genotypes (F508del/F508del, F508del/R1066C, F508del/H1085R). We further characterised the effect of guanabenz and show that it is independent of α-adrenergic receptors, is inhibited by the TRPC family inhibitor SKF-96365 but not by the TRPV family inhibitor ruthenium red. Using western-blotting, Ca2+ measurements and iodide efflux assay, we found that TRPC1 siRNA has no effect on guanabenz induced responses whereas TRPC6 siRNA prevented the guanabenz-dependent Ca2+ influx and the CaCC-dependent activity stimulated by guanabenz. In conclusion, we show that TRPC6 channel is pivotal for the activation of CaCC by guanabenz through a α2-adrenergic-independent pathway in human airway epithelial cells. We suggest propose a functional coupling between TRPC6 and CaCC and guanabenz as a potential TRPC6 activator for exploring TRPC6 and CaCC channel functions and corresponding channelopathies.

  6. Novel expression of a functional glycine receptor chloride channel that attenuates contraction in airway smooth muscle

    PubMed Central

    Yim, Peter D.; Gallos, George; Xu, Dingbang; Zhang, Yi; Emala, Charles W.

    2011-01-01

    Airway smooth muscle (ASM) contraction is an important component of the pathophysiology of asthma. Taurine, an agonist of glycine receptor chloride (GlyR Cl−) channels, was found to relax contracted ASM, which led us to question whether functional GlyR Cl− channels are expressed in ASM. Messenger RNA for β (GLRB), α1 (GLRA1), α2 (GLRA2), and α4 (GLRA4) subunits were found in human (Homo sapiens) and guinea pig (Cavia porcellus) tracheal smooth muscle. Immunoblotting confirmed the protein expression of GLRA1 and GLRB subunits in ASM. Electrical activity of cultured human ASM cells was assessed using a fluorescent potentiometric dye and electrophysiological recordings. Glycine increased current and significantly increased fluorescence in a dose-dependent manner. The GlyR Cl− channel antagonist strychnine significantly blocked the effects of glycine on potentiometric fluorescence in ASM cells. Guinea pig airway ring relaxation of ACh-induced contractions by isoproterenol was significantly left-shifted in the presence of glycine. This effect of glycine was blocked by pretreatment with the GlyR Cl− channel antagonist strychnine. Glycine treatment during tachykinin- and acetylcholine-induced contractions significantly decreased the maintenance of muscle force compared to control. GlyR Cl− channels are expressed on ASM and regulate smooth muscle force and offer a novel target for therapeutic relaxation of ASM.—Yim, P. D., Gallos, G., Xu, D., Zhang, Y., Emala, C. W. Novel expression of a functional glycine receptor chloride channel that attenuates contraction in airway smooth muscle. PMID:21282206

  7. ClC-1 chloride channels: state-of-the-art research and future challenges

    PubMed Central

    Imbrici, Paola; Altamura, Concetta; Pessia, Mauro; Mantegazza, Renato; Desaphy, Jean-François; Camerino, Diana Conte

    2015-01-01

    The voltage-dependent ClC-1 chloride channel belongs to the CLC channel/transporter family. It is a homodimer comprising two individual pores which can operate independently or simultaneously according to two gating modes, the fast and the slow gate of the channel. ClC-1 is preferentially expressed in the skeletal muscle fibers where the presence of an efficient Cl- homeostasis is crucial for the correct membrane repolarization and propagation of action potential. As a consequence, mutations in the CLCN1 gene cause dominant and recessive forms of myotonia congenita (MC), a rare skeletal muscle channelopathy caused by abnormal membrane excitation, and clinically characterized by muscle stiffness and various degrees of transitory weakness. Elucidation of the mechanistic link between the genetic defects and the disease pathogenesis is still incomplete and, at this time, there is no specific treatment for MC. Still controversial is the subcellular localization pattern of ClC-1 channels in skeletal muscle as well as its modulation by some intracellular factors. The expression of ClC-1 in other tissues such as in brain and heart and the possible assembly of ClC-1/ClC-2 heterodimers further expand the physiological properties of ClC-1 and its involvement in diseases. A recent de novo CLCN1 truncation mutation in a patient with generalized epilepsy indeed postulates an unexpected role of this channel in the control of neuronal network excitability. This review summarizes the most relevant and state-of-the-art research on ClC-1 chloride channels physiology and associated diseases. PMID:25964741

  8. TMEM16A(a)/anoctamin-1 Shares a Homodimeric Architecture with CLC Chloride Channels*

    PubMed Central

    Fallah, Ghada; Römer, Thomas; Detro-Dassen, Silvia; Braam, Ursula; Markwardt, Fritz; Schmalzing, Günther

    2011-01-01

    TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca2+-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca2+-induced measured macroscopic Cl− currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl− channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca2+-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel. PMID:20974900

  9. ClC-1 chloride channels: state-of-the-art research and future challenges.

    PubMed

    Imbrici, Paola; Altamura, Concetta; Pessia, Mauro; Mantegazza, Renato; Desaphy, Jean-François; Camerino, Diana Conte

    2015-01-01

    The voltage-dependent ClC-1 chloride channel belongs to the CLC channel/transporter family. It is a homodimer comprising two individual pores which can operate independently or simultaneously according to two gating modes, the fast and the slow gate of the channel. ClC-1 is preferentially expressed in the skeletal muscle fibers where the presence of an efficient Cl(-) homeostasis is crucial for the correct membrane repolarization and propagation of action potential. As a consequence, mutations in the CLCN1 gene cause dominant and recessive forms of myotonia congenita (MC), a rare skeletal muscle channelopathy caused by abnormal membrane excitation, and clinically characterized by muscle stiffness and various degrees of transitory weakness. Elucidation of the mechanistic link between the genetic defects and the disease pathogenesis is still incomplete and, at this time, there is no specific treatment for MC. Still controversial is the subcellular localization pattern of ClC-1 channels in skeletal muscle as well as its modulation by some intracellular factors. The expression of ClC-1 in other tissues such as in brain and heart and the possible assembly of ClC-1/ClC-2 heterodimers further expand the physiological properties of ClC-1 and its involvement in diseases. A recent de novo CLCN1 truncation mutation in a patient with generalized epilepsy indeed postulates an unexpected role of this channel in the control of neuronal network excitability. This review summarizes the most relevant and state-of-the-art research on ClC-1 chloride channels physiology and associated diseases.

  10. The Arabidopsis Thylakoid Chloride Channel AtCLCe Functions in Chloride Homeostasis and Regulation of Photosynthetic Electron Transport

    PubMed Central

    Herdean, Andrei; Nziengui, Hugues; Zsiros, Ottó; Solymosi, Katalin; Garab, Győző; Lundin, Björn; Spetea, Cornelia

    2016-01-01

    Chloride ions can be translocated across cell membranes through Cl− channels or Cl−/H+ exchangers. The thylakoid-located member of the Cl− channel CLC family in Arabidopsis thaliana (AtCLCe) was hypothesized to play a role in photosynthetic regulation based on the initial photosynthetic characterization of clce mutant lines. The reduced nitrate content of Arabidopsis clce mutants suggested a role in regulation of plant nitrate homeostasis. In this study, we aimed to further investigate the role of AtCLCe in the regulation of ion homeostasis and photosynthetic processes in the thylakoid membrane. We report that the size and composition of proton motive force were mildly altered in two independent Arabidopsis clce mutant lines. Most pronounced effects in the clce mutants were observed on the photosynthetic electron transport of dark-adapted plants, based on the altered shape and associated parameters of the polyphasic OJIP kinetics of chlorophyll a fluorescence induction. Other alterations were found in the kinetics of state transition and in the macro-organization of photosystem II supercomplexes, as indicated by circular dichroism measurements. Pre-treatment with KCl but not with KNO3 restored the wild-type photosynthetic phenotype. Analyses by transmission electron microscopy revealed a bow-like arrangement of the thylakoid network and a large thylakoid-free stromal region in chloroplast sections from the dark-adapted clce plants. Based on these data, we propose that AtCLCe functions in Cl− homeostasis after transition from light to dark, which affects chloroplast ultrastructure and regulation of photosynthetic electron transport. PMID:26904077

  11. Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2

    PubMed Central

    Ponissery Saidu, Samsudeen; Stephan, Aaron B.; Talaga, Anna K.

    2013-01-01

    Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca2+-activated Cl− channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5′ rapid amplification of cDNA ends analysis was conducted to characterize the 5′ end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5′ end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca2+ sensitivity and that the exon 4–encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties. PMID:23669718

  12. [Post-translational ligation of split CFTR severed before TMD2 and its chloride channel function].

    PubMed

    Zhu, Fuxiang; Gong, Xiandi; Liu, Zelong; Yang, Shude; Qu, Huige; Chi, Xiaoyan

    2010-12-01

    Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein. Pair of eukaryotic expression vectors pEGFP-NInt and pEYFP-IntC were constructed by inserting them into the vectors pEGFP-N1 and pEYFP-N1 respectively. The transient expression was carried out for observing the ligation of CFTR by Western blotting and recording the chloride current by patch clamps when cotransfection of the pair of vectors into baby hamster kidney (BHK) cells. The results showed that an obvious protein band proven to be ligated intact CFTR can be seen and a higher chloride current and activity of chloride channel were recorded after cotransfection. These data demonstrated that split Ssp DnaB intein could be used as a strategy in delivering CFTR gene by two vectors providing evidence for application of dual adeno-associated virus (AAV) vectors to overcome the limitation of packaging size in cystic fibrosis gene therapy.

  13. The nuclear chloride ion channel NCC27 is involved in regulation of the cell cycle

    PubMed Central

    Valenzuela, Stella M; Mazzanti, Michele; Tonini, Raffaella; Qiu, Min Ru; Warton, Kristina; Musgrove, Elizabeth A; Campbell, Terence J; Breit, Samuel N

    2000-01-01

    NCC27 is a nuclear chloride ion channel, identified in the PMA-activated U937 human monocyte cell line. NCC27 mRNA is expressed in virtually all cells and tissues and the gene encoding NCC27 is also highly conserved. Because of these factors, we have examined the hypothesis that NCC27 is involved in cell cycle regulation. Electrophysiological studies in Chinese hamster ovary (CHO-K1) cells indicated that NCC27 chloride conductance varied according to the stage of the cell cycle, being expressed only on the plasma membrane of cells in G2/M phase. We also demonstrate that Cl− ion channel blockers known to block NCC27 led to arrest of CHO-K1 cells in the G2/M stage of the cell cycle, the same stage at which this ion channel is selectively expressed on the plasma membrane. These data strongly support the hypothesis that NCC27 is involved, in some as yet undetermined manner, in regulation of the cell cycle. PMID:11195932

  14. A proton-activated, outwardly rectifying chloride channel in human umbilical vein endothelial cells

    SciTech Connect

    Ma Zhiyong; Zhang Wei; Chen Liang; Wang Rong; Kan Xiaohong; Sun Guizhen; Liu Chunxi; Li Li Zhang Yun

    2008-07-04

    Extracellular acidic pH-activated chloride channel I{sub Cl,acid}, has been characterized in HEK 293 cells and mammalian cardiac myocytes. This study was designed to characterize I{sub Cl,acid} in human umbilical vein endothelial cells(HUVECs). The activation and deactivation of the current rapidly and repeatedly follows the change of the extracellular solution at pH 4.3, with the threshold pH 5.3. In addition, at very positive potentials, the current displays a time-dependent facilitation. pH-response relationship for I{sub Cl,acid} revealed that EC{sub 50} is pH 4.764 with a threshold pH value of pH 5.3 and nH of 14.545. The current can be blocked by the Cl{sup -} channel inhibitor DIDS (100 {mu}M). In summary, for the first time we report the presence of proton-activated, outwardly rectifying chloride channel in HUVECs. Because an acidic environment can develop in local myocardium under pathological conditions such as myocardial ischemia, I{sub Cl,acid} would play a role in regulation of EC function under these pathological conditions.

  15. Molecular, Biophysical, and Pharmacological Properties of Calcium-Activated Chloride Channels.

    PubMed

    Kamaleddin, Mohammad Amin

    2017-01-25

    Calcium-activated chloride channels (CaCCs) are a family of anionic transmembrane ion channels. They are mainly responsible for the movement of Cl(-) and other anions across the biological membranes and are widely expressed in different tissues. Due to the fact that Cl(-) flow into or out of the cell plays a crucial role in hyperpolarizing or depolarizing the cells, respectively, the impact of intracellular Ca(2+) concentration on these channels is attracting a lot of attentions. After summarizing the molecular, biophysical, and pharmacological properties of CaCCs, the role of CaCCs in normal cellular functions will be discussed, and I will emphasize how dysregulation of CaCCs in pathological conditions can account for different diseases. A better understanding of CaCCs and a pivotal regulatory role of Ca(2+) can shed more light on the therapeutic strategies for different neurological disorders that arise from chloride dysregulation, such as asthma, cystic fibrosis, and neuropathic pain. This article is protected by copyright. All rights reserved.

  16. Requirement of calcium-activated chloride channels in the activation of mouse vomeronasal neurons

    PubMed Central

    Kim, SangSeong; Ma, Limei; Yu, C. Ron

    2011-01-01

    In terrestrial vertebrates, the vomeronasal organ (VNO) detects and transduces pheromone signals. VNO activation is thought to be mediated by the transient receptor potential C2 (TRPC2) channel. The aberrant behavioural phenotypes observed in TRPC2−/− mice are generally attributed to the lost VNO function. Recently, calcium-activated chloride channels have been shown to contribute to VNO activation. Here we show that CACCs can be activated in VNO slice preparations from the TRPC2−/− mice and this activation is blocked by pharmacological agents that inhibit intracellular Ca2+ release. Urine-evoked Cl− current is sufficient to drive spiking changes in VNO neurons from both wild-type (WT) and TRPC2−/− mice. Moreover, blocking Cl− conductance essentially abolishes VNO activation in WT neurons. These results suggest a TRPC2-independent signalling pathway in the VNO and the requirement of calcium-activated chloride channels currents to mediate pheromone activation. Our data further suggest that TRPC2−/− mice retain partial VNO function. PMID:21694713

  17. The ClC-3 chloride channel and osmoregulation in the European sea bass, Dicentrarchus labrax.

    PubMed

    Bossus, Maryline; Charmantier, Guy; Blondeau-Bidet, Eva; Valletta, Bianca; Boulo, Viviane; Lorin-Nebel, Catherine

    2013-07-01

    Dicentrarchus labrax migrates between sea (SW), brackish and fresh water (FW) where chloride concentrations and requirements for chloride handling change: in FW, fish absorb chloride and restrict renal losses; in SW, they excrete chloride. In this study, the expression and localization of ClC-3 and Na(+)/K(+)-ATPase (NKA) were studied in fish adapted to SW, or exposed to FW from 10 min to 30 days. In gills, NKA-α1 subunit expression transiently increased from 10 min and reached a stabilized intermediate expression level after 24 h in FW. ClC-3 co-localized with NKA in the basolateral membrane of mitochondria-rich cells (MRCs) at all conditions. The intensity of MRC ClC-3 immunostaining was significantly higher (by 50 %) 1 h after the transfer to FW, whereas the branchial ClC-3 protein expression was 30 % higher 7 days after the transfer as compared to SW. This is consistent with the increased number of immunopositive MRCs (immunostained for NKA and ClC-3). However, the ClC-3 mRNA expression was significantly lower in FW gills. In the kidney, after FW transfer, a transient decrease in NKA-α1 subunit expression was followed by significantly higher stable levels from 24 h. The low ClC-3 protein expression detected at both salinities was not observed by immunocytochemistry in the SW kidney; ClC-3 was localized in the basal membrane of the collecting ducts and tubules 7 and 30 days after transfer to FW. Renal ClC-3 mRNA expression, however, seemed higher in SW than in FW. The potential role of this chloride channel ClC-3 in osmoregulatory and osmosensing mechanisms is discussed.

  18. Inhibition of GABA-gated chloride channels by 12,14-dichlorodehydroabietic acid in mammalian brain

    PubMed Central

    Nicholson, Russell A; Lees, George; Zheng, Jian; Verdon, Bernard

    1999-01-01

    12,14-dichlorodehydroabietic acid (12,14-Cl2DHA) reduced GABA-stimulated uptake of 36Cl− into mouse brain synaptoneurosomes suggesting inhibition of mammalian GABAA receptor function. 12,14-Cl2DHA did not affect the binding of [3H]-muscimol to brain membranes but displaced specifically bound [3H]-EBOB. The inhibitory effect on [3H]-EBOB binding was not reversible. 12,14-Cl2DHA reduced the availability of [3H]-EBOB binding sites (Bmax) without changing the KD of the radioligand for remaining sites. 12,14-Cl2DHA did not affect the rate of association of [3H]-EBOB with its chloride channel receptor, but increased the initial rate of [3H]-EBOB dissociation. 12,14-Cl2DHA enhanced the incidence of EPSCs when rapidly applied to cultured rat cortical neurones. Longer exposures produced block of IPSCs with marked increases in the frequency of EPSCs and min EPSCs. 12,14-Cl2DHA also irreversibly suppressed chloride currents evoked by pulses of exogenous GABA in these cells. Ultimately, 12,14-Cl2DHA inhibited all synaptic traffic and action currents in current clamped cells indicating that, in contrast to picrotoxinin (which causes paroxysmal bursting), it is not fully selective for the GABAA receptor-chloride channel complex. The depolarizing block seen with 12,14-Cl2DHA in amphotericin-perforated preparations implicates loss of Ca2+ buffering in the polarity change and this may account for inhibition of spontaneous action potentials. Our investigation demonstrates that 12,14-Cl2DHA blocks GABA-dependent chloride entry in mammalian brain and operates as a non-competitive insurmountable GABAA antagonist. The mechanism likely involves either irreversible binding of 12,14-Cl2DHA to the trioxabicyclooctane recognition site or a site that is allosterically coupled to it. We cannot exclude, however, the possibility that 12,14-Cl2DHA causes localized proteolysis or more extensive conformational change within a critical subunit of the chloride channel. PMID:10204999

  19. Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis.

    PubMed

    Bompadre, Silvia G; Hwang, Tzyh-Chang

    2007-08-25

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP-binding cassette (ABC) transporter superfamily. Defective function of CFTR is responsible for cystic fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasian populations. The disease is manifested in defective chloride transport across the epithelial cells in various tissues. To date, more than 1400 different mutations have been identified as CF-associated. CFTR is regulated by phosphorylation in its regulatory (R) domain, and gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBD1 and NBD2). Recent studies reveal that the NBDs of CFTR may dimerize as observed in other ABC proteins. Upon dimerization of CFTR's two NBDs, in a head-to-tail configuration, the two ATP-binding pockets (ABP1 and ABP2) are formed by the canonical Walker A and B motifs from one NBD and the signature sequence from the partner NBD. Mutations of the amino acids that interact with ATP reveal that the two ABPs play distinct roles in controlling ATP-dependent gating of CFTR. It was proposed that binding of ATP to the ABP2, which is formed by the Walker A and B in NBD2 and the signature sequence in NBD1, is critical for catalyzing channel opening. While binding of ATP to the ABP1 alone may not increase the opening rate, it does contribute to the stabilization of the open channel conformation. Several disease-associated mutations of the CFTR channel are characterized by gating defects. Understanding how CFTR's two NBDs work together to gate the channel could provide considerable mechanistic information for future pharmacological studies, which could pave the way for tailored drug design for therapeutical interventions in CF.

  20. Characterization of the mouse ClC-K1/Barttin chloride channel.

    PubMed

    L'Hoste, Sébastien; Diakov, Alexei; Andrini, Olga; Genete, Mathieu; Pinelli, Laurent; Grand, Teddy; Keck, Mathilde; Paulais, Marc; Beck, Laurent; Korbmacher, Christoph; Teulon, Jacques; Lourdel, Stéphane

    2013-11-01

    Several Cl(-) channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl(-) absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl(-)>Br(-)>NO3(-)>I(-). Single-channel recordings revealed a unit conductance of ~40pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~-65mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~20pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~+25mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253-260).

  1. A chloride channel from lobster walking leg nerves. Characterization of single-channel properties in planar bilayers

    PubMed Central

    1990-01-01

    A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers from a plasma membrane preparation of lobster walking leg nerves. Under conditions of symmetrical 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, single Cl- channels exhibit rectifying current-voltage (I-V) behavior with a conductance of 19.2 +/- 0.8 pS at positive voltages and 15.1 +/- 1.6 pS in the voltage range of -40 to 0 mV. The channel exhibits a negligible permeability for Na+ compared with Cl- and displays the following sequence of anion permeability relative to Cl- as measured under near bi-ionic conditions: I- (2.7) greater than NO3- (1.8) greater than Br- (1.5) greater than Cl- (1.0) greater than CH3CO2- (0.18) greater than HCO3- (0.10) greater than gluconate (0.06) greater than F- (0.05). The unitary conductance saturates with increasing Cl- concentration in a Michaelis-Menten fashion with a Km of 100 mM and gamma max = 33 pS at positive voltage. The I-V curve is similar in 10 mM Tris or 10 mM HEPES buffer, but substitution of 100 mM NaCl with 100 mM tetraethylammonium chloride on the cis side results in increased rectification with a 40% reduction in current at negative voltages. The gating of the channel is weakly voltage dependent with an open-state probability of 0.23 at -75 mV and 0.64 at +75 mV. Channel gating is sensitive to cis pH with an increased opening probability observed for a pH change of 7.4 to 11 and nearly complete inhibition for a pH change of 7.4 to 6.0. The lobster Cl- channel is reversibly blocked by the anion transport inhibitors, SITS (4-acetamido, 4'-isothiocyanostilbene-2,2'-disulfonic acid) and NPPB (5- nitro-2-(3-phenylpropylamino)benzoic acid). Many of these characteristics are similar to those previously described for small conductance Cl- channels in various vertebrate cells, including epithelia. These functional comparisons suggest that this invertebrate Cl- channel is an evolutionary prototype of a widely distributed class of small

  2. The Split Personality of Glutamate Transporters: A Chloride Channel and a Transporter.

    PubMed

    Cater, Rosemary J; Ryan, Renae M; Vandenberg, Robert J

    2016-03-01

    Transporters and ion channels are conventionally categorised into distinct classes of membrane proteins. However, some membrane proteins have a split personality and can function as both transporters and ion channels. The excitatory amino acid transporters (EAATs) in particular, function as both glutamate transporters and chloride (Cl(-)) channels. The EAATs couple the transport of glutamate to the co-transport of three Na(+) ions and one H(+) ion into the cell, and the counter-transport of one K(+) ion out of the cell. The EAAT Cl(-) channel is activated by the binding of glutamate and Na(+), but is thermodynamically uncoupled from glutamate transport and involves molecular determinants distinct from those responsible for glutamate transport. Several crystal structures of an EAAT archaeal homologue, GltPh, at different stages of the transport cycle, alongside numerous functional studies and molecular dynamics simulations, have provided extensive insights into the mechanism of substrate transport via these transporters. However, the molecular determinants involved in Cl(-) permeation, and the mechanism by which this channel is activated are not entirely understood. Here we will discuss what is currently known about the molecular determinants involved in EAAT-mediated Cl(-) permeation and the mechanisms that underlie their split personality.

  3. Separate fractions of mRNA from Torpedo electric organ induce chloride channels and acetylcholine receptors in Xenopus oocytes.

    PubMed Central

    Sumikawa, K; Parker, I; Amano, T; Miledi, R

    1984-01-01

    Poly(A)+ mRNA extracted from the electric organ of Torpedo was fractionated by sucrose density gradient centrifugation. After injection into Xenopus oocytes one mRNA fraction induced the appearance of chloride channels in the oocyte membrane. Many of these channels were normally open, and the ensuing chloride current kept the resting potential of injected oocytes close to the chloride equilibrium potential. When the membrane was hyperpolarized, the chloride current was reduced. A separate fraction of mRNA induced the incorporation of acetylcholine receptors into the oocyte membrane. When translated in a cell-free system this fraction directed the synthesis of the alpha, beta, gamma, and delta subunits of the acetylcholine receptor. In contrast, the mRNA fraction that induced the chloride channels caused the synthesis of the delta subunit, a very small amount of alpha, and no detectable beta or gamma subunits. This suggests that the size of the mRNA coding for the chloride channel is similar to the preponderant species of mRNA coding for the delta subunit of the acetylcholine receptor. Images Fig. 1. PMID:6094179

  4. The calcium-activated chloride channel anoctamin 1 acts as a heat sensor in nociceptive neurons.

    PubMed

    Cho, Hawon; Yang, Young Duk; Lee, Jesun; Lee, Byeongjoon; Kim, Tahnbee; Jang, Yongwoo; Back, Seung Keun; Na, Heung Sik; Harfe, Brian D; Wang, Fan; Raouf, Ramin; Wood, John N; Oh, Uhtaek

    2012-05-27

    Nociceptors are a subset of small primary afferent neurons that respond to noxious chemical, thermal and mechanical stimuli. Ion channels in nociceptors respond differently to noxious stimuli and generate electrical signals in different ways. Anoctamin 1 (ANO1 also known as TMEM16A) is a Ca(2+)-activated chloride channel that is essential for numerous physiological functions. We found that ANO1 was activated by temperatures over 44 °C with steep heat sensitivity. ANO1 was expressed in small sensory neurons and was highly colocalized with nociceptor markers, which suggests that it may be involved in nociception. Application of heat ramps to dorsal root ganglion (DRG) neurons elicited robust ANO1-dependent depolarization. Furthermore, knockdown or deletion of ANO1 in DRG neurons substantially reduced nociceptive behavior in thermal pain models. These results indicate that ANO1 is a heat sensor that detects nociceptive thermal stimuli in sensory neurons and possibly mediates nociception.

  5. Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

    PubMed Central

    Larsen, EH; Gabriei, SE; Stutts, MJ; Fullton, J; Price, EM; Boucher, RC

    1996-01-01

    The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1

  6. CFTR chloride channel in the apical compartments: spatiotemporal coupling to its interacting partners.

    PubMed

    Li, Chunying; Naren, Anjaparavanda P

    2010-04-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel located primarily at the apical or luminal surfaces of epithelial cells in the airway, intestine, pancreas, kidney, sweat gland, as well as male reproductive tract, where it plays a crucial role in transepithelial fluid homeostasis. CFTR dysfunction can be detrimental and may result in life-threatening disorders. CFTR hypofunctioning because of genetic defects leads to cystic fibrosis, the most common lethal genetic disease in Caucasians, whereas CFTR hyperfunctioning resulting from various infections evokes secretory diarrhea, the leading cause of mortality in early childhood. Therefore, maintaining a dynamic balance between CFTR up-regulating processes and CFTR down-regulating processes is essential for maintaining fluid and body homeostasis. Accumulating evidence suggests that protein-protein interactions play a critical role in the fine-tuned regulation of CFTR function. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might be coupled spatially and temporally to a wide variety of interacting partners including ion channels, receptors, transporters, scaffolding proteins, enzyme molecules, signaling molecules, and effectors. Most interactions occur primarily between the opposing terminal tails (amino or carboxyl) of CFTR protein and its binding partners, either directly or mediated through various PDZ scaffolding proteins. These dynamic interactions impact the channel function, as well as localization and processing of CFTR protein within cells. This article reviews the most recent progress and findings about the interactions between CFTR and its binding partners through PDZ scaffolding proteins, as well as the spatiotemporal regulation of CFTR-containing macromolecular signaling complexes in the apical compartments of polarized cells lining the secretory epithelia.

  7. Slow conversions among subconductance states of cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed Central

    Tao, T; Xie, J; Drumm, M L; Zhao, J; Davis, P B; Ma, J

    1996-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel exhibits multiple subconductance states. To study the regulation of conductance states of the CFTR channel, we expressed the wild-type CFTR protein in HEK 293 cells, and isolated microsomal membrane vesicles for reconstitution studies in lipid bilayer membranes. A single CFTR channel had a dominant conductance of 7.8 pS (H), plus two sub-open states with conductances of approximately 6 pS (M) and 2.7 pS (L) in 200 mM KCl with 1 mM MgCl2 (intracellular) and 50 mM KCl with no MgCl2 (extracellular), with pH maintained at 7.4 by 10 mM HEPES-Tris on both sides of the channel. In 200 mM KCl, both H and L states could be measured in stable single-channel recordings, whereas M could not. Spontaneous transitions between H and L were slow; it took 4.5 min for L-->H, and 3.2 min for H-->L. These slow conversions among subconductance states of the CFTR channel were affected by extracellular Mg; in the presence of millimolar Mg, the channel remained stable in the H state. Similar phenomena were also observed with endogenous CFTR channels in T84 cells. In high-salt conditions (1.5 M KCl), all three conductance states of the expressed CFTR channel, 12.1 pS, 8.2 pS, and 3.6 pS, became stable and seemed to gate independently from each other. The existence of multiple stable conductance states associated with the CFTR channel suggests two possibilities: either a single CFTR molecule can exist in multiple configurations with different conductance values, or the CFTR channel may contain multimers of the 170-kDa CFTR protein, and different conductance states are due to different aggregation states of the CFTR protein. Images FIGURE 4 FIGURE 8 PMID:8789091

  8. Slow conversions among subconductance states of cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    Tao, T; Xie, J; Drumm, M L; Zhao, J; Davis, P B; Ma, J

    1996-02-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel exhibits multiple subconductance states. To study the regulation of conductance states of the CFTR channel, we expressed the wild-type CFTR protein in HEK 293 cells, and isolated microsomal membrane vesicles for reconstitution studies in lipid bilayer membranes. A single CFTR channel had a dominant conductance of 7.8 pS (H), plus two sub-open states with conductances of approximately 6 pS (M) and 2.7 pS (L) in 200 mM KCl with 1 mM MgCl2 (intracellular) and 50 mM KCl with no MgCl2 (extracellular), with pH maintained at 7.4 by 10 mM HEPES-Tris on both sides of the channel. In 200 mM KCl, both H and L states could be measured in stable single-channel recordings, whereas M could not. Spontaneous transitions between H and L were slow; it took 4.5 min for L-->H, and 3.2 min for H-->L. These slow conversions among subconductance states of the CFTR channel were affected by extracellular Mg; in the presence of millimolar Mg, the channel remained stable in the H state. Similar phenomena were also observed with endogenous CFTR channels in T84 cells. In high-salt conditions (1.5 M KCl), all three conductance states of the expressed CFTR channel, 12.1 pS, 8.2 pS, and 3.6 pS, became stable and seemed to gate independently from each other. The existence of multiple stable conductance states associated with the CFTR channel suggests two possibilities: either a single CFTR molecule can exist in multiple configurations with different conductance values, or the CFTR channel may contain multimers of the 170-kDa CFTR protein, and different conductance states are due to different aggregation states of the CFTR protein.

  9. Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels.

    PubMed

    Reyes, J P; Huanosta-Gutiérrez, A; López-Rodríguez, A; Martínez-Torres, A

    2015-01-01

    We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na(+) permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na(+). Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.

  10. Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

    PubMed Central

    Reyes, JP; Huanosta-Gutiérrez, A; López-Rodríguez, A; Martínez-Torres, A

    2015-01-01

    We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na+ permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na+. Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade. PMID:25853341

  11. Huntington disease skeletal muscle is hyperexcitable owing to chloride and potassium channel dysfunction.

    PubMed

    Waters, Christopher W; Varuzhanyan, Grigor; Talmadge, Robert J; Voss, Andrew A

    2013-05-28

    Huntington disease is a progressive and fatal genetic disorder with debilitating motor and cognitive defects. Chorea, rigidity, dystonia, and muscle weakness are characteristic motor defects of the disease that are commonly attributed to central neurodegeneration. However, no previous study has examined the membrane properties that control contraction in Huntington disease muscle. We show primary defects in ex vivo adult skeletal muscle from the R6/2 transgenic mouse model of Huntington disease. Action potentials in diseased fibers are more easily triggered and prolonged than in fibers from WT littermates. Furthermore, some action potentials in the diseased fibers self-trigger. These defects occur because of decreases in the resting chloride and potassium conductances. Consistent with this, the expression of the muscle chloride channel, ClC-1, in Huntington disease muscle was compromised by improper splicing and a corresponding reduction in total Clcn1 (gene for ClC-1) mRNA. Additionally, the total Kcnj2 (gene for the Kir2.1 potassium channel) mRNA was reduced in disease muscle. The resulting muscle hyperexcitability causes involuntary and prolonged contractions that may contribute to the chorea, rigidity, and dystonia that characterize Huntington disease.

  12. Spatiotemporal Coupling of cAMP Transporter to CFTR Chloride Channel Function in the Gut Epithelia

    PubMed Central

    Li, Chunying; Krishnamurthy, Partha C.; Penmatsa, Himabindu; Marrs, Kevin L.; Wang, Xue Qing; Zaccolo, Manuela; Jalink, Kees; Li, Min; Nelson, Deborah J.; Schuetz, John D.; Naren, Anjaparavanda P.

    2007-01-01

    SUMMARY Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, is functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. MRP4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea. PMID:18045536

  13. Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia.

    PubMed

    Li, Chunying; Krishnamurthy, Partha C; Penmatsa, Himabindu; Marrs, Kevin L; Wang, Xue Qing; Zaccolo, Manuela; Jalink, Kees; Li, Min; Nelson, Deborah J; Schuetz, John D; Naren, Anjaparavanda P

    2007-11-30

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here, we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. Mrp4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea.

  14. Cadmium regulates the expression of the CFTR chloride channel in human airway epithelial cells.

    PubMed

    Rennolds, Jessica; Butler, Susie; Maloney, Kevin; Boyaka, Prosper N; Davis, Ian C; Knoell, Daren L; Parinandi, Narasimham L; Cormet-Boyaka, Estelle

    2010-07-01

    Cadmium is a toxic heavy metal ranked seventh on the Priority List of Hazardous Substances. As a byproduct of smelters, cadmium is a prevalent environmental contaminant. It is also a major component of cigarette smoke, and its inhalation is associated with decreased pulmonary function, lung cancer, and chronic obstructive pulmonary disease. Ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR), play a central role in maintaining fluid homeostasis and lung functions. CFTR is mostly expressed in epithelial cells, and little is known about the effect of cadmium exposure on lung epithelial cell function. We show that exposure to cadmium decreases the expression of the CFTR protein and subsequent chloride transport in human airway epithelial cells in vitro. Impairment of CFTR protein expression was also observed in vivo in the lung of mice after intranasal instillation of cadmium. We established that the inhibitory effect of cadmium was not a nonspecific effect of heavy metals, as nickel had no effect on CFTR protein levels. Finally, we show that selected antioxidants, including alpha-tocopherol (vitamin E), but not N-acetylcysteine, can prevent the cadmium-induced suppression of CFTR. In summary, we have identified cadmium as a regulator of the CFTR chloride channel present in lung epithelial cells. Future strategies to prevent the deleterious effect of cadmium on epithelial cells and lung functions may benefit from the finding that alpha-tocopherol protects CFTR expression and function.

  15. Characteristics of GABA-activated chloride channels in mammalian dorsal root ganglion neurones.

    PubMed

    Robertson, B

    1989-04-01

    1. The properties of gamma-aminobutyric acid (GABA)-activated chloride channels in dorsal root ganglion (DRG) neurones obtained from rats and cats were examined using the single-electrode voltage clamp in conjunction with suction-electrode techniques. 2. GABA-evoked currents showed voltage-sensitive kinetics. Time constants (tau D) were measured from voltage-jump relaxations and tau D became briefer with membrane hyperpolarization. tau D was 33 ms at -120 mV with 60 microM-GABA and changed e-fold for 188 mV. tau D decreased as GABA concentration was increased - the extrapolated tau D at 'zero' GABA concentration was approximately equal to 50 ms at -120 mV. 3. The steady-state current in GABA was curvilinear, rectifying at negative potentials. The instantaneous current was linear with symmetrical chloride concentrations (140 mM) on both sides of the cell membrane. 4. Muscimol was a more effective agonist than GABA, while piperidine-4-sulphonic acid and ethylenediamine monocarbamate were only weakly effective agonists. Taurine and glycine had no detectable agonist activity. 5. Ion substitution experiments revealed the permeability sequence I- greater than Br- greater than Cl- greater than F- greater than propionate (1.88 greater than 1.21 greater than 1.0 approximately equal to 0.1 approximately equal to 0.1). 6. The presence of iodide and bromide ions externally caused an increase in chloride efflux at membrane potentials more negative than -40 mV, and caused a prolongation of voltage-jump relaxations. Relaxations in fluoride and propionate solutions were faster than those seen in chloride.

  16. CLC-3 chloride channels moderate long-term potentiation at Schaffer collateral–CA1 synapses

    PubMed Central

    Farmer, Laurel M; Le, Brandy N; Nelson, Deborah J

    2013-01-01

    The chloride channel CLC-3 is expressed in the brain on synaptic vesicles and postsynaptic membranes. Although CLC-3 is broadly expressed throughout the brain, the CLC-3 knockout mouse shows complete, selective postnatal neurodegeneration of the hippocampus, suggesting a crucial role for the channel in maintaining normal brain function. CLC-3 channels are functionally linked to NMDA receptors in the hippocampus; NMDA receptor-dependent Ca2+ entry, activation of Ca2+/calmodulin kinase II and subsequent gating of CLC-3 link the channels via a Ca2+-mediated feedback loop. We demonstrate that loss of CLC-3 at mature synapses increases long-term potentiation from 135 ± 4% in the wild-type slice preparation to 154 ± 7% above baseline (P < 0.001) in the knockout; therefore, the contribution of CLC-3 is to reduce synaptic potentiation by ∼40%. Using a decoy peptide representing the Ca2+/calmodulin kinase II phosphorylation site on CLC-3, we show that phosphorylation of CLC-3 is required for its regulatory function in long-term potentiation. CLC-3 is also expressed on synaptic vesicles; however, our data suggest functionally separable pre- and postsynaptic roles. Thus, CLC-3 confers Cl− sensitivity to excitatory synapses, controls the magnitude of long-term potentiation and may provide a protective limit on Ca2+ influx. PMID:23165767

  17. Gating the glutamate gate of CLC-2 chloride channel by pore occupancy

    PubMed Central

    De Jesús-Pérez, José J.; Castro-Chong, Alejandra; Shieh, Ru-Chi; Hernández-Carballo, Carmen Y.; De Santiago-Castillo, José A.

    2016-01-01

    CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage–sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H+. Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl−, Br−, SCN−, and I−) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pHe = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl−]i under unlikely protonation conditions (pHe = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H+ plays a minor role in dislodging the glutamate gate. PMID:26666914

  18. Gating the glutamate gate of CLC-2 chloride channel by pore occupancy.

    PubMed

    De Jesús-Pérez, José J; Castro-Chong, Alejandra; Shieh, Ru-Chi; Hernández-Carballo, Carmen Y; De Santiago-Castillo, José A; Arreola, Jorge

    2016-01-01

    CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage-sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H(+). Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl(-), Br(-), SCN(-), and I(-)) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pHe = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl(-)]i under unlikely protonation conditions (pHe = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H(+) plays a minor role in dislodging the glutamate gate.

  19. CLC-3 chloride channels moderate long-term potentiation at Schaffer collateral-CA1 synapses.

    PubMed

    Farmer, Laurel M; Le, Brandy N; Nelson, Deborah J

    2013-02-15

    The chloride channel CLC-3 is expressed in the brain on synaptic vesicles and postsynaptic membranes. Although CLC-3 is broadly expressed throughout the brain, the CLC-3 knockout mouse shows complete, selective postnatal neurodegeneration of the hippocampus, suggesting a crucial role for the channel in maintaining normal brain function. CLC-3 channels are functionally linked to NMDA receptors in the hippocampus; NMDA receptor-dependent Ca(2+) entry, activation of Ca(2+)/calmodulin kinase II and subsequent gating of CLC-3 link the channels via a Ca(2+)-mediated feedback loop. We demonstrate that loss of CLC-3 at mature synapses increases long-term potentiation from 135 ± 4% in the wild-type slice preparation to 154 ± 7% above baseline (P < 0.001) in the knockout; therefore, the contribution of CLC-3 is to reduce synaptic potentiation by ∼40%. Using a decoy peptide representing the Ca(2+)/calmodulin kinase II phosphorylation site on CLC-3, we show that phosphorylation of CLC-3 is required for its regulatory function in long-term potentiation. CLC-3 is also expressed on synaptic vesicles; however, our data suggest functionally separable pre- and postsynaptic roles. Thus, CLC-3 confers Cl(-) sensitivity to excitatory synapses, controls the magnitude of long-term potentiation and may provide a protective limit on Ca(2+) influx.

  20. Chloride channels mediate sodium sulphide-induced relaxation in rat uteri

    PubMed Central

    Mijušković, Ana; Kokić, Aleksandra Nikolić; Dušić, Zorana Oreščanin; Slavić, Marija; Spasić, Mihajlo B; Blagojević, Duško

    2015-01-01

    Background and Purpose Hydrogen sulphide reduces uterine contractility and is of potential interest as a treatment for uterine disorders. The aim of this study was to explore the mechanism of sodium sulphide (Na2S)-induced relaxation of rat uterus, investigate the importance of redox effects and ion channel-mediated mechanisms, and any interactions between these two mechanisms. Experimental Approach Organ bath studies were employed to assess the pharmacological effects of Na2S in uterine strips by exposing them to Na2S with or without Cl− channel blockers (DIDS, NFA, IAA-94, T16Ainh-A01, TA), raised KCl (15 and 75 mM), K+ channel inhibitors (glibenclamide, TEA, 4-AP), L-type Ca2+ channel activator (S-Bay K 8644), propranolol and methylene blue. The activities of antioxidant enzymes were measured in homogenates of treated uteri. The expression of bestrophin channel 1 (BEST-1) was determined by Western blotting and RT-PCR. Key Results Na2S caused concentration-dependent reversible relaxation of spontaneously active and calcium-treated uteri, affecting both amplitude and frequency of contractions. Uteri exposed to 75 mM KCl were less sensitive to Na2S compared with uteri in 15 mM KCl. Na2S-induced relaxations were abolished by DIDS, but unaffected by other modulators or by the absence of extracellular HCO3−, suggesting the involvement of chloride ion channels. Na2S in combination with different modulators provoked specific changes in the anti-oxidant profiles of uteri. The expression of BEST-1, both mRNA and protein, was demonstrated in rat uteri. Conclusions and Implications The relaxant effects of Na2S in rat uteri are mediated mainly via a DIDS-sensitive Cl−-pathway. Components of the relaxation are redox- and Ca2+-dependent. PMID:25857480

  1. Conductance-voltage relations in large-conductance chloride channels in proliferating L6 myoblasts.

    PubMed

    Hurnák, O; Zachar, J

    1994-06-01

    Large-conductance chloride channels (maxi-Cl channels) were studied in cultured myoblasts (L6 rat muscle cell line); in excised (inside-out) and in cell attached membrane patches using a conventional patch clamp method. The incidence of maxi-Cl channels was substantially higher in proliferating myoballs, then in quiescent (bottom-attached) myoblasts (90% and 50% percent of examined cells, respectively). The maxi-Cl channels in myoballs were present both in cell attached and excised patches. The channel conductance at symmetric [Cl] = 150 mmol/l was 359 +/- 42 pS (n = 74) in quiescent cells and 439 +/- 10 pS (n = 6) in proliferating myoballs respectively. The conductance of the channel in quiescent cells increased with chloride concentration in symmetric NaCl rich solutions according to Michaelis-Menten curve with the saturation limiting conductance of about 640 pS (gmax) and Km = 112 mmol/l. The shift of the reversal potential upon increasing the pipette concentration of NaCl from 150 to 250 mmol/l was consistent with PNa/PCl = 0.1. Neither the conductance nor the activation of the channel were dependent on the presence of calcium ions. The bell-shaped steady state channel conductance-voltage relationship is asymmetric and can be fitted by two Boltzmann equations with different Vh and k constants; -25.6 mV and -6.8 mV, respectively, for the negative side and +49.6 mV and +13.7 mV for the positive side in quiescent cells. The corresponding values in proliferating myoballs were as follows: -15.5 mV and -2.4 mV, respectively, for the negative side and +31.4 mV and +6.8 mV for the positive side. From the maximum slopes of the Popen versus V curves an estimate was made of the charges for the gates that close at negative (3.5) or positive (1.7) potentials, respectively, in quiescent cells. The corresponding values in myoballs were 10.6 and 3.7, respectively. The probability of one gate to be open was dependent on the state of activation of the opposite gate as determined

  2. Effect of Trimethyltin Chloride on Slow Vacuolar (SV) Channels in Vacuoles from Red Beet (Beta vulgaris L.) Taproots.

    PubMed

    Trela, Zenon; Burdach, Zbigniew; Siemieniuk, Agnieszka; Przestalski, Stanisław; Karcz, Waldemar

    2015-01-01

    In the present study, patch-clamp techniques have been used to investigate the effect of trimethyltin chloride (Met3SnCl) on the slow vacuolar (SV) channels in vacuoles from red beet (Beta vulgaris L.) taproots. Activity of SV channels has been measured in whole-vacuole and cytosolic side-out patch configurations. It was found that addition of trimethyltin chloride to the bath solution suppressed, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant, τ, increased significantly in the presence of the organotin. When single channel activity was analyzed, only little channel activity could be recorded at 100 μM Met3SnCl. Trimethyltin chloride added to the bath medium significantly decreased (by ca. threefold at 100 μM Met3SnCl and at 100 mV voltage, as compared to the control medium) the open probability of single channels. Single channel recordings obtained in the presence and absence of trimethyltin chloride showed that the organotin only slightly (by <10%) decreased the unitary conductance of single channels. It was also found that Met3SnCl significantly diminished the number of SV channel openings, whereas it did not change the opening times of the channels. Taking into account the above and the fact that under the here applied experimental conditions (pH = 7.5) Met3SnCl is a non-dissociated (more lipophilic) compound, we suggest that the suppression of SV currents observed in the presence of the organotin results probably from its hydrophobic properties allowing this compound to translocate near the selectivity filter of the channel.

  3. Effect of Trimethyltin Chloride on Slow Vacuolar (SV) Channels in Vacuoles from Red Beet (Beta vulgaris L.) Taproots

    PubMed Central

    Trela, Zenon; Burdach, Zbigniew; Siemieniuk, Agnieszka; Przestalski, Stanisław; Karcz, Waldemar

    2015-01-01

    In the present study, patch-clamp techniques have been used to investigate the effect of trimethyltin chloride (Met3SnCl) on the slow vacuolar (SV) channels in vacuoles from red beet (Beta vulgaris L.) taproots. Activity of SV channels has been measured in whole-vacuole and cytosolic side-out patch configurations. It was found that addition of trimethyltin chloride to the bath solution suppressed, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant, τ, increased significantly in the presence of the organotin. When single channel activity was analyzed, only little channel activity could be recorded at 100 μM Met3SnCl. Trimethyltin chloride added to the bath medium significantly decreased (by ca. threefold at 100 μM Met3SnCl and at 100 mV voltage, as compared to the control medium) the open probability of single channels. Single channel recordings obtained in the presence and absence of trimethyltin chloride showed that the organotin only slightly (by <10%) decreased the unitary conductance of single channels. It was also found that Met3SnCl significantly diminished the number of SV channel openings, whereas it did not change the opening times of the channels. Taking into account the above and the fact that under the here applied experimental conditions (pH = 7.5) Met3SnCl is a non-dissociated (more lipophilic) compound, we suggest that the suppression of SV currents observed in the presence of the organotin results probably from its hydrophobic properties allowing this compound to translocate near the selectivity filter of the channel. PMID:26317868

  4. TRPC1 regulates calcium-activated chloride channels in salivary gland cells.

    PubMed

    Sun, Yuyang; Birnbaumer, Lutz; Singh, Brij B

    2015-11-01

    Calcium-activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca(2+) influx that activates ion channels such as CaCC to initiate Cl(-) efflux. However direct evidence as well as the molecular identity of the Ca(2+) channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl(-) current was activated by increasing [Ca(2+)]i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh-A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store-depletion and activates TRPC1-mediated Ca(2+) entry, potentiated the Cl(-) current, which was inhibited by the addition of a non-specific TRPC channel blocker SKF96365 or removal of external Ca(2+). Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca(2+) entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl(-) currents upon increasing [Ca(2+)]i. These Cl(-) currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh-A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl(-) currents without decreasing TMEM16a expression. Together the data suggests that Ca(2+) entry via the TRPC1 channels is essential for the activation of CaCC.

  5. TRPC1 regulates calcium‐activated chloride channels in salivary gland cells

    PubMed Central

    Sun, Yuyang; Birnbaumer, Lutz

    2015-01-01

    Calcium‐activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca2+ influx that activates ion channels such as CaCC to initiate Cl− efflux. However direct evidence as well as the molecular identity of the Ca2+ channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl− current was activated by increasing [Ca2+]i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh‐A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store‐depletion and activates TRPC1‐mediated Ca2+ entry, potentiated the Cl− current, which was inhibited by the addition of a non‐specific TRPC channel blocker SKF96365 or removal of external Ca2+. Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca2+ entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl− currents upon increasing [Ca2+]i. These Cl− currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh‐A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl− currents without decreasing TMEM16a expression. Together the data suggests that Ca2+ entry via the TRPC1 channels is essential for the activation of CaCC. J. Cell. Physiol. 9999: 2848–2856, 2015. © 2015 Wiley Periodicals, Inc. PMID:25899321

  6. Cloning and functional expression of a plant voltage-dependent chloride channel.

    PubMed Central

    Lurin, C; Geelen, D; Barbier-Brygoo, H; Guern, J; Maurel, C

    1996-01-01

    Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants. PMID:8624442

  7. Regulatory-auxiliary subunits of CLC chloride channel-transport proteins.

    PubMed

    Barrallo-Gimeno, Alejandro; Gradogna, Antonella; Zanardi, Ilaria; Pusch, Michael; Estévez, Raúl

    2015-09-15

    The CLC family of chloride channels and transporters is composed by nine members, but only three of them, ClC-Ka/b, ClC-7 and ClC-2, have been found so far associated with auxiliary subunits. These CLC regulatory subunits are small proteins that present few common characteristics among them, both structurally and functionally, and their effects on the corresponding CLC protein are different. Barttin, a protein with two transmembrane domains, is essential for the membrane localization of ClC-K proteins and their activity in the kidney and inner ear. Ostm1 is a protein with a single transmembrane domain and a highly glycosylated N-terminus. Unlike the other two CLC auxiliary subunits, Ostm1 shows a reciprocal relationship with ClC-7 for their stability. The subcellular localization of Ostm1 depends on ClC-7 and not the other way around. ClC-2 is active on its own, but GlialCAM, a transmembrane cell adhesion molecule with two extracellular immunoglobulin (Ig)-like domains, regulates its subcellular localization and activity in glial cells. The common theme for these three proteins is their requirement for a proper homeostasis, since their malfunction leads to distinct diseases. We will review here their properties and their role in normal chloride physiology and the pathological consequences of their improper function.

  8. Ovine congenital myotonia associated with a mutation in the muscle chloride channel gene.

    PubMed

    Monteagudo, Luis Vicente; Tejedor, María Teresa; Ramos, Juan José; Lacasta, Delia; Ferrer, Luis Miguel

    2015-04-01

    Congenital myotonia (CM) is characterised by a delay in muscular relaxation after sudden contractions. In a recent outbreak of ovine CM affecting 1% of new-born lambs in a Spanish flock of Rasa Aragonesa sheep, a comparative pathology approach was taken: because a mutation in the muscle chloride channel gene (CLCN1) was identified as responsible for CM in goats, the same gene was sequenced in the affected lambs. A non-synonymous single nucleotide variation (SNV) in the second exon of CLCN1 was associated with this pathology. Rams carrying this SNV heterozygously were thereafter identified and replaced by wild-type homozygous young males. No additional CM cases were detected in subsequent lambing seasons.

  9. Mutational scanning of potassium, sodium and chloride ion channels in malignant migrating partial seizures in infancy.

    PubMed

    Coppola, Giangennaro; Veggiotti, Pierangelo; Del Giudice, Emanuele Miraglia; Bellini, Giulia; Longaretti, Francesca; Taglialatela, Maurizio; Pascotto, Antonio

    2006-03-01

    The mutational analysis of potassium (KCNQ2, KCNQ3), sodium (SCN1A, SCN2A), and chloride (CLCN2) ion channels was performed in three children with typical features of the recently described syndrome of migrating partial seizures in infancy. Mutational analysis was performed by PCR and automatic sequencing. The coding regions, including the exon-intron boundaries, were amplified in the patients using appropriate primers sets. No mutations associated to migrating partial seizures have been found. Mutational screening of CLCN2 gene, revealed a homozygous mutation G2003C (exon 17), leading to a Ser/Thr substitution at the codon 668, in two of the three patients. The same variation has been found in 38 out of 100 control alleles. The identification of the genetic basis of this new epileptic encephalopathy requires further studies that might be enforced by familial cases.

  10. Functional and molecular identification of a TASK-1 potassium channel regulating chloride secretion through CFTR channels in the shark rectal gland: implications for cystic fibrosis.

    PubMed

    Telles, Connor J; Decker, Sarah E; Motley, William W; Peters, Alexander W; Mehr, Ali Poyan; Frizzell, Raymond A; Forrest, John N

    2016-12-01

    In the shark rectal gland (SRG), apical chloride secretion through CFTR channels is electrically coupled to a basolateral K(+) conductance whose type and molecular identity are unknown. We performed studies in the perfused SRG with 17 K(+) channel inhibitors to begin this search. Maximal chloride secretion was markedly inhibited by low-perfusate pH, bupivicaine, anandamide, zinc, quinidine, and quinine, consistent with the properties of an acid-sensitive, four-transmembrane, two-pore-domain K(+) channel (4TM-K2P). Using PCR with degenerate primers to this family, we identified a TASK-1 fragment in shark rectal gland, brain, gill, and kidney. Using 5' and 3' rapid amplification of cDNA ends PCR and genomic walking, we cloned the full-length shark gene (1,282 bp), whose open reading frame encodes a protein of 375 amino acids that was 80% identical to the human TASK-1 protein. We expressed shark and human TASK-1 cRNA in Xenopus oocytes and characterized these channels using two-electrode voltage clamping. Both channels had identical current-voltage relationships (outward rectifying) and a reversal potential of -90 mV. Both were inhibited by quinine, bupivicaine, and acidic pH. The pKa for current inhibition was 7.75 for shark TASK-1 vs. 7.37 for human TASK-1, values similar to the arterial pH for each species. We identified this protein in SRG by Western blot and confocal immunofluorescent microscopy and detected the protein in SRG and human airway cells. Shark TASK-1 is the major K(+) channel coupled to chloride secretion in the SRG, is the oldest 4TM 2P family member identified, and is the first TASK-1 channel identified to play a role in setting the driving force for chloride secretion in epithelia. The detection of this potassium channel in mammalian lung tissue has implications for human biology and disease.

  11. Myotonia and the muscle chloride channel: dominant mutations show variable penetrance and founder effect.

    PubMed

    Koty, P P; Pegoraro, E; Hobson, G; Marks, H G; Turel, A; Flagler, D; Cadaldini, M; Angelini, C; Hoffman, E P

    1996-10-01

    The delayed relaxation or sustained contraction of skeletal muscle-myotonia-is frequently seen in myotonic dystrophy and sodium channelopathies (hyperkalemic periodic paralysis, paramyotonia congenita). Many cases of congenital myotonia without other clinical symptoms have been associated with mutations in the muscle chloride channel gene. Most cases reported to date show a recessive inheritance pattern, with loss of function of the corresponding protein. Six families have been reported with dominantly inherited myotonia and mutations of the chloride channel gene. Here we report clinical and molecular data on 38 family members from four new families with dominantly inherited myotonia congenita. Three families show a previously characterized G230E mutation, and we show that these three share a common affected ancestor despite living in different regions of the United States (linkage disequilibrium). One Italian family is shown to have a novel dominant mutation-I290M. This is the sixth mutation identified in Thomsen's myotonia. Genotype/phenotype correlations in these four families showed that both of the dominant mutations resulted in a mild clinical picture in 90% of the patients, and no symptoms in 10% of mutation-positive patients. The EMG was the clinical feature that most closely correlated with mutation data; however, 3 of 16 (19%) mutation-positive patients tested negative by electromyography at least once, and 1 (6%) tested negative despite multiple tests. Only about half (55%) of the mutation-positive patients tested positive for percussion myotonia. Most of the clinically symptomatic individuals stated that cold temperatures and stress substantially worsened their myotonia. Our data show that dominantly inherited Thomsen's myotonia is most often a very mild disorder that shows considerable clinical heterogeneity.

  12. Identification and characterization of the zebrafish ClC-2 chloride channel orthologs.

    PubMed

    Pérez-Rius, Carla; Gaitán-Peñas, Héctor; Estévez, Raúl; Barrallo-Gimeno, Alejandro

    2015-08-01

    ClC-2 is a Cl(-) channel that belongs to the CLC family of chloride channel/transport proteins. ClC-2 molecular role is not clear, and Clcn2 knockout mice develop blindness, sterility, and leukodystrophy by unknown reasons. ClC-2 is associated in the brain with the adhesion molecule GlialCAM, which is defective in a type of leukodystrophy, involving ClC-2 in the homeostasis of myelin. To get more insight into the functions of ClC-2, we have identified in this work the three ClC-2 orthologs in zebrafish. clcn2a and clcn2b resulted from the teleost-specific whole genome duplication, while clcn2c arose from a gene duplication from clcn2b. The expression patterns in adult tissues and embryos of zebrafish clcn2 paralogs support their subfunctionalization after the duplications, with clcn2a being enriched in excitable tissues and clcn2c in ionocytes. All three zebrafish clc-2 proteins interact with human GLIALCAM, that is able to target them to cell junctions, as it does with mammalian ClC-2. We could detect clc-2a and clc-2b inward rectified chloride currents with different voltage-dependence and kinetics in Xenopus oocytes, while clc-2c remained inactive. Interestingly, GlialCAM proteins did not modify clc-2b inward rectification. Then, our work extends the repertoire of ClC-2 proteins and provides new tools for structure-function and physiology studies.

  13. The ClC-3 chloride channel associated with microtubules is a target of paclitaxel in its induced-apoptosis.

    PubMed

    Zhang, Haifeng; Li, Huarong; Yang, Lili; Deng, Zhiqin; Luo, Hai; Ye, Dong; Bai, Zhiquan; Zhu, Linyan; Ye, Wencai; Wang, Liwei; Chen, Lixin

    2013-01-01

    Recent evidences show that cationic fluxes play a pivotal role in cell apoptosis. In this study, the roles of Cl(-) channels in paclitaxel-induced apoptosis were investigated in nasopharyngeal carcinoma CNE-2Z cells. Chloride current and apoptosis were induced by paclitaxel and inhibited by chloride channel blockers. Paclitaxel-activated current possessed similar properties to volume-activated chloride current. After ClC-3 was knocked-down by ClC-3-siRNA, hypotonicity-activated and paclitaxel-induced chloride currents were obviously decreased, indicating that the chloride channel involved in paclitaxel-induced apoptosis may be ClC-3. In early apoptotic cells, ClC-3 was up-regulated significantly; over-expressed ClC-3 was accumulated in cell membrane to form intercrossed filaments, which were co-localized with α-tubulins; changes of ultrastructures and decrease of flexibility in cell membrane were detected by atomic force microscopy. These suggest that ClC-3 is a critical target of paclitaxel and the involvement of ClC-3 in apoptosis may be associated with its accumulation with membrane microtubules and its over activation.

  14. Mutations at the signature sequence of CFTR create a Cd(2+)-gated chloride channel.

    PubMed

    Wang, Xiaohui; Bompadre, Silvia G; Li, Min; Hwang, Tzyh-Chang

    2009-01-01

    The canonical sequence LSGGQ, also known as the signature sequence, defines the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail configuration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fibrosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fibrosis. We previously showed that the G551D mutation completely eliminates ATP-dependent gating of the CFTR chloride channel. Here, we report that micromolar [Cd(2+)] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd(2+) is not seen in wild-type channels or in G551A. Pretreatment of G551D-CFTR with the cysteine modification reagent 2-aminoethyl methane thiosulfonate hydrobromide protects the channel from Cd(2+) activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd(2+). The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR's NBD1, show robust response to Cd(2+). On the other hand, negligible effects of Cd(2+) were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd(2+) binding to the gate. Collectively, these results suggest that the effect of Cd(2+) is mediated by a metal bridge formation between yet to be identified cysteine residue(s) and the engineered aspartate or cysteine in the signature sequence. We propose that the signature sequence serves as a switch that transduces the signal of ligand binding to the channel gate.

  15. Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating.

    PubMed

    Wang, Wuyang; Linsdell, Paul

    2012-03-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are controlled by ATP binding and hydrolysis by its nucleotide binding domains (NBDs). This is presumed to control opening of a single "gate" within the permeation pathway, however, the location of such a gate has not been described. We used patch clamp recording to monitor access of cytosolic cysteine reactive reagents to cysteines introduced into different transmembrane (TM) regions in a cysteine-less form of CFTR. The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)(2)(-) ions was reduced when ATP concentration was reduced from 1mM to 10μM, and modification by MTSES was accelerated when 2mM pyrophosphate was applied to prevent channel closure. Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). The rate of modification of Q98C and I344C by both MTSES and Au(CN)(2)(-) was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. These results suggest that access from the cytoplasm to K95 and V345 is similar in open and closed channels. In contrast, modifying ATP-dependent channel gating alters access to Q98 and I344, located further into the pore. We propose that ATP-dependent gating of CFTR is associated with the opening and closing of a gate within the permeation pathway at the level of these pore-lining amino acids.

  16. Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung.

    PubMed

    Gandhi, R; Elble, R C; Gruber, A D; Schreur, K D; Ji, H L; Fuller, C M; Pauli, B U

    1998-11-27

    A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32-38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32-38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32-38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and by in situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl- current that could be activated by Ca2+ and ionomycin and inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.

  17. Novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids as calcium activated chloride channel inhibitors

    PubMed Central

    Kumar, Satish; Namkung, Wan; Verkman, A. S.; Sharma, Pawan K.

    2013-01-01

    Transmembrane protein 16A (TMEM16A) channels are recently discovered membrane proteins that functions as a calcium activated chloride channel (CaCC). CaCCs are major regulators of various physiological processes, such as sensory transduction, epithelial secretion, smooth muscle contraction and oocyte fertilization. Thirty novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids (B01–B30) were synthesized and evaluated for their TMEM16A inhibitory activity by using short circuit current measurements in Fischer rat thyroid (FRT) cells expressing human TMEM16A. IC50 values were calculated using YFP fluorescence plate reader assay. Final compounds, having free carboxylic group displayed significant inhibition. Eight of the novel compounds B02, B13, B21, B23, B25, B27, B28, B29 exhibit excellent CaCCs inhibition with IC50 value <6 μM, with compound B25 exhibiting the lowest IC50 value of 2.8 ± 1.3 μM. None of the tested ester analogs of final benzofuran derivatives displayed TMEM16A/CaCCs inhibition. PMID:22739085

  18. Role of chloride channels in bradykinin-induced guinea pig airway vagal C-fibre activation.

    PubMed

    Lee, Min-Goo; Macglashan, Donald W; Undem, Bradley J

    2005-07-01

    We tested the hypothesis that an ionic current carried by chloride ions contributes to bradykinin (BK)-induced membrane depolarization and activation of vagal afferent C-fibres. In an ex vivo innervated trachea/bronchus preparation, BK (1 microM) consistently produced action potential discharge in vagal afferent C-fibres with receptive fields in the trachea or main stem bronchus. The Ca2+-activated Cl- channel (CLCA) inhibitor, niflumic acid (NFA, 100 microM), significantly reduced BK-induced action potential discharge to 21 +/- 7% of the control BK response. NFA did not inhibit capsaicin-induced or citric-acid-induced action potential discharge in tracheal C-fibres. The inhibitory effect of NFA was mimicked by another CLCA inhibitor, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 100 microM). NFA also inhibited the BK-induced inward current in gramicidin-perforated whole-cell patch-clamp recordings of capsaicin-sensitive jugular ganglion neurones retrogradely labelled from the airways. NFA did not inhibit the BK-induced increase in intracellular free Ca2+. The TRPV1 inhibitor, iodo-resiniferatoxin (1 microM), also partially inhibited BK-induced action potential discharge, and the combination of iodo-resiniferatoxin and NFA virtually abolished the BK-induced action potential discharge. We concluded that in vagal afferent C-fibres, BK evokes membrane depolarization and action potential discharge through the additive effects of TRPV1 and Cl- channel activation.

  19. Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction

    PubMed Central

    Huang, Fen; Zhang, Hongkang; Wu, Meng; Yang, Huanghe; Kudo, Makoto; Peters, Christian J.; Woodruff, Prescott G.; Solberg, Owen D.; Donne, Matthew L.; Huang, Xiaozhu; Sheppard, Dean; Fahy, John V.; Wolters, Paul J.; Hogan, Brigid L. M.; Finkbeiner, Walter E.; Li, Min; Jan, Yuh-Nung; Jan, Lily Yeh; Rock, Jason R.

    2012-01-01

    Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calcium-activated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms. PMID:22988107

  20. Prolactin stimulates sodium and chloride ion channels in A6 renal epithelial cells

    PubMed Central

    Greenlee, Megan M.; Mitzelfelt, Jeremiah D.; Duke, Billie Jeanne; Al-Khalili, Otor; Bao, Hui-Fang

    2015-01-01

    Many hormonal pathways contribute to the regulation of renal epithelial sodium channel (ENaC) function, a key process for maintaining blood volume and controlling blood pressure. In the present study, we examined whether the peptide hormone prolactin (PRL) regulates ENaC function in renal epithelial cells (A6). Basolateral application of several different concentrations of PRL dramatically stimulated the transepithelial current in A6 cells, increasing both amiloride-sensitive (ENaC) and amiloride-insensitive currents. Using cell-attached patch clamp, we determined that PRL increased both the number (N) and open probability (Po) of ENaC present in the apical membrane. Inhibition of PKA with H-89 abolished the effect of PRL on amiloride-sensitive and insensitive transepithelial currents and eliminated the increase in ENaC NPo with PRL exposure. PRL also increased cAMP in A6 cells, consistent with signaling through the cAMP-dependent PKA pathway. We also identified that PRL induced activity of a 2-pS anion channel with outward rectification, electrophysiological properties consistent with ClC4 or ClC5. RT-PCR only detected ClC4, but not ClC5 transcripts. Here, we show for the first time that PRL activates sodium and chloride transport in renal epithelial cells via ENaC and ClC4. PMID:25587116

  1. Identification of a dimerization domain in the TMEM16A calcium-activated chloride channel (CaCC)

    PubMed Central

    Tien, Jason; Lee, Hye Young; Minor, Daniel L.; Jan, Yuh Nung; Jan, Lily Yeh

    2013-01-01

    Transmembrane proteins with unknown function 16 (TMEM16A) is a calcium-activated chloride channel (CaCC) important for neuronal, exocrine, and smooth muscle functions. TMEM16A belongs to a family of integral membrane proteins that includes another CaCC, TMEM16B, responsible for controlling action potential waveform and synaptic efficacy, and a small-conductance calcium-activated nonselective cation channel, TMEM16F, linked to Scott syndrome. We find that these channels in the TMEM16 family share a homodimeric architecture facilitated by their cytoplasmic N termini. This dimerization domain is important for channel assembly in eukaryotic cells, and the in vitro association of peptides containing the dimerization domain is consistent with a homotypic protein–protein interaction. Amino acid substitutions in the dimerization domain affect functional TMEM16A-CaCC channel expression, as expected from its critical role in channel subunit assembly. PMID:23576756

  2. [Identification of a Novel Calcium (Ca^(2+))-Activated Chloride Channel Accessory Gene in Xenopus laevis].

    PubMed

    Lee, R M; Jeong, S M

    2016-01-01

    Calcium (Ca^(2+))-activated chloride channel accessories (CLCAs) are putative anion channel-related proteins with diverse physiological functions. Exploring CLCA diversity is important for prediction of gene structure and function. In an effort to identify novel CLCA genes in Xenopus laevis, we successfully cloned and characterized a Xenopus laevis cDNA predicted to encode the xCLCA3 gene. Cloning of xCLCA3 was achieved by computational analysis, rapid amplification of cDNA ends (RACE), and a tissue distribution analysis by semi-quantitative reverse transcription (RT) PCR or real-time PCR. We obtained a 2958 bp xCLCA3 cDNA sequence with an open reading frame encoding 943 amino acids. According to the primary structure analysis, xCLCA3 contains a predicted signal sequence, multiple sites of N-linked (N-) glycosylation, N-myristoylation, PKA, PKC, and casein kinase II phosphorylation sites, five putative hydrophobic segments, and the HExxH metalloprotease motif. Additionally, the transmembrane prediction server yielded a preserved N-terminal CLCA domain and a von Willebrand factor type A domain with one transmembrane domain in the C-terminal region. Expression analysis showed that xCLCA3 is expressed in a number of tissues, with strong expression in the brain, colon, small intestine, lung, kidney, and spleen, and poor expression in the heart and liver. These results suggest that xCLCA3 may be a candidate CLCA family member as well as a metalloprotease, rather than just an ion channel accessory protein.

  3. Fluid shear stress enhances the cell volume decrease of osteoblast cells by increasing the expression of the ClC-3 chloride channel

    PubMed Central

    LIU, LI; CAI, SIYI; QIU, GUIXING; LIN, JIN

    2016-01-01

    ClC-3 is a volume-sensitive chloride channel that is responsible for cell volume adjustment and regulatory cell volume decrease (RVD). In order to evaluate the effects of fluid shear stress (FSS) stimulation on the osteoblast ClC-3 chloride channel, MC3T3-E1 cells were stimulated by FSS in the experimental group. Fluorescence quantitative polymerase chain reaction was used to detect changes in ClC-3 mRNA expression, the chloride ion fluorescent probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) was used to detect the chloride channel activity, and whole-cell patch clamping was used to monitor the changes in the volume-sensitive chloride current activated by a hypotonic environment following mechanical stimulation. The results show that the expression of the osteoblast chloride channel ClC-3 was significantly higher in the FSS group compared with the control group. MQAE fluorescence intensity was significantly reduced in the FSS group compared to the control group, suggesting that mechanical stimulation increased chloride channel activity and increased the efflux of intracellular chloride ions. Image analysis of osteoblast volume changes showed that osteoblast RVD was enhanced by mechanical stimulation. Whole-cell patch clamping showed that the osteoblast volume-sensitive chloride current was larger in the stimulated group compared to the control group, suggesting that elevated ClC-3 chloride channel expression results in an increased volume-sensitive chloride current. In conclusion, FSS stimulation enhances the RVD of osteoblast cell by increasing the expression of the ClC-3 and enhancing the chloride channel activity. PMID:27073622

  4. Fluid shear stress enhances the cell volume decrease of osteoblast cells by increasing the expression of the ClC-3 chloride channel.

    PubMed

    Liu, L I; Cai, Siyi; Qiu, Guixing; Lin, Jin

    2016-04-01

    ClC-3 is a volume-sensitive chloride channel that is responsible for cell volume adjustment and regulatory cell volume decrease (RVD). In order to evaluate the effects of fluid shear stress (FSS) stimulation on the osteoblast ClC-3 chloride channel, MC3T3-E1 cells were stimulated by FSS in the experimental group. Fluorescence quantitative polymerase chain reaction was used to detect changes in ClC-3 mRNA expression, the chloride ion fluorescent probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) was used to detect the chloride channel activity, and whole-cell patch clamping was used to monitor the changes in the volume-sensitive chloride current activated by a hypotonic environment following mechanical stimulation. The results show that the expression of the osteoblast chloride channel ClC-3 was significantly higher in the FSS group compared with the control group. MQAE fluorescence intensity was significantly reduced in the FSS group compared to the control group, suggesting that mechanical stimulation increased chloride channel activity and increased the efflux of intracellular chloride ions. Image analysis of osteoblast volume changes showed that osteoblast RVD was enhanced by mechanical stimulation. Whole-cell patch clamping showed that the osteoblast volume-sensitive chloride current was larger in the stimulated group compared to the control group, suggesting that elevated ClC-3 chloride channel expression results in an increased volume-sensitive chloride current. In conclusion, FSS stimulation enhances the RVD of osteoblast cell by increasing the expression of the ClC-3 and enhancing the chloride channel activity.

  5. Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels

    PubMed Central

    Maertens, Chantal; Wei, Lin; Tytgat, Jan; Droogmans, Guy; Nilius, Bernd

    2000-01-01

    It was the aim of this study to look for a high-affinity and selective polypeptide toxin, which could serve as a probe for the volume-regulated anion channel (VRAC) or the calcium-activated chloride channel (CaCC). We have partially purified chlorotoxin, including new and homologous short chain insectotoxins, from the crude venom of Leiurus quinquestriatus quinquestriatus (Lqq) by means of gel filtration chromatography. Material eluting between 280 and 420 min, corresponding to fractions 15–21, was lyophilized and tested on VRAC and CaCC, using the whole-cell patch-clamp technique. We have also tested the commercially available chlorotoxin on VRAC, CaCC, the cystic fibrosis transmembrane conductance regulator (CFTR) and on the glioma specific chloride channel (GCC). VRAC and the correspondent current, ICl,swell, was activated in Cultured Pulmonary Artery Endothelial (CPAE) cells by a 25% hypotonic solution. Neither of the fractions 16–21 significantly inhibited ICl,swell (n=4–5). Ca2+-activated Cl− currents, ICl,Ca, activated by loading T84 cells via the patch pipette with 1 μM free Ca2+, were not inhibited by any of the tested fractions (15–21), (n=2–5). Chlorotoxin (625 nM) did neither effect ICl,swell nor ICl,Ca (n=4–5). The CFTR channel, transiently transfected in COS cells and activated by a cocktail containing IBMX and forskolin, was not affected by 1.2 μM chlorotoxin (n=5). In addition, it did not affect currents through GCC. We conclude that submicromolar concentrations of chlorotoxin do not block volume-regulated, Ca2+-activated and CFTR chloride channels and that it can not be classified as a general chloride channel toxin. PMID:10683204

  6. Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function.

    PubMed

    Concepcion, Axel R; Vaeth, Martin; Wagner, Larry E; Eckstein, Miriam; Hecht, Lee; Yang, Jun; Crottes, David; Seidl, Maximilian; Shin, Hyosup P; Weidinger, Carl; Cameron, Scott; Turvey, Stuart E; Issekutz, Thomas; Meyts, Isabelle; Lacruz, Rodrigo S; Cuk, Mario; Yule, David I; Feske, Stefan

    2016-11-01

    Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release-activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel-deficient patients and mice with ectodermal tissue-specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice.

  7. Identification and functional characterization of a voltage-gated chloride channel and its novel splice variant in taste bud cells.

    PubMed

    Huang, Liquan; Cao, Jie; Wang, Hong; Vo, Lynn A; Brand, Joseph G

    2005-10-28

    Taste bud cells are epithelial cells with neuronal properties. Voltage-dependent ion channels have been physiologically described in these cells. Here, we report the molecular identification and functional characterization of a voltage-gated chloride channel (ClC-4) and its novel splice variant (ClC-4A) from taste bud cells. ClC-4A skipped an exon near its 5'-end, incurring the loss of 60 amino acids at the N terminus. In situ hybridization and immunohistochemistry localized these two channels' transcripts and proteins to a subset of taste bud cells. Electrophysiological recordings of the heterologously expressed channels in Xenopus oocytes showed that ClC-4 and ClC-4A have opposite sensitivity to pH and unique ion selectivity. The chloride channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid had a slight or no inhibitory effect on the conductance of ClC-4, but both blockers inhibited ClC-4A, suggesting that ClC-4A is a candidate channel for an acid-induced 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive current. Furthermore, these two channels may play a role in bitter-, sweet-, and umami-mediated taste transmission by regulating transmitter uptake into synaptic vesicles.

  8. The ClC-K2 Chloride Channel Is Critical for Salt Handling in the Distal Nephron.

    PubMed

    Hennings, J Christopher; Andrini, Olga; Picard, Nicolas; Paulais, Marc; Huebner, Antje K; Cayuqueo, Irma Karen Lopez; Bignon, Yohan; Keck, Mathilde; Cornière, Nicolas; Böhm, David; Jentsch, Thomas J; Chambrey, Régine; Teulon, Jacques; Hübner, Christian A; Eladari, Dominique

    2017-01-01

    Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 gene family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause Bartter syndrome, a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule.

  9. Dissecting a regulatory calcium-binding site of CLC-K kidney chloride channels

    PubMed Central

    Gradogna, Antonella; Fenollar-Ferrer, Cristina

    2012-01-01

    The kidney and inner ear CLC-K chloride channels, which are involved in salt absorption and endolymph production, are regulated by extracellular Ca2+ in the millimolar concentration range. Recently, Gradogna et al. (2010. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201010455) identified a pair of acidic residues (E261 and D278) located in the loop between helices I and J as forming a putative intersubunit Ca2+-binding site in hClC-Ka. In this study, we sought to explore the properties of the binding site in more detail. First, we verified that the site is conserved in hClC-Kb and rClC-K1. In addition, we could confer Ca2+ sensitivity to the Torpedo marmorata ClC-0 channel by exchanging its I–J loop with that from ClC-Ka, demonstrating a direct role of the loop in Ca2+ binding. Based on a structure of a bacterial CLC and a new sequence alignment, we built homology models of ClC-Ka. The models suggested additional amino acids involved in Ca2+ binding. Testing mutants of these residues, we could restrict the range of plausible models and positively identify two more residues (E259 and E281) involved in Ca2+ coordination. To investigate cation specificity, we applied extracellular Zn2+, Mg2+, Ba2+, Sr2+, and Mn2+. Zn2+ blocks ClC-Ka as well as its Ca2+-insensitive mutant, suggesting that Zn2+ binds to a different site. Mg2+ does not activate CLC-Ks, but the channels are activated by Ba2+, Sr2+, and Mn2+ with a rank order of potency of Ca2+ > Ba2+ > Sr2+ = Mn2+ for the human CLC-Ks. Dose–response analysis indicates that the less potent Ba2+ has a lower affinity rather than a lower efficacy. Interestingly, rClC-K1 shows an altered rank order (Ca2+ > Sr2+ >> Ba2+), but homology models suggest that residues outside the I–J loop are responsible for this difference. Our detailed characterization of the regulatory Ca2+-binding site provides a solid basis for the understanding of the physiological modulation of CLC-K channel function in the kidney and inner ear. PMID

  10. Calcium-activated chloride channels in bovine pulmonary artery endothelial cells.

    PubMed Central

    Nilius, B; Prenen, J; Szücs, G; Wei, L; Tanzi, F; Voets, T; Droogmans, G

    1997-01-01

    1. We characterized Ca(2+)-activated Cl- currents in calf pulmonary artery endothelial (CPAE) cells by using a combined patch clamp and fura-2 microfluorescence technique to simultaneously measure ionic currents and the intracellular Ca2+ concentration, [Ca2+]i. 2. Various procedures that increased [Ca2+]i, such as stimulation with ATP or ionomycin, or loading the cells with Ca2+ via the patch pipette, activated a strongly outwardly rectifying current with a reversal potential close to the Cl- equilibrium potential. Changing the extracellular Cl- concentration shifted this reversal potential as predicted for a Cl- current. Buffering Ca2+ rises with BAPTA prevented ATP from activating the current. 3. Ca(2+)-activated Cl- currents could be distinguished from volume-activated Cl- currents, which were sometimes coactivated in the same cell. The latter showed much less outward rectification, their activation was voltage independent, and they could be inhibited by exposing the cells to hypertonic solutions. 4. The permeability ratio for the Ca(2+)-activated conductance of the anions iodide:chloride: gluconate was 1.71 +/- 0.06:1:0.39 +/- 0.03 (n = 12). 5. This Ca(2+)-activated Cl- current, ICl, Ca, inactivated rapidly at negative potentials and activated slowly at positive potentials. Outward tail currents were slowly decaying, while inward tail currents decayed much faster. 6. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic-acid (DIDS) and niflumic acid inhibited Icl,Ca in a voltage-dependent manner, i.e. they exerted a more potent block at positive potentials. The block by N-phenylanthracilic acid (NPA), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and tamoxifen was voltage independent. Niflumic acid and tamoxifen were the most potent blockers. 7. The single-channel conductance was 7.9 +/- 0.7 pS (n = 15) at 300 mM extracellular Cl-. The channel open probability was high at positive potentials, but very small at negative potentials. 8. It is concluded that [Ca2+]i

  11. Dissecting a regulatory calcium-binding site of CLC-K kidney chloride channels.

    PubMed

    Gradogna, Antonella; Fenollar-Ferrer, Cristina; Forrest, Lucy R; Pusch, Michael

    2012-12-01

    The kidney and inner ear CLC-K chloride channels, which are involved in salt absorption and endolymph production, are regulated by extracellular Ca(2+) in the millimolar concentration range. Recently, Gradogna et al. (2010. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201010455) identified a pair of acidic residues (E261 and D278) located in the loop between helices I and J as forming a putative intersubunit Ca(2+)-binding site in hClC-Ka. In this study, we sought to explore the properties of the binding site in more detail. First, we verified that the site is conserved in hClC-Kb and rClC-K1. In addition, we could confer Ca(2+) sensitivity to the Torpedo marmorata ClC-0 channel by exchanging its I-J loop with that from ClC-Ka, demonstrating a direct role of the loop in Ca(2+) binding. Based on a structure of a bacterial CLC and a new sequence alignment, we built homology models of ClC-Ka. The models suggested additional amino acids involved in Ca(2+) binding. Testing mutants of these residues, we could restrict the range of plausible models and positively identify two more residues (E259 and E281) involved in Ca(2+) coordination. To investigate cation specificity, we applied extracellular Zn(2+), Mg(2+), Ba(2+), Sr(2+), and Mn(2+). Zn(2+) blocks ClC-Ka as well as its Ca(2+)-insensitive mutant, suggesting that Zn(2+) binds to a different site. Mg(2+) does not activate CLC-Ks, but the channels are activated by Ba(2+), Sr(2+), and Mn(2+) with a rank order of potency of Ca(2+) > Ba(2+) > Sr(2+) = Mn(2+) for the human CLC-Ks. Dose-response analysis indicates that the less potent Ba(2+) has a lower affinity rather than a lower efficacy. Interestingly, rClC-K1 shows an altered rank order (Ca(2+) > Sr(2+) > Ba(2+)), but homology models suggest that residues outside the I-J loop are responsible for this difference. Our detailed characterization of the regulatory Ca(2+)-binding site provides a solid basis for the understanding of the physiological modulation of CLC

  12. Volume-sensitive outwardly rectifying chloride channel blockers protect against high glucose-induced apoptosis of cardiomyocytes via autophagy activation.

    PubMed

    Wang, Lin; Shen, Mingzhi; Guo, Xiaowang; Wang, Bo; Xia, Yuesheng; Wang, Ning; Zhang, Qian; Jia, Lintao; Wang, Xiaoming

    2017-03-16

    Hyperglycemia is a well-characterized contributing factor for cardiac dysfunction and heart failure among diabetic patients. Apoptosis of cardiomyocytes plays a major role during the onset and pathogenesis of diabetic cardiomyopathy (DCM). Nonetheless, the molecular machinery underlying hyperglycemia-induced cardiac damage and cell death remains elusive. In the present study, we found that chloride channel blockers, 4,4'-diisothiocya-natostilbene-2,2'- disulfonic acid (DIDS) and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), inhibited high glucose-activated volume-sensitive outwardly rectifying (VSOR) Cl(-) channel and improved the viability of cardiomyocytes. High glucose induced cardiomyocyte apoptosis by suppressing the autophagic stress, which can be reversed via blockade of VSOR Cl(-) channel. VSOR activation in high glucose-treated cardiomyocytes was attributed to increased intracellular levels of reactive oxygen species (ROS). Taken together, our study unraveled a role of VSOR chloride currents in impaired autophagy and increased apoptosis of high glucose-exposed cardiomyocyte, and has implications for a therapeutic potential of VSOR chloride channel blockers in DCM.

  13. Inhibition of the voltage-dependent chloride channel of Torpedo electric organ by diisopropylfluorophosphate and its reversal by oximes

    SciTech Connect

    Abalis, I.M.; Chiang, P.K.; Wirtz, R.A.; Andre, R.G.

    1986-05-01

    Diisopropylfluorophosphate (DFP), a potent organophosphate inhibitor of cholinesterases, was found to inhibit the specific binding of (/sup 35/S)t-butylbicyclophosphorothionate (TBPS), specific chloride channels ligand, to the electric organ membranes of Torpedo, with a Ki of 21 +/- 3 ..mu..M. The binding sites of (/sup 35/S)TBPS in the Torpedo membranes were found not to be GABA receptors or nicotinic acetylcholine receptors as previously described. Interestingly, a stimulation of the binding of (/sup 35/S)TBPS was observed in the presence of atropine and three oximes, monopyridinium oxime 2-PAM, bispyridinium bis-oxime TMB-4 and H-oxime HI-6. The maximal stimulation was 300-500% of control, after which, the stimulation was reversed at higher concentrations. The three oximes protected by more than 95% the inhibition by 1 mM DFP of the binding of (/sup 35/S)TBPS to the voltage-dependent chloride channel. However, atropine protected only 20% of the inhibited channel. These results, thus, suggest that the protection against the toxic effects of DFP or other anticholinesterase agents by the tested oximes may not be solely a result of the reactivation of cholinesterases but also the protection of the voltage-dependent chloride channel.

  14. Volume-sensitive outwardly rectifying chloride channel blockers protect against high glucose-induced apoptosis of cardiomyocytes via autophagy activation

    PubMed Central

    Wang, Lin; Shen, Mingzhi; Guo, Xiaowang; Wang, Bo; Xia, Yuesheng; Wang, Ning; Zhang, Qian; Jia, Lintao; Wang, Xiaoming

    2017-01-01

    Hyperglycemia is a well-characterized contributing factor for cardiac dysfunction and heart failure among diabetic patients. Apoptosis of cardiomyocytes plays a major role during the onset and pathogenesis of diabetic cardiomyopathy (DCM). Nonetheless, the molecular machinery underlying hyperglycemia-induced cardiac damage and cell death remains elusive. In the present study, we found that chloride channel blockers, 4,4′-diisothiocya-natostilbene-2,2′- disulfonic acid (DIDS) and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), inhibited high glucose-activated volume-sensitive outwardly rectifying (VSOR) Cl− channel and improved the viability of cardiomyocytes. High glucose induced cardiomyocyte apoptosis by suppressing the autophagic stress, which can be reversed via blockade of VSOR Cl− channel. VSOR activation in high glucose-treated cardiomyocytes was attributed to increased intracellular levels of reactive oxygen species (ROS). Taken together, our study unraveled a role of VSOR chloride currents in impaired autophagy and increased apoptosis of high glucose-exposed cardiomyocyte, and has implications for a therapeutic potential of VSOR chloride channel blockers in DCM. PMID:28300155

  15. Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function

    PubMed Central

    Concepcion, Axel R.; Vaeth, Martin; Wagner, Larry E.; Eckstein, Miriam; Hecht, Lee; Yang, Jun; Crottes, David; Seidl, Maximilian; Shin, Hyosup P.; Weidinger, Carl; Cameron, Scott; Turvey, Stuart E.; Issekutz, Thomas; Meyts, Isabelle; Lacruz, Rodrigo S.; Cuk, Mario; Yule, David I.

    2016-01-01

    Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release–activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel–deficient patients and mice with ectodermal tissue–specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice. PMID:27721237

  16. P2Y purinergic receptor regulation of CFTR chloride channels in mouse cardiac myocytes.

    PubMed

    Yamamoto-Mizuma, Shintaro; Wang, Ge-Xin; Hume, Joseph R

    2004-05-01

    The intracellular signalling pathways and molecular mechanisms responsible for P2-purinoceptor-mediated chloride (Cl(-)) currents (I(Cl,ATP)) were studied in mouse ventricular myocytes. In standard NaCl-containing extracellular solutions, extracellular ATP (100 microm) activated two different currents, I(Cl,ATP) with a linear I-V relationship in symmetrical Cl(-) solutions, and an inwardly rectifying cation conductance (cationic I(ATP)). Cationic I(ATP) was selectively inhibited by Gd(3+) and Zn(2+), or by replacement of extracellular NaCl by NMDG; I(Cl,ATP) was Cl(-) selective, and inhibited by replacement of extracellular Cl(-) by Asp(-); both currents were prevented by suramin or DIDS pretreatment. In GTPgammaS-loaded cells, I(Cl,ATP) was irreversibly activated by ATP, but cationic I(ATP) was still regulated reversibly. GDPbetaS prevented activation of the I(Cl,ATP,) even though pertussis toxin pretreatment did not modulate I(Cl,ATP). These results suggest that activation of I(Cl,ATP) occurs via a G-protein coupled P2Y purinergic receptor. The I(Cl,ATP) persistently activated by GTPgammaS, was inhibited by glibenclamide but not by DIDS, thus exhibiting known pharmacological properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. In ventricular cells of cftr(-/-) mice, extracellular ATP activated cationic I(ATP), but failed to activate any detectable I(Cl,ATP). These results provide compelling evidence that activation of CFTR Cl(-) channels in mouse heart are coupled to G-protein coupled P2Y purinergic receptors.

  17. Characterization of the target of ivermectin, the glutamate-gated chloride channel, from Anopheles gambiae

    PubMed Central

    Meyers, Jacob I.; Gray, Meg; Kuklinski, Wojtek; Johnson, Lucas B.; Snow, Christopher D.; Black, William C.; Partin, Kathryn M.; Foy, Brian D.

    2015-01-01

    ABSTRACT The use of insecticide-treated nets and indoor residual insecticides targeting adult mosquito vectors is a key element in malaria control programs. However, mosquito resistance to the insecticides used in these applications threatens malaria control efforts. Recently, the mass drug administration of ivermectin (IVM) has been shown to kill Anopheles gambiae mosquitoes and disrupt Plasmodium falciparum transmission in the field. We cloned the molecular target of IVM from A. gambiae, the glutamate-gated chloride channel (AgGluCl), and characterized its transcriptional patterns, protein expression and functional responses to glutamate and IVM. AgGluCl cloning revealed an unpredicted fourth splice isoform as well as a novel exon and splice site. The predicted gene products contained heterogeneity in the N-terminal extracellular domain and the intracellular loop region. Responses to glutamate and IVM were measured using two-electrode voltage clamp on Xenopus laevis oocytes expressing AgGluCl. IVM induced non-persistent currents in AgGluCl-a1 and did not potentiate glutamate responses. In contrast, AgGluCl-b was insensitive to IVM, suggesting that the AgGluCl gene could produce IVM-sensitive and -insensitive homomultimers from alternative splicing. AgGluCl isoform-specific transcripts were measured across tissues, ages, blood feeding status and sex, and were found to be differentially transcribed across these physiological variables. Lastly, we stained adult, female A. gambiae for GluCl expression. The channel was expressed in the antenna, Johnston's organ, supraesophageal ganglion and thoracic ganglia. In summary, we have characterized the first GluCl from a mosquito, A. gambiae, and described its unique activity and expression with respect to it as the target of the insecticide IVM. PMID:25994631

  18. Characterization of the target of ivermectin, the glutamate-gated chloride channel, from Anopheles gambiae.

    PubMed

    Meyers, Jacob I; Gray, Meg; Kuklinski, Wojtek; Johnson, Lucas B; Snow, Christopher D; Black, William C; Partin, Kathryn M; Foy, Brian D

    2015-05-15

    The use of insecticide-treated nets and indoor residual insecticides targeting adult mosquito vectors is a key element in malaria control programs. However, mosquito resistance to the insecticides used in these applications threatens malaria control efforts. Recently, the mass drug administration of ivermectin (IVM) has been shown to kill Anopheles gambiae mosquitoes and disrupt Plasmodium falciparum transmission in the field. We cloned the molecular target of IVM from A. gambiae, the glutamate-gated chloride channel (AgGluCl), and characterized its transcriptional patterns, protein expression and functional responses to glutamate and IVM. AgGluCl cloning revealed an unpredicted fourth splice isoform as well as a novel exon and splice site. The predicted gene products contained heterogeneity in the N-terminal extracellular domain and the intracellular loop region. Responses to glutamate and IVM were measured using two-electrode voltage clamp on Xenopus laevis oocytes expressing AgGluCl. IVM induced non-persistent currents in AgGluCl-a1 and did not potentiate glutamate responses. In contrast, AgGluCl-b was insensitive to IVM, suggesting that the AgGluCl gene could produce IVM-sensitive and -insensitive homomultimers from alternative splicing. AgGluCl isoform-specific transcripts were measured across tissues, ages, blood feeding status and sex, and were found to be differentially transcribed across these physiological variables. Lastly, we stained adult, female A. gambiae for GluCl expression. The channel was expressed in the antenna, Johnston's organ, supraesophageal ganglion and thoracic ganglia. In summary, we have characterized the first GluCl from a mosquito, A. gambiae, and described its unique activity and expression with respect to it as the target of the insecticide IVM.

  19. Expression of CLC-K chloride channels in the rat cochlea.

    PubMed

    Qu, Chunyan; Liang, Fenghe; Hu, Wei; Shen, Zhijun; Spicer, Samuel S; Schulte, Bradley A

    2006-03-01

    Current models of the lateral K+ recycling pathway in the mammalian cochlea include two multicellular transport networks separated from one another by three interstitial gaps. The first gap is between outer hair cells and Deiters cells, the second is between outer sulcus cells and type II spiral ligament fibrocytes and the third is between intermediate and marginal cells in the stria vascularis. K+ taken up by cells bordering these interstitial spaces is accompanied by Cl-. Maintaining appropriate electrolyte balance and membrane potentials in these cells requires a mechanism for exit of the resorbed Cl-. One possible candidate for regulating this Cl- efflux is ClC-K, a chloride channel previously thought to be kidney specific. Here, we demonstrate the expression of both known isoforms of ClC-K in the organ of Corti, spiral ligament and stria vascularis of the rat cochlea by immunohistochemical, Western blot and RT-PCR analysis. These results indicate a role for ClC-K in mediating Cl- recycling in the cochlea. The widespread expression of both ClC-K isoforms in the cochlea may help to explain the symptoms of Bartter's syndrome Type III, a mutation in the hClC-Kb gene (human homologue of ClC-K2), which results in renal salt wasting without deafness. These data support the hypothesis that both isoforms of ClC-K are co-expressed in some cell membranes and account for the preservation of hearing in the presence of a mutation in only one channel isoform.

  20. MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1.

    PubMed

    Kino, Yoshihiro; Washizu, Chika; Oma, Yoko; Onishi, Hayato; Nezu, Yuriko; Sasagawa, Noboru; Nukina, Nobuyuki; Ishiura, Shoichi

    2009-10-01

    The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families--muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins--are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5' end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.

  1. Myotonia congenita-associated mutations in chloride channel-1 affect zebrafish body wave swimming kinematics.

    PubMed

    Cheng, Wei; Tian, Jing; Burgunder, Jean-Marc; Hunziker, Walter; Eng, How-Lung

    2014-01-01

    Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.

  2. Effect of chloride channel activity on retinal pigment cell proliferation and migration.

    PubMed

    Zhao, Jing; Zhong, Wei; Sun, Lixia; Yin, Yuan; Zheng, Yajuan

    2017-04-01

    The present study aimed to investigate the effects of chloride channels (ClC) on the proliferation and migration of retinal pigment epithelial (RPE) cells, a primary component of proliferative vitreoretinopathy (PVR) membranes. An RPE cell model of phagocytosis was established using fibronectin‑coated latex beads. Cell proliferation was measured by live cell counting. The cell cycle and phagocytosis index was assessed by flow cytometry. Intracellular calcium concentration was quantified using Fura‑2‑acetoxymethyl ester. ClCs were blocked using 5‑nitro‑2‑(3‑phenylpropylamino) benzoic acid (NPPB) and tamoxifen (TAM). NPPB and TAM were identified to inhibit the proliferation of ARPE‑19 human adult RPE cells by arresting them in the G0/G1 phase, inhibit the phagocytosis of fibronectin, and decrease intracellular calcium levels, in a dose‑dependent manner. ClCs serve important roles in mediating human RPE cell proliferation and migration. The underlying mechanisms of action of ClCs are associated with the regulation of calcium. Targeting ClCs may provide a novel strategy to inhibit PVR formation.

  3. Protective effect of a calcium channel blocker "diltiazem" on aluminum chloride-induced dementia in mice.

    PubMed

    Rani, Anu; Neha; Sodhi, Rupinder K; Kaur, Amanpreet

    2015-11-01

    Many studies report that heavy metals such as aluminum are involved in amyloid beta aggregation and neurotoxicity. Further, high concentration of aluminum in the brain deregulates calcium signaling which contributes to synaptic dysfunction and halts neuronal communication which ultimately leads to the development of Alzheimer's disease. Recently, diltiazem, a calcium channel blocker clinically used in angina, is reported to decrease amyloid beta production by inhibiting calcium influx, decreasing inflammation and oxidative stress. However, the probable role of this drug in aluminum chloride (AlCl3)-induced experimental dementia is yet to be explored. Therefore, the present study is designed to investigate the effect of AlCl3-induced dementia in mice. Morris water maze test and elevated plus maze were utilized to evaluate learning and memory. Various biochemical estimations including brain acetylcholinesterase activity (AChE), brain total protein, thiobarbituric acid-reactive species (TBARS) level, reduced glutathione (GSH) level, nitrate/nitrite, and superoxide dismutase (SOD) were measured. AlCl3 significantly impaired learning and memory and increased brain AChE, brain total protein, TBARS, and nitrate/nitrite and decreased brain GSH or SOD. On the other hand, treatment with diltiazem significantly reversed AlCl3-induced behavioral and biochemical deficits. The present study indicates the beneficial role of diltiazem in AlCl3-induced dementia.

  4. Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

    PubMed Central

    Degani-Katzav, Nurit; Gortler, Revital; Gorodetzki, Lilach; Paas, Yoav

    2016-01-01

    The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and β subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds. By electrophysiological characterization of these mutants, we found heteromeric assemblies with two equivalent Glu-binding sites at β/α intersubunit interfaces, where the GluClβ and GluClα subunits, respectively, contribute the “principal” and “complementary” components of the putative Glu-binding pockets. We identified a mutation in the IVM-binding site (far away from the Glu-binding sites), which significantly increased the sensitivity of the heteromeric mutant receptor to both Glu and IVM, and improved the receptor subunits’ cooperativity. We further characterized this heteromeric GluClR mutant as a receptor having a third Glu-binding site at an α/α intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three α and two β subunits arranged in a counterclockwise β-α-β-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces. PMID:26792524

  5. Inducible and titratable silencing of Caenorhabditis elegans neurons in vivo with histamine-gated chloride channels.

    PubMed

    Pokala, Navin; Liu, Qiang; Gordus, Andrew; Bargmann, Cornelia I

    2014-02-18

    Recent progress in neuroscience has been facilitated by tools for neuronal activation and inactivation that are orthogonal to endogenous signaling systems. We describe here a chemical-genetic approach for inducible silencing of Caenorhabditis elegans neurons in intact animals, using the histamine-gated chloride channel HisCl1 from Drosophila and exogenous histamine. Administering histamine to freely moving C. elegans that express HisCl1 transgenes in neurons leads to rapid and potent inhibition of neural activity within minutes, as assessed by behavior, functional calcium imaging, and electrophysiology of neurons expressing HisCl1. C. elegans does not use histamine as an endogenous neurotransmitter, and exogenous histamine has little apparent effect on wild-type C. elegans behavior. HisCl1-histamine silencing of sensory neurons, interneurons, and motor neurons leads to behavioral effects matching their known functions. In addition, the HisCl1-histamine system can be used to titrate the level of neural activity, revealing quantitative relationships between neural activity and behavioral output. We use these methods to dissect escape circuits, define interneurons that regulate locomotion speed (AVA, AIB) and escape-related omega turns (AIB), and demonstrate graded control of reversal length by AVA interneurons and DA/VA motor neurons. The histamine-HisCl1 system is effective, robust, compatible with standard behavioral assays, and easily combined with optogenetic tools, properties that should make it a useful addition to C. elegans neurotechnology.

  6. Species dependent dual modulation of the benzodiazepine/GABA receptor chloride channel by dihydroergosine

    SciTech Connect

    Pericic, D.; Tvrdeic, A. )

    1990-01-01

    Dihydroergosine enhanced the incidence of bicuculline induced convulsions in female rats, while 100 mg/kg of dihydroergosine given to female mice made 45% convulsive dose of bicuculline to be subconvulsive. The same dose of dihydroergosine enhanced in mice the latency of bicuculline-induced convulsions. Although, in in vitro experiments dihydroergosine showed very weak ability to prevent the binding of {sup 3}H-muscimol, the drug was able to diminish and to augment the IC{sub 50} of bicuculline and GABA when added to crude synaptosomal pellet of the rat and mouse brain respectively. Lower concentrations of dihydroergosine stimulated and higher inhibited {sup 3}H-TBOB binding to the crude synaptosomal pellet of the rat brain. In the preparation of mouse brain dihydroergosine produced only inhibition of {sup 3}H-TBOB binding. Only slight quantitative differences were observed in bicuculline-induced stimulation and in GABA- and diazepam-induced inhibition of {sup 3}H-TBOB binding between the two species. The results suggest that the opposite species-dependent effects of dihydroergosine on bicuculline-induced convulsions are due to the ability of this drug to modulate species-dependently the benzodiazepine/GABA receptor chloride channel complex.

  7. Inducible and titratable silencing of Caenorhabditis elegans neurons in vivo with histamine-gated chloride channels

    PubMed Central

    Pokala, Navin; Liu, Qiang; Gordus, Andrew; Bargmann, Cornelia I.

    2014-01-01

    Recent progress in neuroscience has been facilitated by tools for neuronal activation and inactivation that are orthogonal to endogenous signaling systems. We describe here a chemical-genetic approach for inducible silencing of Caenorhabditis elegans neurons in intact animals, using the histamine-gated chloride channel HisCl1 from Drosophila and exogenous histamine. Administering histamine to freely moving C. elegans that express HisCl1 transgenes in neurons leads to rapid and potent inhibition of neural activity within minutes, as assessed by behavior, functional calcium imaging, and electrophysiology of neurons expressing HisCl1. C. elegans does not use histamine as an endogenous neurotransmitter, and exogenous histamine has little apparent effect on wild-type C. elegans behavior. HisCl1-histamine silencing of sensory neurons, interneurons, and motor neurons leads to behavioral effects matching their known functions. In addition, the HisCl1-histamine system can be used to titrate the level of neural activity, revealing quantitative relationships between neural activity and behavioral output. We use these methods to dissect escape circuits, define interneurons that regulate locomotion speed (AVA, AIB) and escape-related omega turns (AIB), and demonstrate graded control of reversal length by AVA interneurons and DA/VA motor neurons. The histamine-HisCl1 system is effective, robust, compatible with standard behavioral assays, and easily combined with optogenetic tools, properties that should make it a useful addition to C. elegans neurotechnology. PMID:24550306

  8. P-glycoprotein Mediates Postoperative Peritoneal Adhesion Formation by Enhancing Phosphorylation of the Chloride Channel-3

    PubMed Central

    Deng, Lulu; Li, Qin; Lin, Guixian; Huang, Dan; Zeng, Xuxin; Wang, Xinwei; Li, Ping; Jin, Xiaobao; Zhang, Haifeng; Li, Chunmei; Chen, Lixin; Wang, Liwei; Huang, Shulin; Shao, Hongwei; Xu, Bin; Mao, Jianwen

    2016-01-01

    P-glycoprotein (P-gp) is encoded by the multidrug resistance (MDR1) gene and is well studied as a multi-drug resistance transporter. Peritoneal adhesion formation following abdominal surgery remains an important clinical problem. Here, we found that P-gp was highly expressed in human adhesion fibroblasts and promoted peritoneal adhesion formation in a rodent model. Knockdown of P-gp expression by intraperitoneal injection of MDR1-targeted siRNA significantly reduced both the peritoneal adhesion development rate and adhesion grades. Additionally, we found that operative injury up-regulated P-gp expression in peritoneal fibroblasts through the TGF-β1/Smad signaling pathway and histone H3 acetylation. The overexpression of P-gp accelerated migration and proliferation of fibroblasts via volume-activated Cl- current and cell volume regulation by enhancing phosphorylation of the chloride channel-3. Therefore, P-gp plays a critical role in postoperative peritoneal adhesion formation and may be a valuable therapeutic target for preventing the formation of peritoneal adhesions. PMID:26877779

  9. Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β

    PubMed Central

    Peng, Yi-Jheng; Huang, Jing-Jia; Wu, Hao-Han; Hsieh, Hsin-Ying; Wu, Chia-Ying; Chen, Shu-Ching; Chen, Tsung-Yu; Tang, Chih-Yung

    2016-01-01

    Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation. PMID:27580824

  10. Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β.

    PubMed

    Peng, Yi-Jheng; Huang, Jing-Jia; Wu, Hao-Han; Hsieh, Hsin-Ying; Wu, Chia-Ying; Chen, Shu-Ching; Chen, Tsung-Yu; Tang, Chih-Yung

    2016-09-01

    Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation.

  11. Benzodiazepine agonist and inverse agonist actions on GABAA receptor-operated chloride channels. II. Chronic effects of ethanol

    SciTech Connect

    Buck, K.J.; Harris, R.A. )

    1990-05-01

    Mice were made tolerant to and dependent on ethanol by administration of a liquid diet. Gamma-aminobutyric acid (GABA) receptor-dependent uptake of 36Cl- by mouse cortical microsacs was used to study the actions of benzodiazepine (BZ) agonists and inverse agonists. Chronic exposure to ethanol attenuated the ability of a BZ agonist, flunitrazepam, to augment muscimol-stimulated uptake of 36Cl- and enhanced the actions of BZ inverse agonists, Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo(1,4)-benzodiazepine - 3-carboxylate) and DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate), to inhibit GABAA receptor-operated chloride channels. Augmentation of chloride flux by pentobarbital was not reduced by chronic ethanol exposure. Attenuation of flunitrazepam efficacy was transient and returned to control levels within 6 to 24 hr after withdrawal from ethanol, but increased sensitivity to Ro15-4513 was observed as long as 8 days after withdrawal. Chronic exposure to ethanol did not alter (3H)SR 95531 (2-(3'-carbethoxy-2'propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide) binding to low-affinity GABAA receptors or muscimol stimulation of chloride flux; and did not alter (3H)Ro15-4513 or (3H)flunitrazepam binding to central BZ receptors or allosteric modulation of this binding by muscimol (i.e., muscimol-shift). These results suggest that chronic exposure to ethanol reduces coupling between BZ agonist sites and the chloride channel, and may be responsible for the development of cross-tolerance between ethanol and BZ agonists. In contrast, coupling between BZ inverse agonist sites and the chloride channel is increased.

  12. Small-molecule activators of TMEM16A, a calcium-activated chloride channel, stimulate epithelial chloride secretion and intestinal contraction

    PubMed Central

    Namkung, Wan; Yao, Zhen; Finkbeiner, Walter E.; Verkman, A. S.

    2011-01-01

    TMEM16A (ANO1) is a calcium-activated chloride channel (CaCC) expressed in secretory epithelia, smooth muscle, and other tissues. Cell-based functional screening of ∼110,000 compounds revealed compounds that activated TMEM16A CaCC conductance without increasing cytoplasmic Ca2+. By patch-clamp, N-aroylaminothiazole “activators” (Eact) strongly increased Cl− current at 0 Ca2+, whereas tetrazolylbenzamide “potentiators” (Fact) were not active at 0 Ca2+ but reduced the EC50 for Ca2+-dependent TMEM16A activation. Of 682 analogs tested, the most potent activator (Eact) and potentiator (Fact) produced large and more sustained CaCC Cl− currents than general agonists of Ca2+ signaling, with EC50 3–6 μM and Cl− conductance comparable to that induced transiently by Ca2+-elevating purinergic agonists. Analogs of activators were identified that fully inhibited TMEM16A Cl− conductance, providing further evidence for direct TMEM16A binding. The TMEM16A activators increased CaCC conductance in human salivary and airway submucosal gland epithelial cells, and IL-4 treated bronchial cells, and stimulated submucosal gland secretion in human bronchi and smooth muscle contraction in mouse intestine. Small-molecule, TMEM16A-targeted activators may be useful for drug therapy of cystic fibrosis, dry mouth, and gastrointestinal hypomotility disorders, and for pharmacological dissection of TMEM16A function.—Namkung, W., Yao, Z., Finkbeiner, W. E., Verkman, A. S. Small-molecule activators of TMEM16A, a calcium-activated chloride channel, stimulate epithelial chloride secretion and intestinal contraction. PMID:21836025

  13. Double Blockade of Glioma Cell Proliferation and Migration by Temozolomide Conjugated with NPPB, a Chloride Channel Blocker.

    PubMed

    Park, Miri; Song, Chiman; Yoon, Hojong; Choi, Kee-Hyun

    2016-03-16

    Glioblastoma is the most common and aggressive primary malignant brain tumor. Temozolomide (TMZ), a chemotherapeutic agent combined with radiation therapy, is used as a standard treatment. The infiltrative nature of glioblastoma, however, interrupts effective treatment with TMZ and increases the tendency to relapse. Voltage-gated chloride channels have been identified as crucial regulators of glioma cell migration and invasion by mediating cell shape and volume change. Accordingly, chloride current inhibition by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), a chloride channel blocker, suppresses cell movement by diminishing the osmotic cell volume regulation. In this study, we developed a novel compound, TMZ conjugated with NPPB (TMZ-NPPB), as a potential anticancer drug. TMZ-NPPB blocked chloride currents in U373MG, a severely invasive human glioma cell line, and suppressed migration and invasion of U373MG cells. Moreover, TMZ-NPPB exhibited DNA modification activity similar to that of TMZ, and surprisingly showed remarkably enhanced cytotoxicity relative to TMZ by inducing apoptotic cell death via DNA damage. These findings indicate that TMZ-NPPB has a dual function in blocking both proliferation and migration of human glioma cells, thereby suggesting its potential to overcome challenges in current glioblastoma therapy.

  14. Lactic acid restores skeletal muscle force in an in vitro fatigue model: are voltage-gated chloride channels involved?

    PubMed

    Bandschapp, Oliver; Soule, Charles L; Iaizzo, Paul A

    2012-04-01

    High interstitial K(+) concentration ([K(+)]) has been reported to impede normal propagation of electrical impulses along the muscle cell membrane (sarcolemma) and then also into the transverse tubule system; this is one considered underlying mechanism associated with the development of muscle fatigue. Interestingly, the extracellular buildup of lactic acid, once considered an additional cause for muscle fatigue, was recently shown to have force-restoring effects in such conditions. Specifically, it was proposed that elevated lactic acid (and intracellular acidosis) may lead to inhibition of voltage-gated chloride channels, thereby reestablishing better excitability of the muscle cell sarcolemma. In the present study, using an in vitro muscle contractile experimental setup to study functionally viable rectus abdominis muscle preparations obtained from normal swine, we examined the effects of 20 mM lactic acid and 512 μM 9-anthracenecarboxylic acid (9-AC; a voltage-gated chloride channel blocker) on the force recovery of K(+)-depressed (10 mM K(+)) twitch forces. We observed a similar muscle contractile restoration after both treatments. Interestingly, at elevated [K(+)], myotonia (i.e., hyperexcitability or afterdepolarizations), usually present in skeletal muscle with inherent or induced chloride channel dysfunctions, was not observed in the presence of either lactic acid or 9-AC. In part, these data confirm previous studies showing a force-restoring effect of lactic acid in high-[K(+)] conditions. In addition, we observed similar restorative effects of lactic acid and 9-AC, implicating a beneficial mechanism via voltage-gated chloride channel modulation.

  15. Calcium-activated chloride channels in müller cells acutely isolated from tiger salamander retina.

    PubMed

    Welch, Nicole C; Lalonde, Melanie R; Barnes, Steven; Kelly, Melanie E M

    2006-01-01

    Ca(2+)-activated chloride channels were identified with whole-cell patch-clamp recording techniques in salamander retinal Müller cells. Cl(Ca) channels were activated by membrane depolarizations that elicited Ca2+ influx or the application of the Ca2+ ionophore, ionomycin. The Ca channel blocker, Cd2+, abolished the Cl(Ca) channel tail currents. Increasing the duration of the depolarizing pulse resulted in enhancement of the Cl(Ca) channel tail current. Repetitive depolarizations with rapid pulses to +20 mV produced a buildup of I(Cl(Ca)), which reversed at 0 mV in symmetrical [Cl-] and at -40 mV when intracellular [Cl-] was reduced to 10% of the external concentration. I(Cl(Ca)) was blocked by the Cl channel blocker niflumic acid, while niflumic acid had no effect on voltage-gated Ca channels. These results offer the first demonstration of Cl(Ca) channels in a nonastrocytic glial cell and expand our understanding of the functional capacities of retinal glial cells.

  16. Chloride intracellular channel-4 is a determinant of native collateral formation in skeletal muscle and brain.

    PubMed

    Chalothorn, Dan; Zhang, Hua; Smith, Jennifer E; Edwards, John C; Faber, James E

    2009-07-02

    The capacity of the collateral circulation to lessen injury in occlusive vascular disease depends on the density and caliber of native (preexisting) collaterals, as well as their ability to outwardly remodel in ischemia. Native collateral conductance varies widely among healthy individuals, yet little is known about what specifies collateral formation. Chloride intracellular channel (CLIC)4 protein is required for endothelial cell hollowing, a process necessary for vessel formation during embryogenesis and ischemia. Whether CLIC4 has other physiological roles in vascular biology is uncertain. We studied collateral formation and remodeling in mice deficient in CLIC1 and CLIC4. Vascular responses to femoral artery ligation were similar in Clic1(-/-) and wild-type mice. In contrast, immediately after ligation perfusion dropped more in Clic4(-/-) than wild-type mice, suggesting fewer preexisting collaterals, a finding confirmed by angiography, greater ischemia, and worse recovery of perfusion; however, collateral remodeling was unaffected. Likewise, native cerebral collateral density in Clic4(-/-) (but not Clic1(-/-)) mice was reduced, resulting in severe infarctions. This was associated with impaired perinatal formation and stabilization of nascent collaterals. Clic4 hemizygous mice had intermediate deficits in the above parameters, suggesting a gene-dose effect. Ischemia augmented CLIC1 and CLIC4 expression similarly in wild-type mice. However, CLIC1 increased 3-fold more in Clic4(-/-) mice, suggesting compensation. Despite greater ischemia in Clic4(-/-) mice, hypoxia-inducible factor-1alpha, vascular endothelial growth factor (VEGF) and angiopoietin-2 increased less compared to wild-type, suggesting CLIC4 exerts influences upstream of hypoxia-inducible factor-1alpha-VEGF signaling. Hence, CLIC4 represents the second gene that, along with VEGF shown by us previously, specifies native collateral formation.

  17. Effect of a chloride channel activator, lubiprostone, on colonic sensory and motor functions in healthy subjects.

    PubMed

    Sweetser, Seth; Busciglio, Irene A; Camilleri, Michael; Bharucha, Adil E; Szarka, Lawrence A; Papathanasopoulos, Athanasios; Burton, Duane D; Eckert, Deborah J; Zinsmeister, Alan R

    2009-02-01

    Lubiprostone, a bicyclic fatty acid chloride channel activator, is efficacious in treatment of chronic constipation and constipation-predominant irritable bowel syndrome. The study aim was to compare effects of lubiprostone and placebo on colonic sensory and motor functions in humans. In double-blind, randomized fashion, 60 healthy adults received three oral doses of placebo or 24 microg lubiprostone per day in a parallel-group, placebo-controlled trial. A barostat-manometry tube was placed in the left colon by flexible sigmoidoscopy and fluoroscopy. We measured treatment effects on colonic sensation and motility with validated methods, with the following end points: colonic compliance, fasting and postprandial tone and motility indexes, pain thresholds, and sensory ratings to distensions. Among participants receiving lubiprostone or placebo, 26 of 30 and 28 of 30, respectively, completed the study. There were no overall effects of lubiprostone on compliance, fasting tone, motility indexes, or sensation. However, there was a treatment-by-sex interaction effect for compliance (P = 0.02), with lubiprostone inducing decreased fasting compliance in women (P = 0.06) and an overall decreased colonic tone contraction after a standard meal relative to fasting tone (P = 0.014), with greater effect in women (P < 0.01). Numerical differences of first sensation and pain thresholds (P = 0.11 in women) in the two groups were not significant. We concluded that oral lubiprostone 24 microg does not increase colonic motor function. The findings of decreased colonic compliance and decreased postprandial colonic tone in women suggest that motor effects are unlikely to cause accelerated colonic transit with lubiprostone, although they may facilitate laxation. Effects of lubiprostone on sensitivity deserve further study.

  18. S-Nitrosylation Regulates Nuclear Translocation of Chloride Intracellular Channel Protein CLIC4*

    PubMed Central

    Malik, Mariam; Shukla, Anjali; Amin, Palak; Niedelman, Wendy; Lee, Jessica; Jividen, Kasey; Phang, Juanita M.; Ding, Jinhui; Suh, Kwang S.; Curmi, Paul M. G.; Yuspa, Stuart H.

    2010-01-01

    Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca2+-induced differentiation, stress-induced apoptosis, and modulating TGF-β signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin α and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor α-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity. Inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor α-induced nitrosylation and association with importin α and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution. PMID:20504765

  19. Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models.

    PubMed

    Contreras-Vite, Juan A; Cruz-Rangel, Silvia; De Jesús-Pérez, José J; Figueroa, Iván A Aréchiga; Rodríguez-Menchaca, Aldo A; Pérez-Cornejo, Patricia; Hartzell, H Criss; Arreola, Jorge

    2016-07-01

    TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca(2+)]i), membrane depolarization, extracellular Cl(-) or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca(2+). (b) The Cl(-) conductance is decreased after reducing extracellular Cl(-) concentration ([Cl(-)]o). (c) ICl is regulated by physiological concentrations of [Cl(-)]o. (d) In cells dialyzed with 0.2 μM [Ca(2+)]i, Cl(-) has a bimodal effect: at [Cl(-)]o <30 mM TMEM16A current activates with a monoexponential time course, but above 30 mM, [Cl(-)]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca(2+) and Cl(-) to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca(2+) ions coupled to a Vm-dependent binding of an external Cl(-) ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl(-) does not alter the apparent Ca(2+) affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl(-) acts by stabilizing the open configuration induced by Ca(2+) and by contributing to the Vm dependence of activation.

  20. Fast and Slow Gating Relaxations in the Muscle Chloride Channel Clc-1

    PubMed Central

    Accardi, Alessio; Pusch, Michael

    2000-01-01

    Gating of the muscle chloride channel CLC-1 involves at least two processes evidenced by double-exponential current relaxations when stepping the voltage to negative values. However, there is little information about the gating of CLC-1 at positive voltages. Here, we analyzed macroscopic gating of CLC-1 over a large voltage range (from −160 to +200 mV). Activation was fast at positive voltages but could be easily followed using envelope protocols that employed a tail pulse to −140 mV after stepping the voltage to a certain test potential for increasing durations. Activation was biexponential, demonstrating the presence of two gating processes. Both time constants became exponentially faster at positive voltages. A similar voltage dependence was also seen for the fast gate time constant of CLC-0. The voltage dependence of the time constant of the fast process of CLC-1, τf, was steeper than that of the slow one, τs (apparent activation valences were zf ∼ −0.79 and zs ∼ −0.42) such that at +200 mV the two processes became kinetically distinct by almost two orders of magnitude (τf ∼ 16 μs, τs ∼ 1 ms). This voltage dependence is inconsistent with a previously published gating model for CLC-1 (Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695–706). The kinetic difference at 200 mV allowed us to separate the steady state open probabilities of the two processes assuming that they reflect two parallel (not necessarily independent) gates that have to be open simultaneously to allow ion conduction. Both open probabilities could be described by Boltzmann functions with gating valences around one and with nonzero “offsets” at negative voltages, indicating that the two “gates” never close completely. For comparison with single channel data and to correlate the two gating processes with the two gates of CLC-0, we characterized their voltage, pHint, and [Cl]ext dependence, and the dominant myotonia inducing

  1. Upregulation of apical sodium-chloride cotransporter and basolateral chloride channels is responsible for the maintenance of salt-sensitive hypertension.

    PubMed

    Capasso, Giovambattista; Rizzo, Maria; Garavaglia, Maria Lisa; Trepiccione, Francesco; Zacchia, Miriam; Mugione, Alessandra; Ferrari, Patrizia; Paulmichl, Markus; Lang, Florian; Loffing, Johannes; Carrel, Monique; Damiano, Sara; Wagner, Carsten A; Bianchi, Giuseppe; Meyer, Giuliano

    2008-08-01

    We investigated which of the NaCl transporters are involved in the maintenance of salt-sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 mo after birth. In MHS, compared with normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged, both for Na+/H+ isoform 3 (NHE3) and Na+-K+-2Cl- (NKCC2), but higher (119%, n = 5, P < 0.005) for Na+-Cl- (NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots, which revealed: 1) unchanged NHE3 in the cortex and NKCC2 in the outer medulla; 2) a significant increase (52%, n = 6, P < 0.001) of NCC in the cortex; 3) alpha- and beta-sodium channels [epithelial Na+ channel (ENaC)] unaffected in renal cortex and slightly reduced in the outer medulla, while gamma-ENaC remained unchanged. Pendrin protein expression was unaffected. The role of NCC was reinforced by immunocytochemical studies showing increased NCC on the apical membrane of DCT cells of MHS animals, and by clearance experiments demonstrating a larger sensitivity (P < 0.001) to bendroflumethiazide in MHS rats. Kidney-specific chloride channels (ClC-K) were studied by Western blot experiments on renal cortex and by patch-clamp studies on primary culture of DCT dissected from MNS and MHS animals. Electrophysiological characteristics of ClC-K channels were unchanged in MHS rats, but the number of active channels in a patch was 0.60 +/- 0.21 (n = 35) in MNS rats and 2.17 +/- 0.59 (n = 23) in MHS rats (P < 0.05). The data indicate that, in salt-sensitive hypertension, there is a strong upregulation, both of NCC and ClC-K along the DCT, which explains the persistence of hypertension.

  2. Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis.

    PubMed

    Cabrero, Pablo; Terhzaz, Selim; Romero, Michael F; Davies, Shireen A; Blumenthal, Edward M; Dow, Julian A T

    2014-09-30

    Epithelia frequently segregate transport processes to specific cell types, presumably for improved efficiency and control. The molecular players underlying this functional specialization are of particular interest. In Drosophila, the renal (Malpighian) tubule displays the highest per-cell transport rates known and has two main secretory cell types, principal and stellate. Electrogenic cation transport is known to reside in the principal cells, whereas stellate cells control the anion conductance, but by an as-yet-undefined route. Here, we resolve this issue by showing that a plasma membrane chloride channel, encoded by ClC-a, is exclusively expressed in the stellate cell and is required for Drosophila kinin-mediated induction of diuresis and chloride shunt conductance, evidenced by chloride ion movement through the stellate cells, leading to depolarization of the transepithelial potential. By contrast, ClC-a knockdown had no impact on resting secretion levels. Knockdown of a second CLC gene showing highly abundant expression in adult Malpighian tubules, ClC-c, did not impact depolarization of transepithelial potential after kinin stimulation. Therefore, the diuretic action of kinin in Drosophila can be explained by an increase in ClC-a-mediated chloride conductance, over and above a resting fluid transport level that relies on other (ClC-a-independent) mechanisms or routes. This key segregation of cation and anion transport could explain the extraordinary fluid transport rates displayed by some epithelia.

  3. Biochemical and biophysical approaches to study the structure and function of the chloride channel (ClC) family of proteins.

    PubMed

    Abeyrathne, Priyanka D; Chami, Mohamed; Stahlberg, Henning

    2016-01-01

    The chloride channel (ClC) protein family comprises both chloride (Cl(-)) channels and chloride/proton (Cl(-)/H(+)) antiporters. In prokaryotes and eukaryotes, these proteins mediate the movement of Cl(-) ions across the membrane. In eukaryotes, ClC proteins play a role in the stabilization of membrane potential, epithelial ion transport, hippocampal neuroprotection, cardiac pacemaker activity and vesicular acidification. Moreover, mutations in the genes encoding ClC proteins can cause genetic disease in humans. In prokaryotes, the Cl(-)/H(+) antiporters, such as ClC-ec1 found in Escherichia coli promote proton expulsion in the extreme acid-resistance response common to enteric bacteria. To date, structural and functional studies of the prokaryotic protein have revealed unique structural features, including complicated transmembrane topology with 18 α-helices in each subunit and an anion-coordinating region in each subunit. Several different approaches such as X-ray crystallography, NMR, biochemical studies, and molecular dynamics simulations have been applied to the study of ClC proteins. Continued study of the unique structure and function of this diverse family of proteins has the potential to lead to the development of novel therapeutic targets for neuronal, renal, bone, and food-borne diseases.

  4. Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation.

    PubMed

    Cubero-Font, Paloma; Maierhofer, Tobias; Jaslan, Justyna; Rosales, Miguel A; Espartero, Joaquín; Díaz-Rueda, Pablo; Müller, Heike M; Hürter, Anna-Lena; Al-Rasheid, Khaled A S; Marten, Irene; Hedrich, Rainer; Colmenero-Flores, José M; Geiger, Dietmar

    2016-08-22

    Higher plants take up nutrients via the roots and load them into xylem vessels for translocation to the shoot. After uptake, anions have to be channeled toward the root xylem vessels. Thereby, xylem parenchyma and pericycle cells control the anion composition of the root-shoot xylem sap [1-6]. The fact that salt-tolerant genotypes possess lower xylem-sap Cl(-) contents compared to salt-sensitive genotypes [7-10] indicates that membrane transport proteins at the sites of xylem loading contribute to plant salinity tolerance via selective chloride exclusion. However, the molecular mechanism of xylem loading that lies behind the balance between NO3(-) and Cl(-) loading remains largely unknown. Here we identify two root anion channels in Arabidopsis, SLAH1 and SLAH3, that control the shoot NO3(-)/Cl(-) ratio. The AtSLAH1 gene is expressed in the root xylem-pole pericycle, where it co-localizes with AtSLAH3. Under high soil salinity, AtSLAH1 expression markedly declined and the chloride content of the xylem sap in AtSLAH1 loss-of-function mutants was half of the wild-type level only. SLAH3 anion channels are not active per se but require extracellular nitrate and phosphorylation by calcium-dependent kinases (CPKs) [11-13]. When co-expressed in Xenopus oocytes, however, the electrically silent SLAH1 subunit gates SLAH3 open even in the absence of nitrate- and calcium-dependent kinases. Apparently, SLAH1/SLAH3 heteromerization facilitates SLAH3-mediated chloride efflux from pericycle cells into the root xylem vessels. Our results indicate that under salt stress, plants adjust the distribution of NO3(-) and Cl(-) between root and shoot via differential expression and assembly of SLAH1/SLAH3 anion channel subunits.

  5. Functional expression of the TMEM16 family of calcium-activated chloride channels in airway smooth muscle

    PubMed Central

    Remy, Kenneth E.; Danielsson, Jennifer; Funayama, Hiromi; Fu, Xiao Wen; Chang, Herng-Yu Sucie; Yim, Peter; Xu, Dingbang; Emala, Charles W.

    2013-01-01

    Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma. PMID:23997176

  6. Interaction between 2 extracellular loops influences the activity of the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    Broadbent, Steven D; Wang, Wuyang; Linsdell, Paul

    2014-10-01

    Activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is thought to be controlled by cytoplasmic factors. However, recent evidence has shown that overall channel activity is also influenced by extracellular anions that interact directly with the extracellular loops (ECLs) of the CFTR protein. Very little is known about the structure of the ECLs or how substances interacting with these ECLs might affect CFTR function. We used patch-clamp recording to investigate the accessibility of cysteine-reactive reagents to cysteines introduced throughout ECL1 and 2 key sites in ECL4. Furthermore, interactions between ECL1 and ECL4 were investigated by the formation of disulfide crosslinks between cysteines introduced into these 2 regions. Crosslinks could be formed between R899C (in ECL4) and a number of sites in ECL1 in a manner that was dependent on channel activity, suggesting that the relative orientation of these 2 loops changes on activation. Formation of these crosslinks inhibited channel function, suggesting that relative movement of these ECLs is important to normal channel function. Implications of these findings for the effects of mutations in the ECLs that are associated with cystic fibrosis and interactions with extracellular substances that influence channel activity are discussed.

  7. Intermediate-conductance Calcium-activated Potassium Channel KCa3.1 and Chloride Channel Modulate Chemokine Ligand (CCL19/CCL21)-induced Migration of Dendritic Cells

    PubMed Central

    Shao, Zhifei; Gaurav, Rohit; Agrawal, Devendra K

    2014-01-01

    The role of ion channels is largely unknown in chemokine-induced migration in non-excitable cells such as dendritic cells. Here, we examined the role of KCa3.1 and chloride channels in lymphatic chemokines-induced migration of dendritic cells. The amplitude and kinetics of CCL19/21-induced Ca2+ influx were associated with CCR7 expression levels, extracellular free Ca2+ and Cl−, and independent of extracellular K+. Chemokines, CCL19 and CCL21, and KCa3.1 activator, 1-EBIO, induced plasma membrane hyperpolarization and K+ efflux, which was blocked by TRAM-34, suggesting that KCa3.1 carried larger conductance than the inward CRAC. Blockade of KCa3.1, low Cl− in the medium, and low dose of DIDS impaired CCL19/CCL21-induced Ca2+ influx, cell volume change, and DC migration. High doses of DIDS completely blocked DC migration possibly by significantly disrupting mitochondrial membrane potential. In conclusion, KCa3.1 and chloride channel are critical in human DC migration by synergistically regulating membrane potential, chemokine-induced Ca2+ influx, and cell volume. PMID:25583444

  8. The AQP-3 water channel and the ClC-3 chloride channel coordinate the hypotonicity-induced swelling volume in nasopharyngeal carcinoma cells.

    PubMed

    Zhang, Haifeng; Li, Huarong; Liu, Enqi; Guang, Yutao; Yang, Lili; Mao, Jianwen; Zhu, Linyan; Chen, Lixin; Wang, Liwei

    2014-12-01

    Cell volume regulation is a fundamental activity to maintain cell survival, and aquaporins and chloride channels play important roles in this process. However, the interactions between these channels are far from clear. In this study, the interactions between AQP-3 and ClC-3 were investigated in CNE-1 and CNE-2Z nasopharyngeal carcinoma cells, which are well and poorly differentiated, respectively. The correlation coefficient of AQP-3 and ClC-3 protein phylogenetic trees was 0.319. In CNE-1 cells, there are overlapping distributions of AQP-3 and ClC-3, mainly in the plasma membrane. This was confirmed by the co-immunoprecipitation of AQP-3 and ClC-3, showing that they could be interlinked and form complexes. AQP-3 over-expression had no significant effects on swelling-induced Cl(-) currents (ICl,swell); however, ICl,swell could be inhibited by aquaporin blockers, anti-AQP-3 antibodies and AQP-3-siRNAs. In addition, the AQP-3 expression was decreased by down-regulation of ClC-3 expression, indicating that ClC-3 can modulate the expression of AQP-3 proteins. The effects of aquaporin blockers, anti-AQP-3 antibodies and AQP-3 over-expression on ICl,swell in CNE-2Z cells were consistent with those in CNE-1 cells. In conclusion, AQP-3 and ClC-3 are functionally-related integral membrane channel proteins, and their interactions are involved in cell volume regulation in CNE-1 and CNE-2Z cells. The opening of ClC-3 transports Cl(-) across the cell membrane and then drives the efflux of water through AQP-3 channels and ion channels; AQP-3 may interact with ClC-3 in order to regulate the effluxes of chloride and water.

  9. Basolateral sorting of chloride channel 2 is mediated by interactions between a dileucine motif and the clathrin adaptor AP-1

    PubMed Central

    de la Fuente-Ortega, Erwin; Gravotta, Diego; Bay, Andres Perez; Benedicto, Ignacio; Carvajal-Gonzalez, Jose Maria; Lehmann, Guillermo L.; Lagos, Carlos F.; Rodríguez-Boulan, Enrique

    2015-01-01

    In spite of the many key cellular functions of chloride channels, the mechanisms that mediate their subcellular localization are largely unknown. ClC-2 is a ubiquitous chloride channel usually localized to the basolateral domain of epithelia that regulates cell volume, ion transport, and acid–base balance; mice knocked out for ClC-2 are blind and sterile. Previous work suggested that CLC-2 is sorted basolaterally by TIFS812LL, a dileucine motif in CLC-2's C-terminal domain. However, our in silico modeling of ClC-2 suggested that this motif was buried within the channel's dimerization interface and identified two cytoplasmically exposed dileucine motifs, ESMI623LL and QVVA635LL, as candidate sorting signals. Alanine mutagenesis and trafficking assays support a scenario in which ESMI623LL acts as the authentic basolateral signal of ClC-2. Silencing experiments and yeast three-hybrid assays demonstrated that both ubiquitous (AP-1A) and epithelium-specific (AP-1B) forms of the tetrameric clathrin adaptor AP-1 are capable of carrying out basolateral sorting of ClC-2 through interactions of ESMI623LL with a highly conserved pocket in their γ1-σ1A hemicomplex. PMID:25739457

  10. Benzopyrimido-pyrrolo-oxazine-dione (R)-BPO-27 Inhibits CFTR Chloride Channel Gating by Competition with ATP

    PubMed Central

    Kim, Yonjung; Anderson, Marc O.; Park, Jinhong; Lee, Min Goo; Namkung, Wan

    2015-01-01

    We previously reported that benzopyrimido-pyrrolo-oxazinedione BPO-27 [6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4′,5′:3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid] inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with low nanomolar potency and reduces cystogenesis in a model of polycystic kidney disease. We used computational chemistry and patch-clamp to show that enantiomerically pure (R)-BPO-27 inhibits CFTR by competition with ATP, whereas (S)-BPO-27 is inactive. Docking computations using a homology model of CFTR structure suggested that (R)-BPO-27 binds near the canonical ATP binding site, and these findings were supported by molecular dynamics simulations showing a lower binding energy for the (R) versus (S) stereoisomers. Three additional lower-potency BPO-27 analogs were modeled in a similar fashion, with the binding energies predicted in the correct order. Whole-cell patch-clamp studies showed linear CFTR currents with a voltage-independent (R)-BPO-27 block mechanism. Single-channel recordings in inside-out patches showed reduced CFTR channel open probability and increased channel closed time by (R)-BPO-27 without altered unitary channel conductance. At a concentration of (R)-BPO-27 that inhibited CFTR chloride current by ∼50%, the EC50 for ATP activation of CFTR increased from 0.27 to 1.77 mM but was not changed by CFTRinh-172 [4-[[4-oxo-2-thioxo-3-[3-trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid], a thiazolidinone CFTR inhibitor that acts at a site distinct from the ATP binding site. Our results suggest that (R)-BPO-27 inhibition of CFTR involves competition with ATP. PMID:26174774

  11. Benzopyrimido-pyrrolo-oxazine-dione (R)-BPO-27 Inhibits CFTR Chloride Channel Gating by Competition with ATP.

    PubMed

    Kim, Yonjung; Anderson, Marc O; Park, Jinhong; Lee, Min Goo; Namkung, Wan; Verkman, A S

    2015-10-01

    We previously reported that benzopyrimido-pyrrolo-oxazinedione BPO-27 [6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid] inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with low nanomolar potency and reduces cystogenesis in a model of polycystic kidney disease. We used computational chemistry and patch-clamp to show that enantiomerically pure (R)-BPO-27 inhibits CFTR by competition with ATP, whereas (S)-BPO-27 is inactive. Docking computations using a homology model of CFTR structure suggested that (R)-BPO-27 binds near the canonical ATP binding site, and these findings were supported by molecular dynamics simulations showing a lower binding energy for the (R) versus (S) stereoisomers. Three additional lower-potency BPO-27 analogs were modeled in a similar fashion, with the binding energies predicted in the correct order. Whole-cell patch-clamp studies showed linear CFTR currents with a voltage-independent (R)-BPO-27 block mechanism. Single-channel recordings in inside-out patches showed reduced CFTR channel open probability and increased channel closed time by (R)-BPO-27 without altered unitary channel conductance. At a concentration of (R)-BPO-27 that inhibited CFTR chloride current by ∼50%, the EC50 for ATP activation of CFTR increased from 0.27 to 1.77 mM but was not changed by CFTRinh-172 [4-[[4-oxo-2-thioxo-3-[3-trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid], a thiazolidinone CFTR inhibitor that acts at a site distinct from the ATP binding site. Our results suggest that (R)-BPO-27 inhibition of CFTR involves competition with ATP.

  12. A solid phase honey-like channel method for synthesizing urea-ammonium chloride cocrystals on industrial scale

    NASA Astrophysics Data System (ADS)

    Xue, Bingchun; Mao, Meiling; Liu, Yanhong; Guo, Jinyu; Li, Jing; Liu, Erbao

    2016-05-01

    Unanticipated a new and simple urea-ammonium chloride cocrystal synthesis method on industrial scale was found during attempts to produce a kind of granulated compound fertilizer. The aggregation of fertilizer powder can make the interaction among particles from loose to close, which generate mechanical pressure and in turn act as the driving force to benefit cocrystal growth. Additionally, the honeycomb-like channels constructed by other coexisting compound make the water evaporates more moderate, which can help the formation of supersaturated solution at suitable rate, further promote the growth of cocrystal. This approach possibly opens a new route toward the developing methodologies for cocrystal synthesis.

  13. A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity

    PubMed Central

    Tien, Jason; Peters, Christian J; Wong, Xiu Ming; Cheng, Tong; Jan, Yuh Nung; Jan, Lily Yeh; Yang, Huanghe

    2014-01-01

    TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. DOI: http://dx.doi.org/10.7554/eLife.02772.001 PMID:24980701

  14. A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase, CDPK.

    PubMed Central

    Pei, Z M; Ward, J M; Harper, J F; Schroeder, J I

    1996-01-01

    Calcium-Dependent Protein Kinases (CDPKs) in higher plants contain a C-terminal calmodulin-like regulatory domain. Little is known regarding physiological CDPK targets. Both kinase activity and multiple Ca2+-dependent signaling pathways have been implicated in the control of stomatal guard cell movements. To determine whether CDPK or other protein kinases could have a role in guard cell signaling, purified and recombinant kinases were applied to Vicia faba guard cell vacuoles during patch-clamp experiments. CDPK activated novel vacuolar chloride (VCL) and malate conductances in guard cells. Activation was dependent on both Ca2+ and ATP. Furthermore, VCL activation occurred in the absence of Ca2+ using a Ca2+-independent, constitutively active, CDPK* mutant. Protein kinase A showed weaker activation (22% as compared with CDPK). Current reversals in whole vacuole recordings shifted with the Nernst potential for Cl-and vanished in glutamate. Single channel recordings showed a CDPK-activated 34 +/- 5 pS Cl- channel. VCL channels were activated at physiological potentials enabling Cl- uptake into vacuoles. VCL channels may provide a previously unidentified, but necessary, pathway for anion uptake into vacuoles required for stomatal opening. CDPK-activated VCL currents were also observed in red beet vacuoles suggesting that these channels may provide a more general mechanism for kinase-dependent anion uptake. PMID:8978683

  15. State-dependent access of anions to the cystic fibrosis transmembrane conductance regulator chloride channel pore.

    PubMed

    Fatehi, Mohammad; Linsdell, Paul

    2008-03-07

    The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is gated by intracellular factors; however, conformational changes in the channel pore associated with channel activation have not been identified. We have used patch clamp recording to investigate the state-dependent accessibility of substituted cysteine residues in the CFTR channel pore to a range of cysteine-reactive reagents applied to the extracellular side of the membrane. Using functional modification of the channel current-voltage relationship as a marker of modification, we find that several positively charged reagents are able to penetrate deeply into the pore from the outside irrespective of whether or not the channels have been activated. In contrast, access of three anionic cysteine-reactive reagents, the methanesulfonate sodium (2-sulfonatoethyl)methanesulfonate, the organic mercurial p-chloromercuriphenylsulfonic acid, and the permeant anion Au(CN)(2)(-), to several different sites in the pore is strictly limited prior to channel activation. This suggests that in nonactivated channels some ion selectivity mechanism exists to exclude anions yet permit cations into the channel pore from the extracellular solution. We suggest that activation of CFTR channels involves a conformational change in the pore that removes a strong selectivity against anion entry from the extracellular solution. We propose further that this conformational change occurs in advance of channel opening, suggesting that multiple distinct closed pore conformations exist.

  16. Low expression of the ClC-2 chloride channel during postnatal development: a mechanism for the paradoxical depolarizing action of GABA and glycine in the hippocampus.

    PubMed Central

    Mladinić, M; Becchetti, A; Didelon, F; Bradbury, A; Cherubini, E

    1999-01-01

    In early postnatal development, during the period of synapse formation, gamma-aminobutyric acid (GABA) and glycine, the main inhibitory transmitters in the adult brain, paradoxically excite and depolarize neuronal membranes by an outward flux of chloride. The mechanisms of chloride homeostasis are not fully understood. It is known that in adult neurons intracellular chloride accumulation is prevented by a particular type of chloride channel, the ClC-2. This channel strongly rectifies in the inward direction at potentials negative to ECl thus ensuring chloride efflux. We have tested the hypothesis that in the developing hippocampus, a differential expression or regulation of ClC-2 channels may contribute to the depolarizing action of GABA and glycine. We have cloned a truncated form of ClC-2 (ClC-2nh) from the neonatal hippocampus which lacks the 157 bp corresponding to exon 2. In situ hybridization experiments show that ClC-2nh is the predominant form of ClC-2 mRNA in the neonatal brain. ClC-2nh mRNA is unable to encode a full-length protein due to a frameshift, consequently it does not induce any currents upon injection into Xenopus oocytes. Low expression of the full-length ClC-2 channel, could alter chloride homeostasis, lead to accumulation of [Cl-]i and thereby contribute to the depolarizing action of GABA and glycine during early development. PMID:10418163

  17. Regulation of the Membrane Insertion and Conductance Activity of the Metamorphic Chloride Intracellular Channel Protein CLIC1 by Cholesterol

    PubMed Central

    Valenzuela, Stella M.; Alkhamici, Heba; Brown, Louise J.; Almond, Oscar C.; Goodchild, Sophia C.; Carne, Sonia; Curmi, Paul M. G.; Holt, Stephen A.; Cornell, Bruce A.

    2013-01-01

    The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer. PMID:23457643

  18. Regulation of the membrane insertion and conductance activity of the metamorphic chloride intracellular channel protein CLIC1 by cholesterol.

    PubMed

    Valenzuela, Stella M; Alkhamici, Heba; Brown, Louise J; Almond, Oscar C; Goodchild, Sophia C; Carne, Sonia; Curmi, Paul M G; Holt, Stephen A; Cornell, Bruce A

    2013-01-01

    The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer.

  19. Arsenic promotes ubiquitinylation and lysosomal degradation of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in human airway epithelial cells.

    PubMed

    Bomberger, Jennifer M; Coutermarsh, Bonita A; Barnaby, Roxanna L; Stanton, Bruce A

    2012-05-18

    Arsenic exposure significantly increases respiratory bacterial infections and reduces the ability of the innate immune system to eliminate bacterial infections. Recently, we observed in the gill of killifish, an environmental model organism, that arsenic exposure induced the ubiquitinylation and degradation of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is essential for the mucociliary clearance of respiratory pathogens in humans. Accordingly, in this study, we tested the hypothesis that low dose arsenic exposure reduces the abundance and function of CFTR in human airway epithelial cells. Arsenic induced a time- and dose-dependent increase in multiubiquitinylated CFTR, which led to its lysosomal degradation, and a decrease in CFTR-mediated chloride secretion. Although arsenic had no effect on the abundance or activity of USP10, a deubiquitinylating enzyme, siRNA-mediated knockdown of c-Cbl, an E3 ubiquitin ligase, abolished the arsenic-stimulated degradation of CFTR. Arsenic enhanced the degradation of CFTR by increasing phosphorylated c-Cbl, which increased its interaction with CFTR, and subsequent ubiquitinylation of CFTR. Because epidemiological studies have shown that arsenic increases the incidence of respiratory infections, this study suggests that one potential mechanism of this effect involves arsenic-induced ubiquitinylation and degradation of CFTR, which decreases chloride secretion and airway surface liquid volume, effects that would be proposed to reduce mucociliary clearance of respiratory pathogens.

  20. Drastic reduction of the slow gate of human muscle chloride channel (ClC-1) by mutation C277S

    PubMed Central

    Accardi, Alessio; Ferrera, Loretta; Pusch, Michael

    2001-01-01

    Single channel measurements suggest that the human muscle chloride channel ClC-1 presumably has a double barrelled structure, with a fast single protopore gate and a slow common pore gate similar to that of ClC-0, the chloride channel from Torpedo. The single point mutation C212S has been shown to abolish the slow gating of ClC-0 locking the slow gate in the open state. In order to test the hypothesis that the slow gating process found in ClC-1 corresponds to the well characterised slow gate found in ClC-0 we investigated the gating effects in ClC-1 of the homologous mutation corresponding to C212S, C277S. We found that the mutation C277S strongly reduced the slow component of macroscopic gating relaxations at negative and at positive voltages. Time constants of the fast gating relaxations were not affected by the mutation but the minimal open probability of the fast gate at negative voltages was slightly reduced to 0.08 compared with the WT value of 0.22. Additionally, we characterised the block of WT ClC-1 and mutant C277S by the S(—) enantiomer of CPB (2-(p-chlorophenoxy) butyric acid), and found that the block is practically unaffected by the mutation suggesting that CPB does not interact with the slow gate of ClC-1. We conclude that the slow and fast gating processes of ClC-1, respectively, reflect the slow common pore gate and the single protopore gate of the double-barrelled ClC-1 channel. PMID:11483705

  1. Drastic reduction of the slow gate of human muscle chloride channel (ClC-1) by mutation C277S.

    PubMed

    Accardi, A; Ferrera, L; Pusch, M

    2001-08-01

    1. Single channel measurements suggest that the human muscle chloride channel ClC-1 presumably has a double barrelled structure, with a fast single protopore gate and a slow common pore gate similar to that of ClC-0, the chloride channel from Torpedo. The single point mutation C212S has been shown to abolish the slow gating of ClC-0 locking the slow gate in the open state. In order to test the hypothesis that the slow gating process found in ClC-1 corresponds to the well characterised slow gate found in ClC-0 we investigated the gating effects in ClC-1 of the homologous mutation corresponding to C212S, C277S. 2. We found that the mutation C277S strongly reduced the slow component of macroscopic gating relaxations at negative and at positive voltages. 3. Time constants of the fast gating relaxations were not affected by the mutation but the minimal open probability of the fast gate at negative voltages was slightly reduced to 0.08 compared with the WT value of 0.22. 4. Additionally, we characterised the block of WT ClC-1 and mutant C277S by the S(-) enantiomer of CPB (2-(p-chlorophenoxy) butyric acid), and found that the block is practically unaffected by the mutation suggesting that CPB does not interact with the slow gate of ClC-1. 5. We conclude that the slow and fast gating processes of ClC-1, respectively, reflect the slow common pore gate and the single protopore gate of the double-barrelled ClC-1 channel.

  2. Chloride channel ClC-3 in gills of the euryhaline teleost, Tetraodon nigroviridis: expression, localization and the possible role of chloride absorption

    PubMed Central

    Tang, Cheng-Hao; Hwang, Lie-Yueh; Lee, Tsung-Han

    2010-01-01

    SUMMARY Previous studies have reported the mechanisms of ion absorption and secretion by diverse membrane transport proteins in gills of various teleostean species. To date, however, the chloride channel expressed in the basolateral membrane of mitochondrion-rich (MR) cells for Cl− uptake in freshwater (FW) fish is still unknown. In this study, the combination of bioinformatics tools [i.e. National Center for Biotechnology Information (NCBI) database, Tetraodon nigroviridis (spotted green pufferfish) genome database (Genoscope), BLAT and BLASTn] were used to identify the gene of ClC-3 (TnClC-3), a member of the CLC chloride channel family in the T. nigroviridis genome. RT-PCR analysis revealed that the gene encoding for the ClC-3 protein was widely expressed in diverse tissues (i.e. gill, kidney, intestine, liver and brain) of FW- and seawater (SW)-acclimated pufferfish. In whole-mount double immunofluorescent staining, branchial ClC-3-like immunoreactive protein was localized to the basolateral membrane of Na+/K+-ATPase (NKA) immunoreactive cells in both the FW- and SW-acclimated pufferfish. In response to salinity, the levels of transcript of branchial TnClC-3 were similar between FW and SW fish. Moreover, the membrane fraction of ClC-3-like protein in gills was 2.7-fold higher in FW compared with SW pufferfish. To identify whether the expression of branchial ClC-3-like protein specifically responded to lower environmental [Cl−], the pufferfish were acclimated to artificial waters either with a normal (control) or lower Cl− concentration (low-Cl). Immunoblotting of membrane fractions of gill ClC-3-like protein showed the expression was about 4.3-fold higher in pufferfish acclimated to the low-Cl environment than in the control group. Furthermore, branchial ClC-3-like protein was rapidly elevated in response to acute changes of environmental salinity or [Cl−]. Taken together, pufferfish ClC-3-like protein was expressed in the basolateral membrane of gill

  3. Intermediate-conductance calcium-activated potassium channel KCa3.1 and chloride channel modulate chemokine ligand (CCL19/CCL21)-induced migration of dendritic cells.

    PubMed

    Shao, Zhifei; Gaurav, Rohit; Agrawal, Devendra K

    2015-07-01

    The role of ion channels is largely unknown in chemokine-induced migration in nonexcitable cells such as dendritic cells (DCs). Here, we examined the role of intermediate-conductance calcium-activated potassium channel (KCa3.1) and chloride channel (CLC3) in lymphatic chemokine-induced migration of DCs. The amplitude and kinetics of chemokine ligand (CCL19/CCL21)-induced Ca(2+) influx were associated with chemokine receptor 7 expression levels, extracellular-free Ca(2+) and Cl(-), and independent of extracellular K(+). Chemokines (CCL19 and CCL21) and KCa3.1 activator (1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) induced plasma membrane hyperpolarization and K(+) efflux, which was blocked by 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, suggesting that KCa3.1 carried larger conductance than the inward calcium release-activated calcium channel. Blockade of KCa3.1, low Cl(-) in the medium, and low dose of 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) impaired CCL19/CCL21-induced Ca(2+) influx, cell volume change, and DC migration. High doses of DIDS completely blocked DC migration possibly by significantly disrupting mitochondrial membrane potential. In conclusion, KCa3.1 and CLC3 are critical in human DC migration by synergistically regulating membrane potential, chemokine-induced Ca(2+) influx, and cell volume.

  4. [Ionic mechanisms of the effects of phenibut and GABA unrelated to changes in the function of chloride channels].

    PubMed

    Komissarov, I V; Abramets, I I

    1985-01-01

    It is established that beta-phenyl-GABA (phenibut) and partly GABA elicit direct depolarization of the isolated spinal cord motoneurons. The depolarizing effect of phenibut and a depolarizing component of GABA action do not alter in the presence of picrotoxin (10(-5) mol/l) and in the chloride-deficient medium. This depolarizing phenibut effect which is not bound with activation of GABAA-receptors and chloride channels coupled with them does not alter in Na+-deficient medium, enhances in the medium with excess of K+ ions (10 mol/l) and in presence of imidazol (5 . 10(-4) mol/l) and is completely abolished in the Ca2+-deficient medium with 2 mmol/l of Mn2+ or in the presence of 10(-4) mol/l theophylline. It is supposed that phenibut and partly GABA diminish intracellular concentration of cAMP via GABAB-receptor activation and decrease functional activity of voltage-dependent Ca2+-ionic channels and Ca2+-activated outward K+-currents.

  5. Effect of a selective chloride channel activator, lubiprostone, on gastrointestinal transit, gastric sensory, and motor functions in healthy volunteers.

    PubMed

    Camilleri, Michael; Bharucha, Adil E; Ueno, Ryuji; Burton, Duane; Thomforde, George M; Baxter, Kari; McKinzie, Sanna; Zinsmeister, Alan R

    2006-05-01

    Chloride channels modulate gastrointestinal neuromuscular functions in vitro. Lubiprostone, a selective type 2 chloride channel (ClC-2) activator, induces intestinal secretion and has been shown to relieve constipation in clinical trials; however, the effects of lubiprostone on gastric function and whole gut transit in humans are unclear. Our aim was to compare the effects of the selective ClC-2 activator lubiprostone on maximum tolerated volume (MTV) of a meal, postprandial symptoms, gastric volumes, and gastrointestinal and colonic transit in humans. We performed a randomized, parallel-group, double-blind, placebo-controlled study evaluating the effects of lubiprostone (24 microg bid) in 30 healthy volunteers. Validated methods were used: scintigraphic gastrointestinal and colonic transit, SPECT to measure gastric volumes, and the nutrient drink ("satiation") test to measure MTV and postprandial symptoms. Lubiprostone accelerated small bowel and colonic transit, increased fasting gastric volume, and retarded gastric emptying. MTV values were reduced compared with placebo; however, the MTV was within the normal range for healthy adults in 13 of 14 participants, and there was no significant change compared with baseline measurements. Lubiprostone had no significant effect on postprandial gastric volume or aggregate symptoms but did decrease fullness 30 min after the fully satiating meal. Thus the ClC-2 activator lubiprostone accelerates small intestinal and colonic transit, which confers potential in the treatment of constipation.

  6. GlialCAM, a CLC-2 Cl(-) channel subunit, activates the slow gate of CLC chloride channels.

    PubMed

    Jeworutzki, Elena; Lagostena, Laura; Elorza-Vidal, Xabier; López-Hernández, Tania; Estévez, Raúl; Pusch, Michael

    2014-09-02

    GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl(-) channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction.

  7. Identifying interacting proteins of a Caenorhabditis elegans voltage-gated chloride channel CLH-1 using GFP-Trap and mass spectrometry.

    PubMed

    Zhou, Zi-Liang; Jiang, Jing; Yin, Jiang-An; Cai, Shi-Qing

    2014-06-25

    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.

  8. An mRNA encoding a putative GABA-gated chloride channel is expressed in the human cardiac conduction system.

    PubMed

    Garret, M; Bascles, L; Boue-Grabot, E; Sartor, P; Charron, G; Bloch, B; Margolskee, R F

    1997-04-01

    GABA-gated chloride channels are the main inhibitory neurotransmitter receptors in the CNS. Conserved domains among members of previously described GABAA receptor subunits were used to design degenerate sense and antisense oligonucleotides. A PCR product from this amplification was used to isolate a full-length cDNA. The predicted protein has many of the features shared by other members of the ligand-gated ion channel family. This channel subunit has significant amino acid identity (25-40%) with members of GABAA and GABAC receptor subunits and thus may represent a new subfamily of the GABA receptor channel. Although we cannot rule out that this clone encodes a receptor for an unidentified ligand, it was termed GABA chi. This gene is mainly expressed in placenta and in heart; however, placenta appears to express only an unspliced mRNA. In situ hybridization reveals that the GABA chi subunit mRNA is present in the electrical conduction system of the human heart. Our results suggest that novel GABA receptors expressed outside of the CNS may regulate cardiac function.

  9. Contribution of a leucine residue in the first transmembrane segment to the selectivity filter region in the CFTR chloride channel.

    PubMed

    Negoda, Alexander; El Hiani, Yassine; Cowley, Elizabeth A; Linsdell, Paul

    2017-05-01

    The anion selectivity and conductance of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are determined predominantly by interactions between permeant anions and the narrow region of the channel pore. This narrow region has therefore been described as functioning as the "selectivity filter" of the channel. Multiple pore-lining transmembrane segments (TMs) have previously been shown to contribute to the selectivity filter region. However, little is known about the three-dimensional organization of this region, or how multiple TMs combine to determine its functional properties. In the present study we have used patch clamp recording to identify changes in channel function associated with the formation of disulfide cross-links between cysteine residues introduced into different TMs within the selectivity filter. Cysteine introduced at position L102 in TM1 was able to form disulfide bonds with F337C and T338C in TM6, two positions that are known to play key roles in determining anion permeation properties. Consistent with this proximal arrangement of L102, F337 and T338, different mutations at L102 altered anion selectivity and conductance properties in a way that suggests that this residue plays an important role in determining selectivity filter function, albeit a much lesser role than that of F337. These results suggest an asymmetric three-dimensional arrangement of the key selectivity filter region of the pore, as well as having important implications regarding the molecular mechanism of anion permeation.

  10. Activation of P2Y1 and P2Y2 receptors induces chloride secretion via calcium-activated chloride channels in kidney inner medullary collecting duct cells

    PubMed Central

    Rajagopal, Madhumitha; Kathpalia, Paru P.; Thomas, Sheela V.

    2011-01-01

    Dysregulation of urinary sodium chloride (NaCl) excretion can result in extracellular fluid (ECF) volume expansion and hypertension. Recent studies demonstrated that urinary nucleotide excretion increases in mice ingesting a high-salt diet and that these increases in extracellular nucleotides can signal through P2Y2 receptors in the kidney collecting duct to inhibit epithelial Na+ channels (ENaC). However, under conditions of ECF volume expansion brought about by high-dietary salt intake, ENaC activity should already be suppressed. We hypothesized that alternative pathways exist by which extracellular nucleotides control renal NaCl excretion. We used an inner medullary collecting duct (mIMCD-K2) cell line in an Ussing chamber system as a model to study additional ion transport pathways that are regulated by extracellular nucleotides. When ENaC was inhibited, the addition of adenosine triphosphate (ATP) to the basal side of cell sheets activated both P2Y1 and P2Y2 receptors, inducing a transient increase in short-circuit current (Isc); addition of ATP to the apical side activated only P2Y2 receptors, inducing first a transient and then a sustained increase in Isc. The ATP-induced increases in Isc were blocked by pretreatment with a phospholipase C (PLC) inhibitor, a calcium (Ca2+) chelator, or Ca2+-activated Cl− channel (CACC) inhibitors, suggesting that ATP signals through both PLC and intracellular Ca2+ to activate CACC. We propose that P2Y1 and P2Y2 receptors operate in tandem in IMCD cells to provide an adaptive mechanism for enhancing urinary NaCl excretion in the setting of high-dietary NaCl intake. PMID:21653634

  11. Computational modeling of anoctamin 1 calcium-activated chloride channels as pacemaker channels in interstitial cells of Cajal

    PubMed Central

    Gibbons, Simon J.; Farrugia, Gianrico; Sneyd, James; Cheng, Leo K.

    2014-01-01

    Interstitial cells of Cajal (ICC) act as pacemaker cells in the gastrointestinal tract by generating electrical slow waves to regulate rhythmic smooth muscle contractions. Intrinsic Ca2+ oscillations in ICC appear to produce the slow waves by activating pacemaker currents, currently thought to be carried by the Ca2+-activated Cl− channel anoctamin 1 (Ano1). In this article we present a novel model of small intestinal ICC pacemaker activity that incorporates store-operated Ca2+ entry and a new model of Ano1 current. A series of simulations were carried out with the ICC model to investigate current controversies about the reversal potential of the Ano1 Cl− current in ICC and to predict the characteristics of the other ion channels that are necessary to generate slow waves. The model results show that Ano1 is a plausible pacemaker channel when coupled to a store-operated Ca2+ channel but suggest that small cyclical depolarizations may still occur in ICC in Ano1 knockout mice. The results predict that voltage-dependent Ca2+ current is likely to be negligible during the slow wave plateau phase. The model shows that the Cl− equilibrium potential is an important modulator of slow wave morphology, highlighting the need for a better understanding of Cl− dynamics in ICC. PMID:24481603

  12. Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore.

    PubMed Central

    Linsdell, P; Evagelidis, A; Hanrahan, J W

    2000-01-01

    Ionic selectivity in many cation channels is achieved over a short region of the pore known as the selectivity filter, the molecular determinants of which have been identified in Ca(2+), Na(+), and K(+) channels. However, a filter controlling selectivity among different anions has not previously been identified in any Cl(-) channel. In fact, because Cl(-) channels are only weakly selective among small anions, and because their selectivity has proved so resistant to site-directed mutagenesis, the very existence of a discrete anion selectivity filter has been called into question. Here we show that mutation of a putative pore-lining phenylalanine residue, F337, in the sixth membrane-spanning region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel, dramatically alters the relative permeabilities of different anions in the channel. Specifically, mutations that reduce the size of the amino acid side chain present at this position virtually abolish the relationship between anion permeability and hydration energy, a relationship that characterizes the anion selectivity not only of wild-type CFTR, but of most classes of Cl(-) channels. These results suggest that the pore of CFTR may indeed contain a specialized region, analogous to the selectivity filter of cation channels, at which discrimination between different permeant anions takes place. Because F337 is adjacent to another amino acid residue, T338, which also affects anion selectivity in CFTR, we suggest that selectivity is predominantly determined over a physically discrete region of the pore located near these important residues. PMID:10827976

  13. Single voltage-dependent chloride-selective channels of large conductance in cultured rat muscle.

    PubMed Central

    Blatz, A L; Magleby, K L

    1983-01-01

    Single-channel currents of an anion-selective channel in the plasma membrane of cultured rat muscle cells (myotubes) were recorded with the patch-clamp technique (Hamill, O.P., A. Marty, E. Neher, B. Sakmann, and F.J. Sigworth, 1981. Pfluegers Arch. Eur. J. Physiol., 391:85-100). The channel is selective for Cl- over cations, and has an unusually large single-channel conductance of approximately 430 pS in symmetrical 143 mM KCl. The channel is often active at 0 mV, opening and closing spontaneously. When active, steps from 0 mV to either negative or positive membrane potentials close the channel to an apparent inactivated state. The mean effective time that a channel is open before it inactivates is approximately 1.19 s for steps to -30 mV and 0.48 s for steps to +30 mV. Returning the membrane potential to 0 mV results in recovery from inactivation. Calcium ions are not required for channel activity. PMID:6311302

  14. Calcium entry via TRPC1 channels activates chloride currents in human glioma cells

    PubMed Central

    Cuddapah, Vishnu Anand; Turner, Kathryn L.; Sontheimer, Harald

    2012-01-01

    Malignant gliomas are highly-invasive brain cancers that carry a dismal prognosis. Recent studies indicate that Cl− channels facilitate glioma cell invasion by promoting hydrodynamic cell shape and volume changes. Here we asked how Cl− channels are regulated in the context of migration. Using patch-clamp recordings we show Cl− currents are activated by physiological increases of [Ca2+]i to 65 and 180 nM. Cl− currents appear to be mediated by ClC-3, a voltage-gated, CaMKII-regulated Cl− channel highly expressed by glioma cells. ClC-3 channels colocalized with TRPC1 on caveolar lipid rafts on glioma cell processes. Using perforated-patch electrophysiological recordings, we demonstrate that inducible knockdown of TRPC1 expression with shRNA significantly inhibited glioma Cl− currents in a Ca2+-dependent fashion, placing Cl− channels under the regulation of Ca2+ entry via TRPC1. In chemotaxis assays epidermal growth factor (EGF)-induced invasion was inhibition by TRPC1 knockdown to the same extent as pharmacological block of Cl− channels. Thus endogenous glioma Cl− channels are regulated by TRPC1. Cl− channels could be an important downstream target of TRPC1 in many other cells types, coupling elevations in [Ca2+]i to the shape and volume changes associated with migrating cells. PMID:23261316

  15. Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation*♦

    PubMed Central

    Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T.; Scheaffer, Suzanne M.; Roswit, William T.; Alevy, Yael G.; Patel, Anand C.; Heier, Richard F.; Romero, Arthur G.; Nichols, Colin G.; Holtzman, Michael J.; Brett, Tom J.

    2012-01-01

    The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface. PMID:23112050

  16. Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

    PubMed

    Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T; Scheaffer, Suzanne M; Roswit, William T; Alevy, Yael G; Patel, Anand C; Heier, Richard F; Romero, Arthur G; Nichols, Colin G; Holtzman, Michael J; Brett, Tom J

    2012-12-07

    The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.

  17. Permeation Mechanisms in the TMEM16B Calcium-Activated Chloride Channels

    PubMed Central

    2017-01-01

    TMEM16A and TMEM16B encode for Ca2+-activated Cl− channels (CaCC) and are expressed in many cell types and play a relevant role in many physiological processes. Here, I performed a site-directed mutagenesis study to understand the molecular mechanisms of ion permeation of TMEM16B. I mutated two positive charged residues R573 and K540, respectively located at the entrance and inside the putative channel pore and I measured the properties of wild-type and mutant TMEM16B channels expressed in HEK-293 cells using whole-cell and excised inside-out patch clamp experiments. I found evidence that R573 and K540 control the ion permeability of TMEM16B depending both on which side of the membrane the ion substitution occurs and on the level of channel activation. Moreover, these residues contribute to control blockage or activation by permeant anions. Finally, R573 mutation abolishes the anomalous mole fraction effect observed in the presence of a permeable anion and it alters the apparent Ca2+-sensitivity of the channel. These findings indicate that residues facing the putative channel pore are responsible both for controlling the ion selectivity and the gating of the channel, providing an initial understanding of molecular mechanism of ion permeation in TMEM16B. PMID:28046119

  18. Permeation Mechanisms in the TMEM16B Calcium-Activated Chloride Channels.

    PubMed

    Pifferi, Simone

    2017-01-01

    TMEM16A and TMEM16B encode for Ca2+-activated Cl- channels (CaCC) and are expressed in many cell types and play a relevant role in many physiological processes. Here, I performed a site-directed mutagenesis study to understand the molecular mechanisms of ion permeation of TMEM16B. I mutated two positive charged residues R573 and K540, respectively located at the entrance and inside the putative channel pore and I measured the properties of wild-type and mutant TMEM16B channels expressed in HEK-293 cells using whole-cell and excised inside-out patch clamp experiments. I found evidence that R573 and K540 control the ion permeability of TMEM16B depending both on which side of the membrane the ion substitution occurs and on the level of channel activation. Moreover, these residues contribute to control blockage or activation by permeant anions. Finally, R573 mutation abolishes the anomalous mole fraction effect observed in the presence of a permeable anion and it alters the apparent Ca2+-sensitivity of the channel. These findings indicate that residues facing the putative channel pore are responsible both for controlling the ion selectivity and the gating of the channel, providing an initial understanding of molecular mechanism of ion permeation in TMEM16B.

  19. Chloride channels are necessary for full platelet phosphatidylserine exposure and procoagulant activity.

    PubMed

    Harper, M T; Poole, A W

    2013-12-19

    Platelets enhance thrombin generation at sites of vascular injury by exposing phosphatidylserine during necrosis-like cell death. Anoctamin 6 (Ano6) is required for Ca(2+)-dependent phosphatidylserine exposure and is defective in patients with Scott syndrome, a rare bleeding disorder. Ano6 may also form Cl(-) channels, though the role of Cl(-) fluxes in platelet procoagulant activity has not been explored. We found that Cl(-) channel blockers or removal of extracellular Cl(-) inhibited agonist-induced phosphatidylserine exposure. However, this was not due to direct inhibition of Ca(2+)-dependent scrambling since Ca(2+) ionophore-induced phosphatidylserine exposure was normal. This implies that the role of Ano6 in Ca(2+-)dependent PS exposure is likely to differ from any putative function of Ano6 as a Cl(-) channel. Instead, Cl(-) channel blockade inhibited agonist-induced Ca(2+) entry. Importantly, Cl(-) channel blockers also prevented agonist-induced membrane hyperpolarization, resulting in depolarization. We propose that Cl(-) entry through Cl(-) channels is required for this hyperpolarization, maintaining the driving force for Ca(2+) entry and triggering full phosphatidylserine exposure. This demonstrates a novel role for Cl(-) channels in controlling platelet death and procoagulant activity.

  20. Variomics screen identifies the re-entrant loop of the calcium-activated chloride channel ANO1 that facilitates channel activation.

    PubMed

    Bill, Anke; Popa, M Oana; van Diepen, Michiel T; Gutierrez, Abraham; Lilley, Sarah; Velkova, Maria; Acheson, Kathryn; Choudhury, Hedaythul; Renaud, Nicole A; Auld, Douglas S; Gosling, Martin; Groot-Kormelink, Paul J; Gaither, L Alex

    2015-01-09

    The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases.

  1. Variomics Screen Identifies the Re-entrant Loop of the Calcium-activated Chloride Channel ANO1 That Facilitates Channel Activation*

    PubMed Central

    Bill, Anke; Popa, M. Oana; van Diepen, Michiel T.; Gutierrez, Abraham; Lilley, Sarah; Velkova, Maria; Acheson, Kathryn; Choudhury, Hedaythul; Renaud, Nicole A.; Auld, Douglas S.; Gosling, Martin; Groot-Kormelink, Paul J.; Gaither, L. Alex

    2015-01-01

    The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases. PMID:25425649

  2. Molecular and functional analyses of two new calcium-activated chloride channel family members from mouse eye and intestine.

    PubMed

    Evans, Stella R; Thoreson, Wallace B; Beck, Carol L

    2004-10-01

    Two new calcium-activated chloride channel (CLCA) family members, mCLCA5 and mCLCA6, have been cloned from mouse eye and intestine, respectively. mCLCA5 is highly homologous to hCLCA2, and mCLCA6 is highly homologous to hCLCA4. mCLCA5 is widely expressed with strong expression in eye and spleen, whereas mCLCA6 is primarily expressed in intestine and stomach. mCLCA6 is also expressed as a splice variant lacking exon 8 and part of exon 10 in intestine and stomach. Transfection of tsA201 cells with enhanced green fluorescent protein-tagged versions of the three cDNAs reveals protein products of 155 and 65 kDa for mCLCA5 and mCLCA6 and 145 and 65 kDa for the mCLCA6 splice variant. In vitro translation of mCLCA5 generates a 90-kDa protein that does not appear to be glycosylated. mCLCA6 also generates a 90-kDa protein that is glycosylated to a 110-kDa product, whereas the mCLCA6 splice variant generates an 80-kDa product that is 100 kDa after glycosylation. Treatment of enhanced green fluorescent protein-tagged mCLCA6 with PNGase F (peptide: N-glycosidase F) to remove N-linked glycosyl groups shows a reduction in size of the 65 kDa product to 60 kDa. Consistent with the hypothesis that mCLCA5, mCLCA6, and its splice variant encode calcium-activated chloride channels, in HEK293 cells expressing CLCAs ionomycin-evoked increases in intracellular calcium stimulated a current that reversed near Cl(-) equilibrium potential, E(Cl). Furthermore, these currents were inhibited by the chloride channel blocker niflumic acid. Given the prominent role of hCLCA2 in cancer cell adhesion and the unique high level of expression of hCLCA4 in brain, the identification of their murine counterparts presents the opportunity to clarify the role of CLCAs in disease and normal cell physiology.

  3. Novel residues lining the CFTR chloride channel pore identified by functional modification of introduced cysteines.

    PubMed

    Fatehi, Mohammad; Linsdell, Paul

    2009-04-01

    Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current-voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl(-) permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.

  4. Calcium-Activated Chloride Channels (CaCCs) Regulate Action Potential and Synaptic Response in Hippocampal Neurons

    PubMed Central

    Huang, Wendy C.; Xiao, Shaohua; Huang, Fen; Harfe, Brian D.; Jan, Yuh Nung; Jan, Lily Yeh

    2012-01-01

    SUMMARY Central neurons respond to synaptic inputs from other neurons by generating synaptic potentials. Once the summated synaptic potentials reach threshold for action potential firing, the signal propagates leading to transmitter release at the synapse. The calcium influx accompanying such signaling opens calcium-activated ion channels for feedback regulation. Here we report a novel mechanism for modulating hippocampal neuronal signaling that involves calcium-activated chloride channels (CaCCs). We present the first evidence that CaCCs reside in hippocampal neurons and are in close proximity of calcium channels and NMDA receptors to shorten action potential duration, dampen excitatory synaptic potentials, impede temporal summation, and raise the threshold for action potential generation by synaptic potential. Having recently identified TMEM16A and TMEM16B as CaCCs, we further show that TMEM16B but not TMEM16A is important for hippocampal CaCC, laying the groundwork for deciphering the dynamic CaCC modulation of neuronal signaling in neurons important for learning and memory. PMID:22500639

  5. Dissection of the Mechanical Impedance Components of the Outer Hair Cell Using a Chloride-Channel Blocker

    NASA Astrophysics Data System (ADS)

    Harasztosi, Csaba; Gummer, Anthony W.

    2011-11-01

    The voltage-dependent chloride-channel blocker anthracene-9-carboxylic acid (9AC) has been found to reduce the imaginary but not the real part of the mechanical impedance of the organ of Corti, suggesting that the effective stiffness of outer hair cells (OHCs) is reduced by 9AC. To examine whether 9AC interacts directly with the motor protein prestin to reduce the membrane component of the impedance, the patch-clamp technique in whole-cell configuration was used to measure the nonlinear capacitance (NLC) of isolated OHCs and, as control, prestin-transfected human embryonic kidney 293 (HEK293) cells. Extracellular application of 9AC significantly reduced the NLC of both OHCs and HEK293 cells. Intracellular 9AC did not influence the blocking effect of the extracellular applied drug. These results suggest that 9AC interacts directly with prestin, reducing the effective stiffness of the motor, and that the interaction is extracellular.

  6. Synthesis of 4-thiophen-2'-yl-1,4-dihydropyridines as potentiators of the CFTR chloride channel.

    PubMed

    Cateni, Francesca; Zacchigna, Marina; Pedemonte, Nicoletta; Galietta, Luis J V; Mazzei, Marco T; Fossa, Paola; Giampieri, Michele; Mazzei, Mauro

    2009-12-01

    The gating of the CFTR chloride channel is altered by a group of mutations that cause cystic fibrosis. This gating defect may be corrected by small molecules called potentiators. Some 1,4-dihydropyridine (DHP) derivatives, bearing a thiophen-2-yl and a furanyl ring at the 4-position of the nucleus, were prepared and tested as CFTR potentiators. In particular, we evaluated the ability of novel DHPs to enhance the activity of the rescued DeltaF508-CFTR as measured with a functional assay based on the halide-sensitive yellow fluorescent protein. Most DHPs showed an effect comparable to or better than that of the reference compound genistein. The potency was instead significantly improved, with some compounds, such as 3g, 3h, 3n, 4a, 4b, and 4d, having a half effective concentration in the submicromolar range. CoMFA analysis gave helpful suggestions to improve the activity of DHPs.

  7. A voltage-dependent chloride channel fine-tunes photosynthesis in plants

    PubMed Central

    Herdean, Andrei; Teardo, Enrico; Nilsson, Anders K.; Pfeil, Bernard E.; Johansson, Oskar N.; Ünnep, Renáta; Nagy, Gergely; Zsiros, Ottó; Dana, Somnath; Solymosi, Katalin; Garab, Győző; Szabó, Ildikó; Spetea, Cornelia; Lundin, Björn

    2016-01-01

    In natural habitats, plants frequently experience rapid changes in the intensity of sunlight. To cope with these changes and maximize growth, plants adjust photosynthetic light utilization in electron transport and photoprotective mechanisms. This involves a proton motive force (PMF) across the thylakoid membrane, postulated to be affected by unknown anion (Cl−) channels. Here we report that a bestrophin-like protein from Arabidopsis thaliana functions as a voltage-dependent Cl− channel in electrophysiological experiments. AtVCCN1 localizes to the thylakoid membrane, and fine-tunes PMF by anion influx into the lumen during illumination, adjusting electron transport and the photoprotective mechanisms. The activity of AtVCCN1 accelerates the activation of photoprotective mechanisms on sudden shifts to high light. Our results reveal that AtVCCN1, a member of a conserved anion channel family, acts as an early component in the rapid adjustment of photosynthesis in variable light environments. PMID:27216227

  8. The secret life of CFTR as a calcium-activated chloride channel

    PubMed Central

    Billet, Arnaud; Hanrahan, John W

    2013-01-01

    cAMP-stimulated anion conductance is defective in cystic fibrosis (CF). The regulatory domain of CFTR, the anion channel protein encoded by the CF gene, possesses an unusually high density of consensus sequences for phosphorylation by protein kinase A (14 in a stretch of <200 amino acids). Thus it is not surprising that CFTR is viewed primarily as a cAMP-stimulated anion channel, and most studies have focused on this mode of activation. However, there is growing evidence that CFTR also responds to Ca2+-mobilizing secretagogues and contributes substantially to cholinergic and purinergic responses in native tissues. G protein-coupled receptors that signal through Gαq can stimulate CFTR channels by activating Ca2+-dependent adenylyl cyclase and tyrosine kinases, and also by inhibiting protein phosphatase type 2A. Here we review evidence for these novel mechanisms of CFTR activation and discuss how they may help explain previous observations. PMID:23959675

  9. The secret life of CFTR as a calcium-activated chloride channel.

    PubMed

    Billet, Arnaud; Hanrahan, John W

    2013-11-01

    cAMP-stimulated anion conductance is defective in cystic fibrosis (CF). The regulatory domain of CFTR, the anion channel protein encoded by the CF gene, possesses an unusually high density of consensus sequences for phosphorylation by protein kinase A (14 in a stretch of <200 amino acids). Thus it is not surprising that CFTR is viewed primarily as a cAMP-stimulated anion channel, and most studies have focused on this mode of activation. However, there is growing evidence that CFTR also responds to Ca(2+)-mobilizing secretagogues and contributes substantially to cholinergic and purinergic responses in native tissues. G protein-coupled receptors that signal through Gαq can stimulate CFTR channels by activating Ca(2+)-dependent adenylyl cyclase and tyrosine kinases, and also by inhibiting protein phosphatase type 2A. Here we review evidence for these novel mechanisms of CFTR activation and discuss how they may help explain previous observations.

  10. Phosphorylation and IGF-1-mediated dephosphorylation pathways control the activity and the pharmacological properties of skeletal muscle chloride channels

    PubMed Central

    De Luca, Annamaria; Pierno, Sabata; Liantonio, Antonella; Camerino, Claudia; Conte Camerino, Diana

    1998-01-01

    In the present study we tested the hypothesis that insulin-like growth factor-1 (IGF-1) modulates resting chloride conductance (GCl) of rat skeletal muscle by activating a phosphatase and that the chloride channel, based on the activity of phosphorylating-dephosphorylating pathways, has different sensitivity to specific ligands, such as the enantiomers of 2-(p-chlorophenoxy) propionic acid (CPP).For this purpose GCl in EDL muscle isolated from adult rat was first lowered by treatment with 5 nM 4-β-phorbol 12,13 dibutyrate (4-β-PDB), presumably activating protein kinase C (PKC). The effects of IGF-1 and of the enantiomers of CPP on GCl were then tested.IGF-1 (3.3 nM) had no effect of GCl on EDL muscle fibres in normal physiological solution, whereas it completely counteracted the 30% decrease of GCl induced by 4-β-PDB. No effects of IGF-1 were observed on GCl lowered by the phosphatase inhibitor okadaic acid (0.25 μM).Ceramide, reported to activate on okadaic acid-sensitive phosphatase, mimicked the effects of IGF-1. In fact, N-acetyl-sphingosine (2.5–5 μM), not very effective in control conditions, increased the GCl lowered by the phorbol ester, but not the GCl lowered by okadaic acid.In the presence of 4-β-PDB, GCl was differently affected by the enantiomers of CPP. The S(−)-CPP was remarkably less potent in producing the concentration-dependent reduction of GCl, whereas the R(+)-CPP caused an increase of GCl at all the concentrations tested.In conclusion, the PKC-induced lowering of GCl is counteracted by IGF-1 through an okadaic acid sensitive phosphatase, and this effect can have therapeutic relevance in situations characterized by excessive channel phosphorylation. In turn the phosphorylation state of the channel can modulate the effects and the therapeutic potential of direct channel ligands. PMID:9806330

  11. Interactions between permeant and blocking anions inside the CFTR chloride channel pore.

    PubMed

    Linsdell, Paul

    2015-07-01

    Binding of cytoplasmic anionic open channel blockers within the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is antagonized by extracellular Cl(-). In the present work, patch clamp recording was used to investigate the interaction between extracellular Cl(-) (and other anions) and cytoplasmic Pt(NO2)4(2-) ions inside the CFTR channel pore. In constitutively open (E1371Q-CFTR) channels, these different anions bind to two separate sites, located in the outer and inner vestibules of the pore respectively, in a mutually antagonistic fashion. A mutation in the inner vestibule (I344K) that greatly increased Pt(NO2)4(2-) binding affinity also greatly strengthened antagonistic Cl(-):blocker interactions as well as the voltage-dependence of block. Quantitative analysis of ion binding affinity suggested that the I344K mutation strengthened interactions not only with intracellular Pt(NO2)4(2-) ions but also with extracellular Cl(-), and that altered blocker Cl(-)- and voltage-dependence were due to the introduction of a novel type of antagonistic ion:ion interaction inside the pore that was independent of Cl(-) binding in the outer vestibule. It is proposed that this mutation alters the arrangement of anion binding sites inside the pore, allowing both Cl(-) and Pt(NO2)4(2-) to bind concurrently within the inner vestibule in a strongly mutually antagonistic fashion. However, the I344K mutation does not increase single channel conductance following disruption of Cl(-) binding in the outer vestibule in R334Q channels. Implications for the arrangement of ion binding sites in the pore, and their functional consequences for blocker binding and for rapid Cl(-) permeation, are discussed.

  12. Presynaptic Localization and Possible Function of Calcium-Activated Chloride Channel Anoctamin 1 in the Mammalian Retina

    PubMed Central

    Chun, Myung-Hoon; Oh, Uhtaek; Kim, In-Beom

    2013-01-01

    Calcium (Ca2+)-activated chloride (Cl−) channels (CaCCs) play a role in the modulation of action potentials and synaptic responses in the somatodendritic regions of central neurons. In the vertebrate retina, large Ca2+-activated Cl− currents (ICl(Ca)) regulate synaptic transmission at photoreceptor terminals; however, the molecular identity of CaCCs that mediate ICl(Ca) remains unclear. The transmembrane protein, TMEM16A, also called anoctamin 1 (ANO1), has been recently validated as a CaCC and is widely expressed in various secretory epithelia and nervous tissues. Despite the fact that tmem16a was first cloned in the retina, there is little information on its cellular localization and function in the mammalian retina. In this study, we found that ANO1 was abundantly expressed as puncta in 2 synaptic layers. More specifically, ANO1 immunoreactivity was observed in the presynaptic terminals of various retinal neurons, including photoreceptors. ICl(Ca) was first detected in dissociated rod bipolar cells expressing ANO1. ICl(Ca) was abolished by treatment with the Ca2+ channel blocker Co2+, the L-type Ca2+ channel blocker nifedipine, and the Cl− channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and niflumic acid (NFA). More specifically, a recently discovered ANO1-selective inhibitor, T16Ainh-A01, and a neutralizing antibody against ANO1 inhibited ICl(Ca) in rod bipolar cells. Under a current-clamping mode, the suppression of ICl(Ca) by using NPPB and T16Ainh-A01 caused a prolonged Ca2+ spike-like depolarization evoked by current injection in dissociated rod bipolar cells. These results suggest that ANO1 confers ICl(Ca) in retinal neurons and acts as an intrinsic regulator of the presynaptic membrane potential during synaptic transmission. PMID:23840801

  13. Emodin augments calcium activated chloride channel in colonic smooth muscle cells by Gi/Go protein.

    PubMed

    Xu, Long; Ting-Lou; Lv, Nonghua; Zhu, Xuan; Chen, Youxiang; Yang, Jing

    2009-08-01

    Emodin is a natural anthraquinone in rhubarb. It has been identified as a prokinetic drug for gastrointestinal motility in Chinese traditional medicine. Emodin contracts smooth muscle by increasing the concentration of intracellular Ca(2+). In many smooth muscles, increasing intracellular Ca(2+) activates Ca(2+)-activated Cl(-) channels (ClCA). The study was aimed to investigate the effects of emodin on ClCA channels in colonic smooth muscle. 4 channel physiology signal acquire system was used to measure isometric contraction of smooth muscle strips. ClCA currents were recorded by EPC10 with perforated whole cell model. Emodin contracted strips and cells in colonic smooth muscle and augmented ClCA currents. Niflumic acid (NFA) and 4', 4'-diisothiostilbene-2, 2-disulfonic acid (DIDS) blocked the effects. Gi/Go protein inhibits protein kinase A (PKA) and protein kinase C (PKC), and PKA and PKC reduced ClCA currents. Pertussis toxin (PTX, a special inhibitor of Gi/Go protein), 8-bromoadenosine 38, 58-cyclic monophosphate (8-BrcAMP, a membrane-permeant protein kinase A activator) and Phorbol-12-myristate-13-acetate (PMA, a membrane-permeant protein kinase C activator) inhibited the effects on ClCA currents significantly. Our findings suggest that emodin augments ClCA channels to contract smooth muscle in colon, and the effect is induced mostly by enhancement of membrane Gi/Go protein signal transducer pathway.

  14. Assignment of the genes encoding the human chloride channels, CLCNKA and CLCNKB, to 1p36 and of CLCN3 to 4q32-q33 by in situ hybridization

    SciTech Connect

    Saito-Ohara, Fumiko; Uchida, Shinichi; Takeuchi, Yasuo

    1996-09-01

    This report describes the localization of the genes encoding the human chloride channels, CLCNKA and CLCNKB, to human chromosome 1p36 and of CLCN3 to human chromosome 4q32-33 using fluorescence in situ hybridization. Mutations in these voltage-gated chloride channel genes have been implicated in various hereditary diseases. 18 refs., 1 fig.

  15. Downregulation of chloride channel ClC-2 by Janus kinase 3.

    PubMed

    Warsi, Jamshed; Elvira, Bernat; Hosseinzadeh, Zohreh; Shumilina, Ekaterina; Lang, Florian

    2014-05-01

    Janus kinase-3 (JAK3) fosters proliferation and counteracts apoptosis of lymphocytes and tumor cells. The gain of function mutation (A572V)JAK3 has been discovered in acute megakaryoplastic leukemia. JAK3 is inactivated by replacement of lysine by alanine in the catalytic subunit ((K855A)JAK3). Regulation of cell proliferation and apoptosis involves altered activity of Cl(-) channels. The present study, thus, explored whether JAK3 modifies the function of the small conductance Cl(-) channel ClC-2. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild-type JAK3, (A568V)JAK3 or (K851A)JAK3, and the Cl(-) channel activity determined by dual-electrode voltage clamp. Channel protein abundance in the cell membrane was determined utilizing chemiluminescence. As a result, expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK3 or (A568V)JAK3, but not by coexpression of (K851A)JAK3. Exposure of the oocytes expressing ClC-2 together with (A568V)JAK3 to the JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 μM) increased the conductance. Coexpression of (A568V)JAK3 decreased the ClC-2 protein abundance in the cell membrane of ClC-2 expressing oocytes. The decline of conductance in ClC-2 and (A568V)JAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 μM) was similar in oocytes expressing ClC-2 with (A568V)JAK3 and oocytes expressing ClC-2 alone, indicating that (A568V)JAK3 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK3 downregulates ClC-2 activity and thus counteracts Cl(-) exit-an effect possibly influencing cell proliferation and apoptosis.

  16. Expression of calcium-activated chloride channels Ano1 and Ano2 in mouse taste cells.

    PubMed

    Cherkashin, Alexander P; Kolesnikova, Alisa S; Tarasov, Michail V; Romanov, Roman A; Rogachevskaja, Olga A; Bystrova, Marina F; Kolesnikov, Stanislav S

    2016-02-01

    Specialized Ca(2+)-dependent ion channels ubiquitously couple intracellular Ca(2+) signals to a change in cell polarization. The existing physiological evidence suggests that Ca(2+)-activated Cl(-) channels (CaCCs) are functional in taste cells. Because Ano1 and Ano2 encode channel proteins that form CaCCs in a variety of cells, we analyzed their expression in mouse taste cells. Transcripts for Ano1 and Ano2 were detected in circumvallate (CV) papillae, and their expression in taste cells was confirmed using immunohistochemistry. When dialyzed with CsCl, taste cells of the type III exhibited no ion currents dependent on cytosolic Ca(2+). Large Ca(2+)-gated currents mediated by TRPM5 were elicited in type II cells by Ca(2+) uncaging. When TRPM5 was inhibited by triphenylphosphine oxide (TPPO), ionomycin stimulated a small but resolvable inward current that was eliminated by anion channel blockers, including T16Ainh-A01 (T16), a specific Ano1 antagonist. This suggests that CaCCs, including Ano1-like channels, are functional in type II cells. In type I cells, CaCCs were prominently active, blockable with the CaCC antagonist CaCCinh-A01 but insensitive to T16. By profiling Ano1 and Ano2 expressions in individual taste cells, we revealed Ano1 transcripts in type II cells only, while Ano2 transcripts were detected in both type I and type II cells. P2Y agonists stimulated Ca(2+)-gated Cl(-) currents in type I cells. Thus, CaCCs, possibly formed by Ano2, serve as effectors downstream of P2Y receptors in type I cells. While the role for TRPM5 in taste transduction is well established, the physiological significance of expression of CaCCs in type II cells remains to be elucidated.

  17. The CLC-2 Chloride Channel Modulates ECM Synthesis, Differentiation, and Migration of Human Conjunctival Fibroblasts via the PI3K/Akt Signaling Pathway.

    PubMed

    Sun, Lixia; Dong, Yaru; Zhao, Jing; Yin, Yuan; Zheng, Yajuan

    2016-06-09

    Recent evidence suggests that chloride channels are critical for cell proliferation, migration, and differentiation. We examined the effects of transforming growth factor (TGF)-β1 on chloride channel expression and associations with human conjunctival fibroblast (HConF) biology. To investigate the potential role of chloride channel (CLC)-2 in migration, transition to myofibroblasts and extracellular matrix (ECM) synthesis of HconF, a small interfering RNA (siRNA) approach was applied. TGF-β1-induced migration and transition of fibroblasts to myofibroblasts characterized by α-smooth muscle actin (α-SMA) expression, supported by increased endogenous expression of CLC-2 protein and mRNA transcripts. ECM (collagen I and fibronectin) synthesis in HConF was enhanced by TGF-β1. CLC-2 siRNA treatment reduced TGF-β1-induced cell migration, transition of fibroblasts to myofibroblasts, and ECM synthesis of HConF. CLC-2 siRNA treatment in the presence of TGF-β1 inhibited phosphorylation of PI3K and Akt in HConF. These findings demonstrate that CLC-2 chloride channels are important for TGF-β1-induced migration, differentiation, and ECM synthesis via PI3K/Akt signaling in HConF.

  18. Voltage-dependent gating mechanism for single fast chloride channels from rat skeletal muscle.

    PubMed Central

    Weiss, D S; Magleby, K L

    1992-01-01

    1. A voltage-dependent gating mechanism for the fast Cl- channel was developed from the analysis of single-channel current records obtained with the patch clamp technique from primary cultures of rat skeletal muscle. Up to 10(6) open and shut intervals were analysed from each of five different excised patches of membrane containing a single fast Cl- channel. 2. Rate constants for a kinetic scheme with six closed and two open states (scheme I) were estimated at a given voltage by maximum likelihood fitting of open and closed dwell-time distributions obtained at that voltage. This procedure was then repeated for data obtained at each of three to eight different membrane potentials for each channel. 3. Plots of the estimated rate constants against membrane potential typically appeared linear on semilogarithmic co-ordinates, consistent with rate constants that are exponentially dependent on voltage. 4. Regression analysis of these plots yielded two parameters for each rate constant: the value of the rate constant at -50 mV (B) and its voltage sensitivity (A). The dwell-time distributions predicted with these parameters and scheme I gave a good description of the experimental dwell-time distributions at all the studied voltages, lending further support for an exponential dependence of rate constants on membrane potential. 5. Estimates of A and B were also obtained by simultaneously fitting dwell-time distributions obtained at three to eight different voltages, in order to better define these parameters. Predicted dwell-time distributions obtained with these estimates and scheme I could approach the theoretical best description of the data for discrete-state Markov models. 6. Eight to twelve of the fourteen rate constants in scheme I appeared voltage sensitive, with effective gating charges ranging from about -1.5 to +1.0 units of electronic charge. 7. The estimated rate constants and their voltage sensitivities for the five analysed channels were generally similar, but

  19. Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions.

    PubMed

    Linsdell, P; Tabcharani, J A; Rommens, J M; Hou, Y X; Chang, X B; Tsui, L C; Riordan, J R; Hanrahan, J W

    1997-10-01

    Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (P/P) sequence NO > Cl > HCO > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (P/P < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of approximately 5.3 A. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.

  20. Ginsenoside Rb1, a novel activator of the TMEM16A chloride channel, augments the contraction of guinea pig ileum.

    PubMed

    Guo, Shuai; Chen, Yafei; Pang, Chunli; Wang, Xuzhao; Qi, Jinlong; Mo, Li; Zhang, Hailin; An, Hailong; Zhan, Yong

    2017-01-25

    Calcium-activated chloride channels (CaCCs) play important roles in many physiological processes, and the molecular basis of CaCCs has been identified as TMEM16A in many cell types. It is well established that TMEM16A is a drug target in many diseases, including cystic fibrosis, hypertension, asthma, and various tumors. Therefore, identifying potent and specific modulators of the TMEM16A channel is crucial. In this study, we identified the first natural activator of TMEM16A from traditional Chinese medicine and explored its mechanism. Our data showed that Ginsenoside Rb1 (GRb1) can activate TMEM16A directly from the intracellular side in a dose-dependent manner at an EC50 of 38.4 ± 2.14 μM. GRb1 specifically activated TMEM16A/B, but not the other previously proposed CaCC mediators such as CFTR and bestrophin. Moreover, GRb1 promoted proliferation of CHO cells stably expressing TMEM16A, in a concentration-dependent manner. Finally, we showed that GRb1 increased the amplitude and frequency of contractions in an isolated guinea pig ileum assay in vivo. In summary, GRb1 can be considered a lead compound for the development of novel drugs for the treatment of diseases caused by TMEM16A dysfunction.

  1. Clcn2 encodes the hyperpolarization-activated chloride channel in the ducts of mouse salivary glands

    PubMed Central

    Romanenko, Victor G.; Nakamoto, Tetsuji; Catalán, Marcelo A.; Gonzalez-Begne, Mireya; Schwartz, George J.; Jaramillo, Yasna; Sepúlveda, Francisco V.; Figueroa, Carlos D.; Melvin, James E.

    2008-01-01

    Transepithelial Cl− transport in salivary gland ducts is a major component of the ion reabsorption process, the final stage of saliva production. It was previously demonstrated that a Cl− current with the biophysical properties of ClC-2 channels dominates the Cl− conductance of unstimulated granular duct cells in the mouse submandibular gland. This inward-rectifying Cl− current is activated by hyperpolarization and elevated intracellular Cl− concentration. Here we show that ClC-2 immunolocalized to the basolateral region of acinar and duct cells in mouse salivary glands, whereas its expression was most robust in granular and striated duct cells. Consistent with this observation, nearly 10-fold larger ClC-2-like currents were observed in granular duct cells than the acinar cells obtained from submandibular glands. The loss of inward-rectifying Cl− current in cells from Clcn2−/− mice confirmed the molecular identity of the channel responsible for these currents as ClC-2. Nevertheless, both in vivo and ex vivo fluid secretion assays failed to identify significant changes in the ion composition, osmolality, or salivary flow rate of Clcn2−/− mice. Additionally, neither a compensatory increase in Cftr Cl− channel protein expression nor in Cftr-like Cl− currents were detected in Clcn2 null mice, nor did it appear that ClC-2 was important for blood-organ barrier function. We conclude that ClC-2 is the inward-rectifying Cl− channel in duct cells, but its expression is not apparently required for the ion reabsorption or the barrier function of salivary ductal epithelium. PMID:18801913

  2. Mechanism of interaction of niflumic acid with heterologously expressed kidney CLC-K chloride channels.

    PubMed

    Picollo, Alessandra; Liantonio, Antonella; Babini, Elena; Camerino, Diana Conte; Pusch, Michael

    2007-04-01

    CLC-K Cl(-) channels belong to the CLC protein family. In kidney and inner ear, they are involved in transepithelial salt transport. Mutations in ClC-Kb lead to Bartter's syndrome, and mutations in the associated subunit barttin produce Bartter's syndrome and deafness. We have previously found that 3-phenyl-CPP blocks hClC-Ka and rClC-K1 from the extracellular side in the pore entrance. Recently, we have shown that niflumic acid (NFA), a nonsteroidal anti-inflammatory fenamate, produces biphasic behavior on human CLC-K channels that suggests the presence of two functionally different binding sites: an activating site and a blocking site. Here, we investigate in more detail the interaction of NFA on CLC-K channels. Mutants that altered block by 3-phenyl-2-(p-chlorophenoxy)propionic acid (CPP) had no effect on NFA block, indicating that the inhibition binding site of NFA is different from that of 3-phenyl-CPP and flufenamic acid. Moreover, NFA does not compete with extracellular Cl(-) ions, suggesting that the binding sites of NFA are not located deep in the pore. Differently from ClC-Ka, on the rat homologue ClC-K1, NFA has only an inhibitory effect. We developed a quantitative model to describe the complex action of NFA on ClC-Ka. The model predicts that ClC-Ka possesses two NFA binding sites: when only one site is occupied, NFA increases ClC-Ka currents, whereas the occupation of both binding sites leads to channel block.

  3. Flow-activated chloride channels in vascular endothelium. Shear stress sensitivity, desensitization dynamics, and physiological implications.

    PubMed

    Gautam, Mamta; Shen, Yue; Thirkill, Twanda L; Douglas, Gordon C; Barakat, Abdul I

    2006-12-01

    Although activation of outward rectifying Cl(-) channels is one of the fastest responses of endothelial cells (ECs) to shear stress, little is known about these channels. In this study, we used whole-cell patch clamp recordings to characterize the flow-activated Cl(-) current in bovine aortic ECs (BAECs). Application of shear stress induced rapid development of a Cl(-) current that was effectively blocked by the Cl(-) channel antagonist 5-nitro-2-(3-phenopropylamino)benzoic acid (100 microM). The current initiated at a shear stress as low as 0.3 dyne/cm(2), attained its peak within minutes of flow onset, and saturated above 3.5 dynes/cm(2) approximately 2.5-3.5-fold increase over pre-flow levels). The Cl(-) current desensitized slowly in response to sustained flow, and step increases in shear stress elicited increased current only if the shear stress levels were below the 3.5 dynes/cm(2) saturation level. Oscillatory flow with a physiological oscillation frequency of 1 Hz, as occurs in disturbed flow zones prone to atherosclerosis, failed to elicit the Cl(-) current, whereas lower oscillation frequencies led to partial recovery of the current. Nonreversing pulsatile flow, generally considered protective of atherosclerosis, was as effective in eliciting the current as steady flow. Measurements using fluids of different viscosities indicated that the Cl(-) current is responsive to shear stress rather than shear rate. Blocking the flow-activated Cl(-) current abolished flow-induced Akt phosphorylation in BAECs, whereas blocking flow-sensitive K(+) currents had no effect, suggesting that flow-activated Cl(-) channels play an important role in regulating EC flow signaling.

  4. Inhibition of collateral artery growth by mibefradil: possible role of volume-regulated chloride channels.

    PubMed

    Ziegelhoeffer, Tibor; Scholz, Dimitri; Friedrich, Christian; Helisch, Armin; Wagner, Shawn; Fernandez, Borja; Schaper, Wolfgang

    2003-01-01

    Endothelial cell swelling is one of the earliest hallmarks of arteriogenesis, the growth and maturation of collaterals. Mibefradil was found to block endothelial Cl(-) channels that control the volume of endothelial cells. Thus the authors investigated whether the blockade of volume-controlling endothelial cell channels would translate into an inhibition of arteriogenesis. In BALB/c mice, the right femoral artery was ligated and the animals received either mibefradil or solvent (phosphate-buffered saline [PBS]) via osmotic minipumps. Laser Doppler perfusion ratio (R/L) of ligated versus nonligated distal hindlimb increased from 0.06 +/- 0.01 (immediately after ligation) to 0.25 +/- 0.02 (day 7) in the PBS group and only from 0.07 +/- 0.02 to 0.13 +/- 0.02 in the mibefradil group (p <.01). Collateral artery diameters were significantly smaller in the mibefradil group (61 +/- 4.7 microm) versus controls (77.3 +/- 0.9 microm) (p <.05). Relative hemoglobin oxygen saturation measurements confirmed these findings (p <.02). The inhibition of arteriogenesis in the mibefradil group suggests that endothelial Cl(-) channels are involved in the initiation of arteriogenesis.

  5. Structural dynamics of soluble chloride intracellular channel protein CLIC1 examined by amide hydrogen-deuterium exchange mass spectrometry.

    PubMed

    Stoychev, Stoyan H; Nathaniel, Christos; Fanucchi, Sylvia; Brock, Melissa; Li, Sheng; Asmus, Kyle; Woods, Virgil L; Dirr, Heini W

    2009-09-08

    Chloride intracellular channel protein 1 (CLIC1) functions as an anion channel in plasma and nuclear membranes when its soluble monomeric form converts to an integral-membrane form. The transmembrane region of CLIC1 is located in its thioredoxin-like domain 1, but the mechanism whereby the protein converts to its membrane conformation has yet to be determined. Since channel formation in membranes is enhanced at low pH (5 to 5.5), a condition that is found at the surface of membranes, the structural dynamics of soluble CLIC1 was studied at pH 7 and at pH 5.5 in the absence of membranes by amide hydrogen-deuterium exchange mass spectrometry (DXMS). Rapid hydrogen exchange data indicate that CLIC1 displays a similar core structure at these pH values. Domain 1 is less stable than the all-helical domain 2, and, while the structure of domain 1 remains intact, its conformational flexibility is further increased in an acidic environment (pH 5.5). In the absence of membrane, an acidic environment appears to prime the solution structure of CLIC1 by destabilizing domain 1 in order to lower the activation energy barrier for its conversion to the membrane-insertion conformation. The significantly enhanced H/D-exchange rates at pH 5.5 displayed by two segments (peptides 11-31 and 68-82) could be due to the protonation of acidic residues in salt bridges. One of these segments (peptide 11-31) includes part of the transmembrane region which, in the solution structure, consists of helix alpha1. This helix is intrinsically stable and is most likely retained in the membrane conformation. Strand beta2, another element of the transmembrane region, displays a propensity to form a helical structure and has putative N- and C-capping motifs, suggesting that it too most likely forms a helix in a lipid bilayer.

  6. Interaction of hydrophobic anions with the rat skeletal muscle chloride channel ClC-1: effects on permeation and gating

    PubMed Central

    Rychkov, Grigori Y; Pusch, Michael; Roberts, Michael L; Bretag, Allan H

    2001-01-01

    Permeation of a range of hydrophobic anions through the rat skeletal muscle chloride channel, rClC-1, expressed in Sf-9 (a Spodoptera frugiperda insect cell line) cells has been studied using the whole-cell patch-clamp technique. Bi-ionic reversal potentials measured with external application of foreign anions gave the following permeability sequence: Cl− (1) ≫ benzoate (0·15) ≫ hexanoate (0·12) ≫ butyrate (0·09) ≫ propionate (0·047) x∼ formate (0·046). Anions with larger hydrophobic moieties were more permeant, which suggested that ClC-1 selectivity for hydrophobic anions is dominated by their interaction with a hydrophobic region in the external mouth of the pore. All anions studied when applied from outside show an apparently paradoxical voltage-dependent block of inward currents; this voltage-dependent block could be qualitatively described by a discrete-state permeation model with two binding sites and three barriers. Effects of the external anions with aliphatic side-chains on the apparent open probability (Po) suggested that they are unable to gate the channel, but can modulate ClC-1 gating, probably, by changing Cl− affinity to the gating site. Effects of internal application of benzoate, hexanoate or propionate mimicked those of increasing internal pH, and similarly depended on the channel protonation from the external side. Results for internal benzoate support the concept of a negatively charged cytoplasmic particle being involved in the ClC-1 gating mechanism sensitive to the internal pH. PMID:11158270

  7. Effects of alternative splicing on function of Bestrophin-1 calcium-activated chloride channels

    PubMed Central

    Kuo, Yu-Hung; Abdullaev, Iskandar F.; Hyzinski-García, María C.; Mongin, Alexander A.

    2014-01-01

    Synopsis The proposed Ca2+-activated Cl− channel protein Bestrophin 1 (Best1) is expressed and functionally important in the retina and in the brain. Human BEST1 has two known splice variants, Best1V1 and Best1V2, which arise from alternative splicing of two exons: exon 2 splicing results in a unique N-terminal domain, whereas alternative splicing of exon 11 produces two mutually exclusive C-termini. Prior studies were limited to Best1V1 and its clinically relevant mutations. In the present work, we cloned a novel splice variant of Best1V1 missing exon 2 (Best1V1Δex2) and differing from each of the two previously identified isoforms by one alternatively spliced domain. This finding allowed us to determine the role for alternative splicing of the Best1 N- and C-termini. We heteroexpressed Best1V1Δex2 in HEK293 cells, and compared its properties to Best1V1 and Best1V2. Western blot analysis confirmed protein expression from all three splice variants. Both Best1V1 and Best1V1Δex2 successfully formed Ca2+-activated Cl− channels, demonstrating that the N-terminus encoded by exon 2 is not essential for channel function. In contrast, Best1V2 expressing cells had no detectable Ca2+-activated Cl− currents, pointing to a critical role for splicing of the C-terminus. Surface protein biotinylation demonstrated that Best1V1 and Best1V1Δex2 are trafficked to the plasma membrane, whereas Best1V2 is not. These results define the impact of alternative splicing on Best1 function, and should be taken into consideration in future modeling of the Best1 protein structure. PMID:24341532

  8. The ClC-7 Chloride Channel Is Downregulated by Hypoosmotic Stress in Human Chondrocytes.

    PubMed

    Kurita, Takashi; Yamamura, Hisao; Suzuki, Yoshiaki; Giles, Wayne R; Imaizumi, Yuji

    2015-07-01

    Articular chondrocytes in osteoarthritis (OA) patients are exposed to hypoosmotic stress because the osmolality of this synovial fluid is significantly decreased. Hypoosmotic stress can cause an efflux of Cl(-) and an associated decrease of cell volume. We have previously reported that a Cl(-) conductance contributes to the regulation of resting membrane potential and thus can alter intracellular Ca(2+) concentration ([Ca(2+)]i) in human chondrocytes. The molecular identity and pathologic function of these Cl(-) channels, however, remained to be determined. Here, we show that the ClC-7 Cl(-) channel is strongly expressed in a human chondrocyte cell line (OUMS-27) and that it is responsible for Cl(-) currents that are activated by extracellular acidification (pH 5.0). These acid-sensitive currents are inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; IC50 = 13 μM) and are markedly reduced by small-interfering RNA-induced knockdown of ClC-7. DIDS hyperpolarized these chondrocytes, and this was followed by an increase in [Ca(2+)]i. ClC-7 knockdown caused a similar hyperpolarization of the membrane potential. Short-term culture (48 hours) in hypoosmotic medium (270 mOsm) reduced the expression of ClC-7 and decreased the acid-sensitive currents. Interestingly, these hypoosmotic culture conditions, or ClC-7 knockdown, resulted in enhanced cell death. Taken together, our results show that the significant hyperpolarization due to ClC-7 impairment in chondrocytes can significantly increase [Ca(2+)]i and cell death. Thus, downregulation of ClC-7 channels during the hypoosmotic stress that accompanies OA progression is one important concept of the complex etiology of OA. These findings suggest novel targets for therapeutic intervention(s) and drug development for OA.

  9. Molecular diversity of GABA-gated chloride channels in the rat anterior pituitary.

    PubMed

    Boué-Grabot, E; Dufy, B; Garret, M

    1995-12-15

    mRNA expression of GABA-gated Cl(-)-channels in rat antepituitary was evaluated by using an reverse-transcribed (RT)-polymerase chain reaction (RT-PCR) method with degenerate and specific oligonucleotides. The main result of our findings is that the antepituitary expresses mRNAs encoding alpha 4 and rho 1 GABA receptor subunits. These two subunits are believed to be, respectively, constituents of benzodiazepine-insensitive GABAA and GABAC receptors in the CNS. This molecular analysis is consistent with the pharmacological diversity of GABA receptors in pituitary cells.

  10. The Transition from Proliferation to Differentiation in Colorectal Cancer Is Regulated by the Calcium Activated Chloride Channel A1

    PubMed Central

    Liu, Bin; McCaig, Colin D.; Pu, Jin

    2013-01-01

    Breaking the balance between proliferation and differentiation in animal cells can lead to cancer, but the mechanisms maintaining this balance remain largely undefined. The calcium activated chloride channel A1 (CLCA1) is a member of the calcium sensitive chloride conductance family of proteins and is expressed mainly in the colon, small intestine and appendix. We show that CLCA1 plays a functional role in differentiation and proliferation of Caco-2 cells and of intestinal tissue. Caco-2 cells spontaneously differentiate either in confluent culture or when treated with butyrate, a molecule present naturally in the diet. Here, we compared CLCA1 expressional levels between patients with and without colorectal cancer (CRC) and determined the functional role of CLCA1 in differentiation and proliferation of Caco-2 cells. We showed that: 1) CLCA1 and CLCA4 expression were down-regulated significantly in CRC patients; 2) CLCA1 expression was up-regulated in Caco-2 cells induced to differentiate by confluent culture or by treatment with sodium butyrate (NaBT); 3) Knockdown of CLCA1 with siRNA significantly inhibited cell differentiation and promoted cell proliferation in Caco-2 confluent cultures, and 4) In Caco-2 3D culture, suppression of CLCA1 significantly increased cell proliferation and compromised NaBT-induced inhibition of proliferation. In conclusion, CLCA1 may contribute to promoting spontaneous differentiation and reducing proliferation of Caco-2 cells and may be a target of NaBT-induced inhibition of proliferation and therefore a potential diagnostic marker for CRC prognosis. PMID:23593331

  11. Benzodiazepine agonist and inverse agonist actions on GABAA receptor-operated chloride channels. I. Acute effects of ethanol

    SciTech Connect

    Buck, K.J.; Harris, R.A. )

    1990-05-01

    Acute exposure to ethanol was found to enhance the ability of a benzodiazepine (BZ) inverse agonist, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), to reduce muscimol-activated 36Cl- uptake by membranes isolated from mouse cerebral cortex. Pretreatment in vivo with a hypnotic dose of ethanol (but not a subhypnotic dose), or exposure to a corresponding concentration in vitro, was effective. This increase in sensitivity of gamma-aminobutyric acid receptor-operated chloride channels to the actions of DMCM was due to an increase in both the potency and efficacy of DMCM. Sensitization to DMCM was reversible and was not observed 24 hr after a single injection of ethanol. Pretreatment with ethanol (10, 50 and 100 mM) in vitro produced sensitization to DMCM in a concentration-dependent manner, similar to that produced by in vivo exposure; this increase in sensitivity did not develop if the membranes were pretreated with ethanol at 0 degrees C. Similarly, in vitro exposure to pentobarbital (200 microM) or flunitrazepam (1 microM) enhanced the actions of the inverse agonist Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo(1,5a)(1,4)BZ-3- carboxylate). Acute ethanol exposure did not alter low-affinity gamma-aminobutyric acidA receptor binding or muscimol action, or the ability of a BZ agonist, flunitrazepam, to augment muscimol-activated chloride flux. Ethanol exposure did not alter (3H)flumazenil (Ro15-1788) binding to central BZ receptors, its displacement by DMCM or allosteric modulation of DMCM binding by muscimol (muscimol-shift).

  12. Spectrum of mutations in the major human skeletal muscle chloride channel gene (CLCN1) leading to myotonia.

    PubMed Central

    Meyer-Kleine, C; Steinmeyer, K; Ricker, K; Jentsch, T J; Koch, M C

    1995-01-01

    Autosomal dominant myotonia congenita and autosomal recessive generalized myotonia (GM) are genetic disorders characterized by the symptom of myotonia, which is based on an electrical instability of the muscle fiber membrane. Recently, these two phenotypes have been associated with mutations in the major muscle chloride channel gene CLCN1 on human chromosome 7q35. We have systematically screened the open reading frame of the CLCN1 gene for mutations by SSC analysis (SSCA) in a panel of 24 families and 17 single unrelated patients with human myotonia. By direct sequencing of aberrant SSCA conformers were revealed 15 different mutations in a total of 18 unrelated families and 13 single patients. Of these, 10 were novel (7 missense mutations, 2 mutations leading to frameshift, and 1 mutation predicted to affect normal splicing). In our overall sample of 94 GM chromosomes we were able to detect 48 (51%) mutant GM alleles. Three mutations (F413C), R894X, and a 14-bp deletion in exon 13) account for 32% of the GM chromosomes in the German population. Our finding that A437T is probably a polymorphism is in contrast to a recent report that the recessive phenotype GM is associated with this amino acid change. We also demonstrate that the R894X mutation may act as a recessive or a dominant mutation in the CLCN1 gene, probably depending on the genetic background. Functional expression of the R894X mutant in Xenopus oocytes revealed a large reduction, but not complete abolition, of chloride currents. Further, it had a weak dominant negative effect on wild-type currents in coexpression studies. Reduction of currents predicted for heterozygous carriers are close to the borderline value, which is sufficient to elicit myotonia. Images Figure 4 PMID:8533761

  13. Spectrum of mutations in the major human skeletal muscle chloride channel gene (CLCN1) leading to myotonia

    SciTech Connect

    Meyer-Kleine, C.; Koch, M.C.; Steinmeyer, K.

    1995-12-01

    Autosomal dominant myotonia congenita and autosomal recessive generalized myotonia (GM) are genetic disorders characterized by the symptom of myotonia, which is based on an electrical instability of the muscle fiber membrane. Recently, these two phenotypes have been associated with mutations in the major muscle chloride channel gene CLCN1 on human chromosome 7q35. We have systematically screened the open reading frame of the CLCN1 gene for mutations by SSC analysis (SSCA) in a panel of 24 families and 17 single unrelated patients with human myotonia. By direct sequencing of aberrant SSCA conformers we revealed 15 different mutations in a total of 18 unrelated families and 13 single patients. Of these, 10 were novel (7 missense mutations, 2 mutations leading to frameshift, and 1 mutation predicted to affect normal splicing). In our overall sample of 94 GM chromsomes we were able to detect 48 (50%) mutant GM alleles. Three mutations (F413C, R894X, and a 14-bp deletion in exon 13) account for 32% of the GM chromosomes in the German population. Our finding that A437T is probably a polymorphism is in contrast to a recent report that the recessive phenotype GM is associated with this amino acid change. We also demonstrate that the R894X mutation may act as a recessive or a dominant mutation in the CLCN1 gene, probably depending on the genetic background. Functional expression of the R894X mutant in Xenopus oocytes revealed a large reduction, but not complete abolition, of chloride currents. Further, it had a weak dominant negative effect on wild-type currents in coexpression studies. Reduction of currents predicted for heterozygous carriers are close to the borderline value, which is sufficient to elicit myotonia. 31 refs., 6 figs., 3 tabs.

  14. Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore.

    PubMed

    Zhou, Jing-Jun; Fatehi, Mohammad; Linsdell, Paul

    2008-11-01

    We have used site-directed mutagenesis and functional analysis to identify positively charged amino acid residues in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that interact with extracellular anions. Mutation of two positively charged arginine residues in the first extracellular loop (ECL) of CFTR, R104, and R117, as well as lysine residue K335 in the sixth transmembrane region, leads to inward rectification of the current-voltage relationship and decreased single channel conductance. These effects are dependent on the charge of the substituted side chain and on the Cl(-) concentration, suggesting that these positive charges normally act to concentrate extracellular Cl(-) ions near the outer mouth of the pore. Side chain charge-dependent effects are mimicked by manipulating charge in situ by mutating these amino acids to cysteine followed by covalent modification with charged cysteine-reactive reagents, confirming the location of these side chains within the pore outer vestibule. State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore. We suggest that ECL1 contributes to the CFTR pore external vestibule and that positively charged amino acid side chains in this region act to attract Cl(-) ions into the pore. In contrast, we find no evidence that fixed positive charges in other ECLs contribute to the permeation properties of the pore.

  15. Calcium dependence and distribution of calcium-activated chloride channels in Xenopus oocytes.

    PubMed Central

    Gomez-Hernandez, J M; Stühmer, W; Parekh, A B

    1997-01-01

    1. The Ca(2+)-dependent Cl- current (ICl,Ca), expressed in the plasma membrane of Xenopus oocytes, was examined in excised inside-out macropatches using a rapid perfusion system. 2. Application of Ca(2+)-containing Ringer solution resulted in the activation of a current whose reversal potential shifted to the right by 51 +/- 5.2 mV when Cl- in the pipette solution was lowered from 119.3 to 10 mM. No currents were generated when Ca2+ was omitted from the solution. The current is therefore a Ca(2+)-activated Cl- one. 3. Following exposure to Ca2+, the half-time for activation of ICl,Ca was not voltage dependent, whereas deactivation was strongly so. 4. ICl,Ca was stable in the continuous presence of Ca2+ and showed no sign of inactivation or adaptation. 5. Comparison of the size of the currents (normalized to pipette resistance) from the animal and vegetal poles revealed that ICl,Ca had a highly polarized distribution. The current density was almost 10 times higher in the animal pole. 6. The results suggest that Cl- channels provide a continuous and reliable indication of submembranous Ca2+, at least in an excised patch, and the clustering of the Cl- channels renders it necessary to exert caution in interpreting results involving the kinetics of Ca2+ signalling, when ICl,Ca is used as the sole monitor of calcium. PMID:9279809

  16. Cytoplasmic pathway followed by chloride ions to enter the CFTR channel pore.

    PubMed

    El Hiani, Yassine; Negoda, Alexander; Linsdell, Paul

    2016-05-01

    Most ATP-binding cassette (ABC) proteins function as ATP-dependent membrane pumps. One exception is the cystic fibrosis transmembrane conductance regulator (CFTR), an ABC protein that functions as a Cl(-) ion channel. As such, the CFTR protein must form a continuous pathway for the movement of Cl(-) ions from the cytoplasm to the extracellular solution when in its open channel state. Extensive functional investigations have characterized most parts of this Cl(-) permeation pathway. However, one region remains unexplored-the pathway connecting the cytoplasm to the membrane-spanning pore. We used patch clamp recording and extensive substituted cysteine accessibility mutagenesis to identify amino acid side-chains in cytoplasmic regions of CFTR that lie close to the pathway taken by Cl(-) ions as they pass from the cytoplasm through this pathway. Our results suggest that Cl(-) ions enter the permeation pathway via a single lateral tunnel formed by the cytoplasmic parts of the protein, and then follow a fairly direct central pathway towards the membrane-spanning parts of the protein. However, this pathway is not lined continuously by any particular part of the protein; instead, the contributions of different cytoplasmic regions of the protein appear to change as the permeation pathway approaches the membrane, which appears to reflect the ways in which different cytoplasmic regions of the protein are oriented towards its central axis. Our results allow us to define for the first time the complete Cl(-) permeation pathway in CFTR, from the cytoplasm to the extracellular solution.

  17. Donnan effect on chloride ion distribution as a determinant of body fluid composition that allows action potentials to spread via fast sodium channels.

    PubMed

    Kurbel, Sven

    2011-05-30

    Proteins in any solution with a pH value that differs from their isoelectric point exert both an electric Donnan effect (DE) and colloid osmotic pressure. While the former alters the distribution of ions, the latter forces water diffusion. In cells with highly Cl--permeable membranes, the resting potential is more dependent on the cytoplasmic pH value, which alters the Donnan effect of cell proteins, than on the current action of Na/K pumps. Any weak (positive or negative) electric disturbances of their resting potential are quickly corrected by chloride shifts.In many excitable cells, the spreading of action potentials is mediated through fast, voltage-gated sodium channels. Tissue cells share similar concentrations of cytoplasmic proteins and almost the same exposure to the interstitial fluid (IF) chloride concentration. The consequence is that similar intra- and extra-cellular chloride concentrations make these cells share the same Nernst value for Cl-.Further extrapolation indicates that cells with the same chloride Nernst value and high chloride permeability should have similar resting membrane potentials, more negative than -80 mV. Fast sodium channels require potassium levels >20 times higher inside the cell than around it, while the concentration of Cl- ions needs to be >20 times higher outside the cell.When osmotic forces, electroneutrality and other ions are all taken into account, the overall osmolarity needs to be near 280 to 300 mosm/L to reach the required resting potential in excitable cells. High plasma protein concentrations keep the IF chloride concentration stable, which is important in keeping the resting membrane potential similar in all chloride-permeable cells. Probable consequences of this concept for neuron excitability, erythrocyte membrane permeability and several features of circulation design are briefly discussed.

  18. Chloride intracellular channel 4 is required for maturation of the cerebral collateral circulation.

    PubMed

    Lucitti, Jennifer L; Tarte, Natalie J; Faber, James E

    2015-10-01

    The number and diameter of native collaterals in tissues of healthy mice vary widely, resulting in large differences in tissue injury in occlusive diseases. Recent studies suggest similar variation may exist in humans. Collateral variation in mice is determined by genetic background-dependent differences in embryonic collateral formation, by variation in maturation of the nascent collaterals, and by environmental factors such as aging that cause collateral rarefaction in the adult. Recently, formation of the collateral circulation in the brain was found to involve a unique VEGF-A-dependent "arteriolar" angiogenic sprouting-like mechanism. Elsewhere, chloride intracellular protein 4 (CLIC4) was implicated but not investigated directly, prompting the present study. Deletion of Clic4 had no effect on embryonic collaterogenesis. However, during collateral maturation from embryonic day 18.5 to postnatal day 7, reduced mural cell investment was observed and excessive pruning of collaterals occurred. Growth in collateral diameter was reduced. This resulted in 50% fewer collaterals of smaller diameter in the adult and thus larger infarct volume after middle cerebral artery occlusion. During collateral maturation, CLIC4 deficiency resulted in reduced expression of Vegfr2, Vegfr1, Vegfc, and mural cell markers, but not notch-pathway genes. Overexpression of VEGF-A in Clic4(-/-) mice had no effect on collaterogenesis, but rescued the above defects in collateral maturation by preventing mural cell loss and collateral pruning, thus restoring collateral number and diameter and reducing stroke severity in the adult. CLIC4 is not required for collaterogenesis but is essential for perinatal maturation of nascent collaterals through a mechanism that supports VEGF signaling.

  19. Chloride intracellular channel 4 is required for maturation of the cerebral collateral circulation

    PubMed Central

    Lucitti, Jennifer L.; Tarte, Natalie J.

    2015-01-01

    The number and diameter of native collaterals in tissues of healthy mice vary widely, resulting in large differences in tissue injury in occlusive diseases. Recent studies suggest similar variation may exist in humans. Collateral variation in mice is determined by genetic background-dependent differences in embryonic collateral formation, by variation in maturation of the nascent collaterals, and by environmental factors such as aging that cause collateral rarefaction in the adult. Recently, formation of the collateral circulation in the brain was found to involve a unique VEGF-A-dependent “arteriolar” angiogenic sprouting-like mechanism. Elsewhere, chloride intracellular protein 4 (CLIC4) was implicated but not investigated directly, prompting the present study. Deletion of Clic4 had no effect on embryonic collaterogenesis. However, during collateral maturation from embryonic day 18.5 to postnatal day 7, reduced mural cell investment was observed and excessive pruning of collaterals occurred. Growth in collateral diameter was reduced. This resulted in 50% fewer collaterals of smaller diameter in the adult and thus larger infarct volume after middle cerebral artery occlusion. During collateral maturation, CLIC4 deficiency resulted in reduced expression of Vegfr2, Vegfr1, Vegfc, and mural cell markers, but not notch-pathway genes. Overexpression of VEGF-A in Clic4−/− mice had no effect on collaterogenesis, but rescued the above defects in collateral maturation by preventing mural cell loss and collateral pruning, thus restoring collateral number and diameter and reducing stroke severity in the adult. CLIC4 is not required for collaterogenesis but is essential for perinatal maturation of nascent collaterals through a mechanism that supports VEGF signaling. PMID:26276819

  20. Chloride channel ClC-5 binds to aspartyl aminopeptidase to regulate renal albumin endocytosis.

    PubMed

    Lee, Aven; Slattery, Craig; Nikolic-Paterson, David J; Hryciw, Deanne H; Wilk, Sherwin; Wilk, Elizabeth; Zhang, Yuan; Valova, Valentina A; Robinson, Phillip J; Kelly, Darren J; Poronnik, Philip

    2015-04-01

    ClC-5 is a chloride/proton exchanger that plays an obligate role in albumin uptake by the renal proximal tubule. ClC-5 forms an endocytic complex with the albumin receptor megalin/cubilin. We have identified a novel ClC-5 binding partner, cytosolic aspartyl aminopeptidase (DNPEP; EC 3.4.11.21), that catalyzes the release of N-terminal aspartate/glutamate residues. The physiological role of DNPEP remains largely unresolved. Mass spectrometric analysis of proteins binding to the glutathione-S-transferase (GST)-ClC-5 C terminus identified DNPEP as an interacting partner. Coimmunoprecipitation confirmed that DNPEP and ClC-5 also associated in cells. Further experiments using purified GST-ClC-5 and His-DNPEP proteins demonstrated that the two proteins bound directly to each other. In opossum kidney (OK) cells, confocal immunofluorescence studies revealed that DNPEP colocalized with albumin-containing endocytic vesicles. Overexpression of wild-type DNPEP increased cell-surface levels of ClC-5 and albumin uptake. Analysis of DNPEP-immunoprecipitated products from rat kidney lysate identified β-actin and tubulin, suggesting a role for DNPEP in cytoskeletal maintenance. A DNase I inhibition assay showed a significant decrease in the amount of G actin when DNPEP was overexpressed in OK cells, suggesting a role for DNPEP in stabilizing the cytoskeleton. DNPEP was not present in the urine of healthy rats; however, it was readily detected in the urine in rat models of mild and heavy proteinuria (diabetic nephropathy and anti-glomerular basement membrane disease, respectively). Urinary levels of DNPEP were found to correlate with the severity of proteinuria. Therefore, we have identified another key molecular component of the albumin endocytic machinery in the renal proximal tubule and describe a new role for DNPEP in stabilizing the actin cytoskeleton.

  1. Calcium-activated chloride channels as a new target to control the spiking pattern of neurons.

    PubMed

    Ha, Go Eun; Cheong, Eunji

    2017-03-03

    Neuronal firing patterns and frequencies determine the nature of encoded information in the neural circuits. Here we discuss the molecular identity and cellular mechanisms of spike-frequency adaptation in central nervous system (CNS). Spike-frequency adaptation in thalamocortical (TC) and CA1 hippocampal neurons is mediated by the Ca2+-activated Cl- channel (CACC) anoctamin-2 (ANO2). Knockdown of ANO2 in these neurons results in significantly reduced spike-frequency adaptation along with increased number of spikes. No previous study has described the finding that CACCs mediate afterhyperpolarization currents, which result in the modulation of neuronal spike patterns in the central nervous system. Therefore, our study proposes a novel role for ANO2 in spike-frequency adaptation and transmission of information in the brain.

  2. ClC Channels and Transporters: Structure, Physiological Functions, and Implications in Human Chloride Channelopathies

    PubMed Central

    Poroca, Diogo R.; Pelis, Ryan M.; Chappe, Valérie M.

    2017-01-01

    The discovery of ClC proteins at the beginning of the 1990s was important for the development of the Cl- transport research field. ClCs form a large family of proteins that mediate voltage-dependent transport of Cl- ions across cell membranes. They are expressed in both plasma and intracellular membranes of cells from almost all living organisms. ClC proteins form transmembrane dimers, in which each monomer displays independent ion conductance. Eukaryotic members also possess a large cytoplasmic domain containing two CBS domains, which are involved in transport modulation. ClC proteins function as either Cl- channels or Cl-/H+ exchangers, although all ClC proteins share the same basic architecture. ClC channels have two gating mechanisms: a relatively well-studied fast gating mechanism, and a slow gating mechanism, which is poorly defined. ClCs are involved in a wide range of physiological processes, including regulation of resting membrane potential in skeletal muscle, facilitation of transepithelial Cl- reabsorption in kidneys, and control of pH and Cl- concentration in intracellular compartments through coupled Cl-/H+ exchange mechanisms. Several inherited diseases result from C1C gene mutations, including myotonia congenita, Bartter’s syndrome (types 3 and 4), Dent’s disease, osteopetrosis, retinal degeneration, and lysosomal storage diseases. This review summarizes general features, known or suspected, of ClC structure, gating and physiological functions. We also discuss biophysical properties of mammalian ClCs that are directly involved in the pathophysiology of several human inherited disorders, or that induce interesting phenotypes in animal models. PMID:28386229

  3. ClC Channels and Transporters: Structure, Physiological Functions, and Implications in Human Chloride Channelopathies.

    PubMed

    Poroca, Diogo R; Pelis, Ryan M; Chappe, Valérie M

    2017-01-01

    The discovery of ClC proteins at the beginning of the 1990s was important for the development of the Cl(-) transport research field. ClCs form a large family of proteins that mediate voltage-dependent transport of Cl(-) ions across cell membranes. They are expressed in both plasma and intracellular membranes of cells from almost all living organisms. ClC proteins form transmembrane dimers, in which each monomer displays independent ion conductance. Eukaryotic members also possess a large cytoplasmic domain containing two CBS domains, which are involved in transport modulation. ClC proteins function as either Cl(-) channels or Cl(-)/H(+) exchangers, although all ClC proteins share the same basic architecture. ClC channels have two gating mechanisms: a relatively well-studied fast gating mechanism, and a slow gating mechanism, which is poorly defined. ClCs are involved in a wide range of physiological processes, including regulation of resting membrane potential in skeletal muscle, facilitation of transepithelial Cl(-) reabsorption in kidneys, and control of pH and Cl(-) concentration in intracellular compartments through coupled Cl(-)/H(+) exchange mechanisms. Several inherited diseases result from C1C gene mutations, including myotonia congenita, Bartter's syndrome (types 3 and 4), Dent's disease, osteopetrosis, retinal degeneration, and lysosomal storage diseases. This review summarizes general features, known or suspected, of ClC structure, gating and physiological functions. We also discuss biophysical properties of mammalian ClCs that are directly involved in the pathophysiology of several human inherited disorders, or that induce interesting phenotypes in animal models.

  4. Explaining Calcium-Dependent Gating of Anoctamin-1 Chloride Channels Requires a Revised Topology

    PubMed Central

    Yu, Kuai; Duran, Charity; Qu, Zhiqiang; Cui, Yuan-Yuan; Hartzell, H. Criss

    2013-01-01

    Rationale Ca2+-activated Cl channels (CaCCs) play pivotal roles in the cardiovascular system: they regulate vascular smooth muscle tone and participate in cardiac action potential repolarization in some species. CaCCs were recently discovered to be encoded by members of the Anoctamin (Ano, also called Tmem16) superfamily, but the mechanisms of Ano1 gating by Ca2+ remain enigmatic. Objective The objective was to identify regions of Ano1 involved in channel gating by Ca2+. Methods and results The Ca2+ sensitivity of Ano1 was estimated from rates of current activation and deactivation in excised patches rapidly switched between zero and high Ca2+ on the cytoplasmic side. Mutation of glutamates E702 and E705 dramatically altered Ca2+ sensitivity. E702 and E705 are predicted to be in an extracellular loop, but antigenic epitopes introduced into this loop are not accessible to extracellular antibodies, suggesting this loop is intracellular. Cytoplasmically-applied membrane-impermeant sulfhydryl reagents alter the Ca2+ sensitivity of Ano1 E702C and E705C, as expected if E702 and E705 are intracellular. Substituted cysteine accessibility mutagenesis of the putative re-entrant loop suggests that E702 and E705 are located adjacent to the Cl conduction pathway. Conclusions We propose an alternative model of Ano1 topology based on mutagenesis, epitope accessibility, and cysteine-scanning accessibility. These data contradict the popular re-entrant loop model by showing that the putative 4th extracellular loop (ECL 4) is intracellular and may contain a Ca2+ binding site. These studies provide new perspectives on regulation of Ano1 by Ca2+. PMID:22394518

  5. Phylogenetic shadowing of a histamine-gated chloride channel involved in insect vision.

    PubMed

    Iovchev, Mladen; Boutanaev, Alexander; Ivanov, Ivaylo; Wolstenholme, Adrian; Nurminsky, Dmitry; Semenov, Eugene

    2006-01-01

    A recently identified gene, hclA (synonym: ort), codes for an ionotrophic histamine receptor subunit in Drosophila melanogaster, and known hclA mutations lead to defects in the visual system, neurologic disorders and changed responsiveness to neurotoxins. To investigate whether this novel class of receptors is common across the Insecta, we analysed the genomes of 15 other insect species (Diptera, Hymenoptera, Coleoptera, Lepidoptera) and revealed orthologs of hclA in all of them. The predicted receptor domain of HCLA is extensively conserved (86-100% of identity) among the 16 proteins. Minor changes in the amino acid sequence that includes the putative transmembrane domains (TMs) 1-3 were found in non-drosophilid species only. Substantial amino acid variability was observed in the signal polypeptides, the intracellular loop domains and in TM4, in good accordance with known data on sequence variations in ligand-gated ion channels. Pairwise comparisons revealed three consensus sequences for N-glycosylation, conserved in HCLAs of all species studied, as well as a drosophilid-specific putative phosphorylation site. Real-time PCR analysis demonstrated that hclA-mRNA is abundant in heads of adult Drosophila. However, species- and sex-specific variations of the hclA expression levels were also observed.

  6. Inhibition of the gravitropic response of snapdragon spikes by the calcium-channel blocker lanthanum chloride.

    PubMed

    Friedman, H; Meir, S; Rosenberger, I; Halevy, A H; Kaufman, P B; Philosoph-Hadas, S

    1998-10-01

    The putative Ca(2+)-channel blocker LaCl3 prevented the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes (S. Philosoph-Hadas, S. Meir, I. Rosenberger, A.H. Halevy [1996] Plant Physiol 110: 301-310) and inhibited stem curvature to a greater extent than vertical and horizontal stem elongation at the bending zone. This might indicate that LaCl3, which modulates cytosolic Ca2+, does not influence general stem-growth processes but may specifically affect other gravity-associated processes occurring at the stem-bending zone. Two such specific gravity-dependent events were found to occur in the bending zone of snapdragon spikes: sedimentation of starch-containing chloroplasts at the bottom of stem cortex cells, as seen in cross-sections, and establishment of an ethylene gradient across the stem. Our results show that the lateral sedimentation of chloroplasts associated with gravity sensing was prevented in cross-sections taken from the bending zone of LaCl3-treated and subsequently gravistimulated spikes and that LaCl3 completely prevented the gravity-induced, asymmetric ethylene production established across the stem-bending zone. These data indicate that LaCl3 inhibits stem curvature of snapdragon spikes by preventing several gravity-dependent processes. Therefore, we propose that the gravitropic response of shoots could be mediated through a Ca(2+)-dependent pathway involving modulation of cytosolic Ca2+ at various stages.

  7. Unique Contributions of an Arginine Side Chain to Ligand Recognition in a Glutamate-gated Chloride Channel*

    PubMed Central

    Lynagh, Timothy; Komnatnyy, Vitaly V.

    2017-01-01

    Glutamate recognition by neurotransmitter receptors often relies on Arg residues in the binding site, leading to the assumption that charge-charge interactions underlie ligand recognition. However, assessing the precise chemical contribution of Arg side chains to protein function and pharmacology has proven to be exceedingly difficult in such large and complex proteins. Using the in vivo nonsense suppression approach, we report the first successful incorporation of the isosteric, titratable Arg analog, canavanine, into a neurotransmitter receptor in a living cell, utilizing a glutamate-gated chloride channel from the nematode Haemonchus contortus. Our data unveil a surprisingly small contribution of charge at a conserved arginine side chain previously suggested to form a salt bridge with the ligand, glutamate. Instead, our data show that Arg contributes crucially to ligand sensitivity via a hydrogen bond network, where Arg interacts both with agonist and with a conserved Thr side chain within the receptor. Together, the data provide a new explanation for the reliance of neurotransmitter receptors on Arg side chains and highlight the exceptional capacity of unnatural amino acid incorporation for increasing our understanding of ligand recognition. PMID:28096462

  8. Inhibition of calcium-activated chloride channel ANO1 suppresses proliferation and induces apoptosis of epithelium originated cancer cells.

    PubMed

    Guan, Lizhao; Song, Yan; Gao, Jian; Gao, Jianjun; Wang, KeWei

    2016-11-29

    ANO1, a calcium-activated chloride channel, has been reported to be amplified or overexpressed in tissues of several cancers. However, reports on its roles in tumor progression obtained from cancer cell lines are inconsistent, suggesting that the role of ANO1 in tumorigenesis is likely dependent on either its expression level or cell-type expressing ANO1. To investigate the biological roles of ANO1 in different tumor cells, we, in this study, selected several cancer cell lines and a normal HaCaT cell line with high expression levels of ANO1, and examined the function of ANO1 in these cells using approaches of lentiviral knockdown and pharmacological inhibition. We found that ANO1 knockdown significantly inhibited cell proliferation and induced cell apoptosis in either tumor cell lines or normal HaCaT cell line. Moreover, silencing ANO1 arrested cancer cells at G1 phase of cell cycle. Treatment with ANO1 inhibitor CaCCinh-A01 reduced cell viability in a dose-dependent manner. Furthermore, both ANO1 inhibitors CaCCinh-A01 and T16Ainh-A01 significantly suppressed cell migration. Our findings show that ANO1 overexpression promotes cancer cell proliferation and migration; and genetic or pharmacological inhibition of ANO1 induces apoptosis and cell cycle arrest at G1 phase in different types of epithelium-originated cancer cells.

  9. Inhibition of calcium-activated chloride channel ANO1 suppresses proliferation and induces apoptosis of epithelium originated cancer cells

    PubMed Central

    Guan, Lizhao; Song, Yan; Gao, Jian; Gao, Jianjun; Wang, KeWei

    2016-01-01

    ANO1, a calcium-activated chloride channel, has been reported to be amplified or overexpressed in tissues of several cancers. However, reports on its roles in tumor progression obtained from cancer cell lines are inconsistent, suggesting that the role of ANO1 in tumorigenesis is likely dependent on either its expression level or cell-type expressing ANO1. To investigate the biological roles of ANO1 in different tumor cells, we, in this study, selected several cancer cell lines and a normal HaCaT cell line with high expression levels of ANO1, and examined the function of ANO1 in these cells using approaches of lentiviral knockdown and pharmacological inhibition. We found that ANO1 knockdown significantly inhibited cell proliferation and induced cell apoptosis in either tumor cell lines or normal HaCaT cell line. Moreover, silencing ANO1 arrested cancer cells at G1 phase of cell cycle. Treatment with ANO1 inhibitor CaCCinh-A01 reduced cell viability in a dose-dependent manner. Furthermore, both ANO1 inhibitors CaCCinh-A01 and T16Ainh-A01 significantly suppressed cell migration. Our findings show that ANO1 overexpression promotes cancer cell proliferation and migration; and genetic or pharmacological inhibition of ANO1 induces apoptosis and cell cycle arrest at G1 phase in different types of epithelium-originated cancer cells. PMID:27732935

  10. Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction.

    PubMed

    Hoegg-Beiler, Maja B; Sirisi, Sònia; Orozco, Ian J; Ferrer, Isidre; Hohensee, Svea; Auberson, Muriel; Gödde, Kathrin; Vilches, Clara; de Heredia, Miguel López; Nunes, Virginia; Estévez, Raúl; Jentsch, Thomas J

    2014-03-19

    Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2's biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.

  11. Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.

    PubMed

    El Hiani, Yassine; Linsdell, Paul

    2015-06-19

    As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm.

  12. Human spermatozoa possess a calcium-dependent chloride channel that may participate in the acrosomal reaction

    PubMed Central

    Orta, Gerardo; Ferreira, Gonzalo; José, Omar; Treviño, Claudia L; Beltrán, Carmen; Darszon, Alberto

    2012-01-01

    Motility, maturation and the acrosome reaction (AR) are fundamental functions of mammalian spermatozoa. While travelling through the female reproductive tract, spermatozoa must mature through a process named capacitation, so that they can reach the egg and undergo the AR, an exocytotic event necessary to fertilize the egg. Though Cl− is important for sperm capacitation and for the AR, not much is known about the molecular identity of the Cl− transporters involved in these processes. We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa. Our whole cell recordings revealed the presence of a Ca2+-dependent Cl− current. The biophysical characteristics of this current and its sensitivity to niflumic acid (NFA) and 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid (DIDIS) are consistent with those displayed by the Ca2+-dependent Cl− channel from the anoctamin family (TMEM16). Whole cell patch clamp recordings in the cytoplasmic droplet of human spermatozoa corroborated the presence of these currents, which were sensitive to NFA and to a small molecule TMEM16A inhibitor (TMEM16Ainh, an aminophenylthiazole). Importantly, the human sperm AR induced by a recombinant human glycoprotein from the zona pellucida, rhZP3, displayed a similar sensitivity to NFA, DIDS and TMEM16Ainh as the sperm Ca2+-dependent Cl− currents. Our findings indicate the presence of Ca2+-dependent Cl− currents in human spermatozoa, that TMEM16A may contribute to these currents and also that sperm Ca2+-dependent Cl− currents may participate in the rhZP3-induced AR. PMID:22473777

  13. Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore*

    PubMed Central

    El Hiani, Yassine; Linsdell, Paul

    2015-01-01

    As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl− and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl− ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl− ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl− permeation, demonstrating their functional role in maximization of Cl− flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl−. The location of these Cl−-attractive residues suggests that cytoplasmic Cl− ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl− ions from the cytoplasm. PMID:25944907

  14. Effects of Small Molecule Calcium-Activated Chloride Channel Inhibitors on Structure and Function of Accessory Cholera Enterotoxin (Ace) of Vibrio cholerae

    PubMed Central

    Chatterjee, Tanaya; Sheikh, Irshad Ali; Chakravarty, Devlina; Chakrabarti, Pinak; Sarkar, Paramita; Saha, Tultul; Chakrabarti, Manoj K.; Hoque, Kazi Mirajul

    2015-01-01

    Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders. PMID:26540279

  15. Effects of Small Molecule Calcium-Activated Chloride Channel Inhibitors on Structure and Function of Accessory Cholera Enterotoxin (Ace) of Vibrio cholerae.

    PubMed

    Chatterjee, Tanaya; Sheikh, Irshad Ali; Chakravarty, Devlina; Chakrabarti, Pinak; Sarkar, Paramita; Saha, Tultul; Chakrabarti, Manoj K; Hoque, Kazi Mirajul

    2015-01-01

    Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders.

  16. Superoxide radicals increase transforming growth factor-{beta}1 and collagen release from human lung fibroblasts via cellular influx through chloride channels

    SciTech Connect

    Qi Shufan Hartog, Gertjan J.M. den; Bast, Aalt

    2009-05-15

    Reactive oxygen species (ROS) have been implicated in the pathogenesis of fibrosis. However, it remains unclear which ROS is the major cause. We hypothesize that superoxide elicits specific toxicity to human lung fibroblasts and plays an important role in the development of pulmonary fibrosis. In this study, superoxide generated from xanthine and xanthine oxidase activated lung fibroblasts by increasing the release of TGF-{beta}1 and collagen. This was associated with increased levels of intracellular superoxide. SOD and tempol, by scavenging respectively extracellular and intracellular superoxide, prevented the activation of fibroblasts induced by exposure to exogenous superoxide, whereas catalase did not. Moreover, hydrogen peroxide did not activate fibroblasts. Apparently, superoxide rather than hydrogen peroxide is involved in the regulation of TGF-{beta}1 and collagen release in lung fibroblasts. The chloride channel blocker, DIDS, inhibited the increase of intracellular superoxide levels induced by exogenous superoxide and consequently prevented the activation of fibroblasts. This suggests that the cellular influx of superoxide through chloride channels is essential for superoxide-induced activation of fibroblasts. ERK1/2 and p38 MAPKs are involved in the intracellular pathway leading to superoxide-induced fibroblasts activation. Superoxide possesses until now undiscovered specific pro-fibrotic properties in human lung fibroblasts. This takes place via the cellular influx of superoxide through chloride channels rather than via the formation of hydrogen peroxide.

  17. MiR-101 and miR-144 Regulate the Expression of the CFTR Chloride Channel in the Lung

    PubMed Central

    Hassan, Fatemat; Nuovo, Gerard J.; Crawford, Melissa; Boyaka, Prosper N.; Kirkby, Stephen; Nana-Sinkam, Serge P.; Cormet-Boyaka, Estelle

    2012-01-01

    The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that plays a critical role in the lung by maintaining fluid homeostasis. Absence or malfunction of CFTR leads to Cystic Fibrosis, a disease characterized by chronic infection and inflammation. We recently reported that air pollutants such as cigarette smoke and cadmium negatively regulate the expression of CFTR by affecting several steps in the biogenesis of CFTR protein. MicroRNAs (miRNAs) have recently received a great deal of attention as both biomarkers and therapeutics due to their ability to regulate multiple genes. Here, we show that cigarette smoke and cadmium up-regulate the expression of two miRNAs (miR-101 and miR-144) that are predicted to target CFTR in human bronchial epithelial cells. When premature miR-101 and miR-144 were transfected in human airway epithelial cells, they directly targeted the CFTR 3′UTR and suppressed the expression of the CFTR protein. Since miR-101 was highly up-regulated by cigarette smoke in vitro, we investigated whether such increase also occurred in vivo. Mice exposed to cigarette smoke for 4 weeks demonstrated an up-regulation of miR-101 and suppression of CFTR protein in their lungs. Finally, we show that miR-101 is highly expressed in lung samples from patients with severe chronic obstructive pulmonary disease (COPD) when compared to control patients. Taken together, these results suggest that chronic cigarette smoking up-regulates miR-101 and that this miRNA could contribute to suppression of CFTR in the lungs of COPD patients. PMID:23226399

  18. Cloning and characterization of genes encoding alpha and beta subunits of glutamate-gated chloride channel protein in Cylicocyclus nassatus.

    PubMed

    Tandon, Ritesh; LePage, Keith T; Kaplan, Ray M

    2006-11-01

    The invertebrate glutamate-gated chloride channels (GluCls) are receptor molecules and targets for the avermectin-milbemycin (AM) group of anthelmintics. Mutations in GluCls are associated with ivermectin resistance in the soil dwelling nematode Caenorhabditis elegans and the parasitic nematode Cooperia oncophora. In this study, full-length cDNAs encoding alpha and beta subunits of GluCl were cloned and sequenced in Cylicocyclus nassatus, a common and important cyathostomin nematode parasite of horses. Both genes possess the sequence characteristics typical of GluCls, and phylogenetic analysis confirms that these genes are evolutionarily closely related to GluCls of other nematodes and flies. Complete coding sequences of C. nassatus GluCl-alpha and GluCl-beta were subcloned into pTL1 mammalian expression vector, and proteins were expressed in COS-7 cells. Ivermectin-binding characteristics were determined by incubating COS-7 cell membranes expressing C. nassatus GluCl-alpha and GluCl-beta proteins with [(3)H]ivermectin. In competitive binding experiments, fitting the data to a one site competition model, C. nassatus GluCl-alpha was found to bind [(3)H]ivermectin with a high amount of displaceable binding (IC(50)=208 pM). Compared to the mock-transfected COS-7 cells, the means of [(3)H]ivermectin binding were significantly different for C. nassatus GluCl-alpha and the Haemonchus contortus GluCl (HcGluCla) (p=0.018 and 0.023, respectively) but not for C. nassatus GluCl-beta (p=0.370). This is the first report of orthologs of GluCl genes and in vitro expression of an ivermectin-binding protein in a cyathostomin species. These data suggest the likelihood of a similar mechanism of action of AM drugs in these parasites, and suggest that mechanisms of resistance may also be similar.

  19. Evidence for the participation of Ca(2+)-activated chloride channels in formalin-induced acute and chronic nociception.

    PubMed

    García, Guadalupe; Martínez-Rojas, Vladimir A; Rocha-González, Héctor I; Granados-Soto, Vinicio; Murbartián, Janet

    2014-09-04

    In this study we determined the role of Ca(2+)-activated chloride channels (CaCC) in acute and chronic nociceptive responses elicited by 1% formalin. Formalin injection produced a typical pattern of flinching behavior for about 1h. Moreover, it produced secondary allodynia and hyperalgesia in the ipsilateral and contralateral paws for at least 6 days. Local peripheral and intrathecal pre-treatment (-10 min) with the non-selective and selective CaCC blockers niflumic acid and CaCCinh-A01, respectively, prevented formalin-induced flinching behavior mainly during phase 2 of the formalin test. Furthermore, niflumic acid and CaCCinh-A01 also prevented in a dose-dependent manner the long-lasting evoked secondary mechanical allodynia and hyperalgesia in the ipsilateral and contralateral paws. Moreover, local peripheral and intrathecal post-treatment (on day 6) with both CaCC blockers decreased the established formalin-induced secondary mechanical allodynia and hyperalgesia behavior in both paws. CaCC anoctamin-1 and bestrophin-1 were detected in the dorsal root ganglia. Formalin injection increased anoctamin-1, but not bestrophin-1 protein levels at 6 days. Intrathecal injection of the CaCC inhibitor CaCCinh-A01 prevented formalin-induced anoctamin-1 increase. Data suggest that peripheral and spinal CaCC, and particularly anoctamin-1, participates in the acute nociception induced by formalin as well as in the development and maintenance of secondary mechanical allodynia and hyperalgesia. Thus, CaCC activity contributes to neuronal excitability in the process of nociception induced by formalin.

  20. Bile acids stimulate chloride secretion through CFTR and calcium-activated Cl- channels in Calu-3 airway epithelial cells.

    PubMed

    Hendrick, Siobhán M; Mroz, Magdalena S; Greene, Catherine M; Keely, Stephen J; Harvey, Brian J

    2014-09-01

    Bile acids resulting from the aspiration of gastroesophageal refluxate are often present in the lower airways of people with cystic fibrosis and other respiratory distress diseases. Surprisingly, there is little or no information on the modulation of airway epithelial ion transport by bile acids. The secretory effect of a variety of conjugated and unconjugated secondary bile acids was investigated in Calu-3 airway epithelial cells grown under an air-liquid interface and mounted in Ussing chambers. Electrogenic transepithelial ion transport was measured as short-circuit current (Isc). The taurine-conjugated secondary bile acid, taurodeoxycholic acid (TDCA), was found to be the most potent modulator of basal ion transport. Acute treatment (5 min) of Calu-3 cells with TDCA (25 μM) on the basolateral side caused a stimulation of Isc, and removal of extracellular Cl(-) abolished this response. TDCA produced an increase in the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent current that was abolished by pretreatment with the CFTR inhibitor CFTRinh172. TDCA treatment also increased Cl(-) secretion through calcium-activated chloride (CaCC) channels and increased the Na(+)/K(+) pump current. Acute treatment with TDCA resulted in a rapid cellular influx of Ca(2+) and increased cAMP levels in Calu-3 cells. Bile acid receptor-selective activation with INT-777 revealed TGR5 localized at the basolateral membrane as the receptor involved in TDCA-induced Cl(-) secretion. In summary, we demonstrate for the first time that low concentrations of bile acids can modulate Cl(-) secretion in airway epithelial cells, and this effect is dependent on both the duration and sidedness of exposure to the bile acid.

  1. A point mutation in the glutamate-gated chloride channel of Plutella xylostella is associated with resistance to abamectin.

    PubMed

    Wang, X; Wang, R; Yang, Y; Wu, S; O'Reilly, A O; Wu, Y

    2016-04-01

    The diamondback moth, Plutella xylostella, is a global pest of cruciferous vegetables. Abamectin resistance in a field population of P. xylostella was introgressed into the susceptible Roth strain. The resulting introgression strain Roth-Abm showed 11 000-fold resistance to abamectin compared with Roth. An A309V substitution at the N-terminus of the third transmembrane helix (M3) of the glutamate-gated chloride channel of P. xylostella (PxGluCl) was identified in Roth-Abm. The frequency of the V309 allele of PxGluCl was 94.7% in Roth-Abm, whereas no such allele was detected in Roth. A subpopulation of Roth-Abm was kept without abamectin selection for 20 generations to produce a revertant strain, Roth-Abm-D. Abamectin resistance in Roth-Abm-D declined to 1150-fold compared with Roth, with the V309 allele frequency decreased to 9.6%. After treatment of the Roth-Abm-D strain with 80 mg/l abamectin the V309 allele frequency in the survivors increased to 55%. This demonstrates that the A309V mutation in PxGluCl is strongly associated with a 10-fold increase in abamectin resistance in Roth-Abm relative to Roth-Abm-D. Homology modelling and automated ligand docking results suggest that the A309V substitution allosterically modifies the abamectin-binding site, as opposed to directly eliminating a key binding contact. Other resistance mechanisms to abamectin in Roth-Abm are discussed besides the A309V mutation of PxGluCl.

  2. Characterization of Cardiac Anoctamin1 Ca2+-Activated Chloride Channels and Functional Role in Ischemia-Induced Arrhythmias

    PubMed Central

    Ye, Zhen; Wu, Ming-Ming; Wang, Chun-Yu; Li, Yan-Chao; Yu, Chang-Jiang; Gong, Yuan-Feng; Zhang, Jun; Wang, Qiu-Shi; Song, Bin-Lin; Yu, Kuai; Hartzell, H. Criss; Duan, Dayue Darrel; Zhao, Dan; Zhang, Zhi-Ren

    2015-01-01

    Anoctamin1 (ANO1) encodes a Ca2+-activated chloride (Cl−) channel (CaCC) in variety tissues of many species. Whether ANO1 expresses and functions as a CaCC in cardiomyocytes remain unknown. The objective of this study is to characterize the molecular and functional expression of ANO1 in cardiac myocytes and the role of ANO1-encoded CaCCs in ischemia-induced arrhythmias in the heart. Quantitative real-time RT-PCR, immunofluorescence staining assays, and immunohistochemistry identified the molecular expression, location, and distribution of ANO1 in mouse ventricular myocytes (mVMs). Patch-clamp recordings combined with pharmacological analyses found that ANO1 was responsible for a Ca2+-activated Cl− current (ICl.Ca) in cardiomyocytes. Myocardial ischemia led to a significant increase in the current density of ICl.Ca, which was inhibited by a specific ANO1 inhibitor, T16Ainh-A01, and an antibody targeting at the pore area of ANO1. Moreover, cardiomyocytes isolated from mice with ischemia-induced arrhythmias had an accelerated early phase 1 repolarization of action potentials (APs) and a deeper “spike and dome” compared to control cardiomyocytes from non-ischemia mice. Application of the antibody targeting at ANO1 pore prevented the ischemia-induced early phase 1 repolarization acceleration and caused a much shallower “spike and dome”. We conclude that ANO1 encodes CaCC and plays a significant role in the phase 1 repolarization of APs in mVMs. The ischemia-induced increase in ANO1 expression may be responsible for the increased density of ICl.Ca in the ischemic heart and may contribute, at least in part, to ischemia-induced arrhythmias. PMID:24962810

  3. MiR-101 and miR-144 regulate the expression of the CFTR chloride channel in the lung.

    PubMed

    Hassan, Fatemat; Nuovo, Gerard J; Crawford, Melissa; Boyaka, Prosper N; Kirkby, Stephen; Nana-Sinkam, Serge P; Cormet-Boyaka, Estelle

    2012-01-01

    The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that plays a critical role in the lung by maintaining fluid homeostasis. Absence or malfunction of CFTR leads to Cystic Fibrosis, a disease characterized by chronic infection and inflammation. We recently reported that air pollutants such as cigarette smoke and cadmium negatively regulate the expression of CFTR by affecting several steps in the biogenesis of CFTR protein. MicroRNAs (miRNAs) have recently received a great deal of attention as both biomarkers and therapeutics due to their ability to regulate multiple genes. Here, we show that cigarette smoke and cadmium up-regulate the expression of two miRNAs (miR-101 and miR-144) that are predicted to target CFTR in human bronchial epithelial cells. When premature miR-101 and miR-144 were transfected in human airway epithelial cells, they directly targeted the CFTR 3'UTR and suppressed the expression of the CFTR protein. Since miR-101 was highly up-regulated by cigarette smoke in vitro, we investigated whether such increase also occurred in vivo. Mice exposed to cigarette smoke for 4 weeks demonstrated an up-regulation of miR-101 and suppression of CFTR protein in their lungs. Finally, we show that miR-101 is highly expressed in lung samples from patients with severe chronic obstructive pulmonary disease (COPD) when compared to control patients. Taken together, these results suggest that chronic cigarette smoking up-regulates miR-101 and that this miRNA could contribute to suppression of CFTR in the lungs of COPD patients.

  4. Antagonists of the TMEM16A Calcium-Activated Chloride Channel Modulate Airway Smooth Muscle Tone and Intracellular Calcium

    PubMed Central

    Danielsson, Jennifer; Perez-Zoghbi, Jose; Bernstein, Kyra; Barajas, Matthew B.; Zhang, Yi; Kumar, Satish; Sharma, Pawan K.; Gallos, George; Emala, Charles W.

    2015-01-01

    Background Perioperative bronchospasm refractory to β-agonists continues to challenge anesthesiologists and intensivists. The TMEM16A calcium-activated chloride channel modulates airway smooth muscle (ASM) contraction. We hypothesized that TMEM16A antagonists would relax ASM contraction by modulating membrane potential and calcium flux. Methods Human ASM, guinea pig tracheal rings or mouse peripheral airways were contracted with acetylcholine (Ach) or leukotriene D4 (LTD4) and then treated with the TMEM16A antagonists: benzbromarone, T16Ainh-A01, MONNA or B25. In separate studies, guinea pig tracheal rings were contracted with Ach and then exposed to increasing concentrations of isoproterenol (0.01nM-10μM) ± benzbromarone. Plasma membrane potential and intracellular calcium concentrations were measured in human ASM cells. Results Benzbromarone was the most potent TMEM16A antagonist tested for relaxing an Ach-induced contraction in guinea pig tracheal rings (n=6). Further studies were done to investigate benzbromarone’s clinical utility. In human ASM, benzbromarone relaxed either an acetylcholine- or LTD4-induced contraction (n=8). Benzbromarone was also effective in relaxing peripheral airways (n=9) and potentiating relaxation by β-agonists (n=5–10). In cellular mechanistic studies, benzbromarone hyperpolarized human ASM cells (n=9–12) and attenuated intracellular calcium flux from both the plasma membrane and sarcoplasmic reticulum (n=6–12). Conclusions TMEM16A antagonists work synergistically with β-agonists and through a novel pathway of interrupting ion flux both at the plasma membrane and sarcoplasmic reticulum to acutely relax human airway smooth muscle. PMID:26181339

  5. Adenosine regulates a chloride channel via protein kinase C and a G protein in a rabbit cortical collecting duct cell line.

    PubMed Central

    Schwiebert, E M; Karlson, K H; Friedman, P A; Dietl, P; Spielman, W S; Stanton, B A

    1992-01-01

    We examined the regulation by adenosine of a 305-pS chloride (Cl-) channel in the apical membrane of a continuous cell line derived from rabbit cortical collecting duct (RCCT-28A) using the patch clamp technique. Stimulation of A1 adenosine receptors by N6-cyclohexyladenosine (CHA) activated the channel in cell-attached patches. Phorbol 12,13-didecanoate and 1-oleoyl 2-acetylglycerol, activators of protein kinase C (PKC), mimicked the effect of CHA, whereas the PKC inhibitor H7 blocked the action of CHA. Stimulation of A1 adenosine receptors also increased the production of diacylglycerol, an activator of PKC. Exogenous PKC added to the cytoplasmic face of inside-out patches also stimulated the Cl- channel. Alkaline phosphatase reversed PKC activation. These results show that stimulation of A1 adenosine receptors activates a 305-pS Cl-channel in the apical membrane by a phosphorylation-dependent pathway involving PKC. In previous studies, we showed that the protein G alpha i-3 activated the 305-pS Cl- channel (Schwiebert et al. 1990. J. Biol. Chem. 265:7725-7728). We, therefore, tested the hypothesis that PKC activates the channel by a G protein-dependent pathway. In inside-out patches, pertussis toxin blocked PKC activation of the channel. In contrast, H7 did not prevent G protein activation of the channel. We conclude that adenosine activates a 305-pS Cl- channel in the apical membrane of RCCT-28A cells by a membrane-delimited pathway involving an A1 adenosine receptor, phospholipase C, diacylglycerol, PKC, and a G protein. Because we have shown, in previous studies, that this Cl- channel participates in the regulatory volume decrease subsequent to cell swelling, adenosine release during ischemic cell swelling may activate the Cl-channel and restore cell volume. Images PMID:1311718

  6. New selective inhibitors of calcium-activated chloride channels … T16Ainh-A01, CaCCinh-A01 and MONNA … what do they inhibit?

    PubMed Central

    Boedtkjer, D M B; Kim, S; Jensen, A B; Matchkov, V M; Andersson, K E

    2015-01-01

    Background and Purpose T16Ainh-A01, CaCCinh-A01 and MONNA are identified as selective inhibitors of the TMEM16A calcium-activated chloride channel (CaCC). The aim of this study was to examine the chloride-specificity of these compounds on isolated resistance arteries in the presence and absence (±) of extracellular chloride. Experimental Approach Isolated resistance arteries were maintained in a myograph and tension recorded, in some instances combined with microelectrode impalement for membrane potential measurements or intracellular calcium monitoring using fura-2. Voltage-dependent calcium currents (VDCC) were measured in A7r5 cells with voltage-clamp electrophysiology using barium as a charge carrier. Key Results Rodent arteries preconstricted with noradrenaline or U46619 were concentration-dependently relaxed by T16Ainh-A01 (0.1–10 μM): IC50 and maximum relaxation were equivalent in ±chloride (30 min aspartate substitution) and the T16Ainh-A01-induced vasorelaxation ±chloride were accompanied by membrane hyperpolarization and lowering of intracellular calcium. However, agonist concentration–response curves ±chloride, with 10 μM T16Ainh-A01 present, achieved similar maximum constrictions although agonist-sensitivity decreased. Contractions induced by elevated extracellular potassium were concentration-dependently relaxed by T16Ainh-A01 ±chloride. Moreover, T16Ainh-A01 inhibited VDCCs in A7r5 cells in a concentration-dependent manner. CaCCinh-A01 and MONNA (0.1–10 μM) induced vasorelaxation ±chloride and both compounds lowered maximum contractility. MONNA, 10 μM, induced substantial membrane hyperpolarization under resting conditions. Conclusions and Implications T16Ainh-A01, CaCCinh-A01 and MONNA concentration-dependently relax rodent resistance arteries, but an equivalent vasorelaxation occurs when the transmembrane chloride gradient is abolished with an impermeant anion. These compounds therefore display poor selectivity for TMEM16A

  7. The novel isoxazoline ectoparasiticide fluralaner: selective inhibition of arthropod γ-aminobutyric acid- and L-glutamate-gated chloride channels and insecticidal/acaricidal activity.

    PubMed

    Gassel, Michael; Wolf, Christian; Noack, Sandra; Williams, Heike; Ilg, Thomas

    2014-02-01

    Isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and L-glutamate-gated chloride channels (GluCls). In this study, the effects of the isoxazoline drug fluralaner on insect and acarid GABACl (RDL) and GluCl and its parasiticidal potency were investigated. We report the identification and cDNA cloning of Rhipicephalus (R.) microplus RDL and GluCl genes, and their functional expression in Xenopus laevis oocytes. The generation of six clonal HEK293 cell lines expressing Rhipicephalus microplus RDL and GluCl, Ctenocephalides felis RDL-A285 and RDL-S285, as well as Drosophila melanogaster RDLCl-A302 and RDL-S302, combined with the development of a membrane potential fluorescence dye assay allowed the comparison of ion channel inhibition by fluralaner with that of established insecticides addressing RDL and GluCl as targets. In these assays fluralaner was several orders of magnitude more potent than picrotoxinin and dieldrin, and performed 5-236 fold better than fipronil on the arthropod RDLs, while a rat GABACl remained unaffected. Comparative studies showed that R. microplus RDL is 52-fold more sensitive than R. microplus GluCl to fluralaner inhibition, confirming that the GABA-gated chloride channel is the primary target of this new parasiticide. In agreement with the superior RDL on-target activity, fluralaner outperformed dieldrin and fipronil in insecticidal screens on cat fleas (Ctenocephalides felis), yellow fever mosquito larvae (Aedes aegypti) and sheep blowfly larvae (Lucilia cuprina), as well as in acaricidal screens on cattle tick (R. microplus) adult females, brown dog tick (Rhipicephalus sanguineus) adult females and Ornithodoros moubata nymphs. These findings highlight the potential of fluralaner as a novel ectoparasiticide.

  8. Sequential interaction of chloride and proton ions with the fast gate steer the voltage-dependent gating in ClC-2 chloride channels

    PubMed Central

    Sánchez-Rodríguez, Jorge E; De Santiago-Castillo, José A; Contreras-Vite, Juan Antonio; Nieto-Delgado, Pablo G; Castro-Chong, Alejandra; Arreola, Jorge

    2012-01-01

    The interaction of either H+ or Cl− ions with the fast gate is the major source of voltage (Vm) dependence in ClC Cl− channels. However, the mechanism by which these ions confer Vm dependence to the ClC-2 Cl− channel remains unclear. By determining the Vm dependence of normalized conductance (Gnorm(Vm)), an index of open probability, ClC-2 gating was studied at different [H+]i, [H+]o and [Cl−]i. Changing [H+]i by five orders of magnitude whilst [Cl−]i/[Cl−]o = 140/140 or 10/140 mm slightly shifted Gnorm(Vm) to negative Vm without altering the onset kinetics; however, channel closing was slower at acidic pHi. A similar change in [H+]o with [Cl−]i/[Cl−]o = 140/140 mm enhanced Gnorm in a bell-shaped manner and shifted Gnorm(Vm) curves to positive Vm. Importantly, Gnorm was >0 with [H+]o = 10−10 m but channel closing was slower when [H+]o or [Cl−]i increased implying that ClC-2 was opened without protonation and that external H+ and/or internal Cl− ions stabilized the open conformation. The analysis of kinetics and steady-state properties at different [H+]o and [Cl−]i was carried out using a gating Scheme coupled to Cl− permeation. Unlike previous results showing Vm-dependent protonation, our analysis revealed that fast gate protonation was Vm and Cl− independent and the equilibrium constant for closed–open transition of unprotonated channels was facilitated by elevated [Cl−]i in a Vm-dependent manner. Hence a Vm dependence of pore occupancy by Cl− induces a conformational change in unprotonated closed channels, before the pore opens, and the open conformation is stabilized by Cl− occupancy and Vm-independent protonation. PMID:22753549

  9. Mosquitocidal properties of IgG targeting the glutamate-gated chloride channel in three mosquito disease vectors (Diptera: Culicidae).

    PubMed

    Meyers, Jacob I; Gray, Meg; Foy, Brian D

    2015-05-15

    The glutamate-gated chloride channel (GluCl) is a highly sensitive insecticide target of the avermectin class of insecticides. As an alternative to using chemical insecticides to kill mosquitoes, we tested the effects of purified immunoglobulin G (IgG) targeting the extracellular domain of GluCl from Anopheles gambiae (AgGluCl) on the survivorship of three key mosquito disease vectors: Anopheles gambiae s.s., Aedes aegypti and Culex tarsalis. When administered through a single blood meal, anti-AgGluCl IgG reduced the survivorship of A. gambiae in a dose-dependent manner (LC50: 2.82 mg ml(-1), range 2.68-2.96 mg ml(-1)) but not A. aegypti or C. tarsalis. We previously demonstrated that AgGluCl is only located in tissues of the head and thorax of A. gambiae. To verify that AgGluCl IgG is affecting target antigens found outside the midgut, we injected it directly into the hemocoel via intrathoracic injection. A single, physiologically relevant concentration of anti-AgGluCl IgG injected into the hemocoel equally reduced mosquito survivorship of all three species. To test whether anti-AgGluCl IgG was entering the hemocoel of each of these mosquitoes, we fed mosquitoes a blood meal containing anti-AgGluCl IgG and subsequently extracted their hemolymph. We only detected IgG in the hemolymph of A. gambiae, suggesting that resistance of A. aegypti and C. tarsalis to anti-AgGluCl IgG found in blood meals is due to deficient IgG translocation across the midgut. We predicted that anti-AgGluCl IgG's mode of action is by antagonizing GluCl activity. To test this hypothesis, we fed A. gambiae blood meals containing anti-AgGluCl IgG and the GluCl agonist ivermectin (IVM). Anti-AgGluCl IgG attenuated the mosquitocidal effects of IVM, suggesting that anti-AgGluCl IgG antagonizes IVM-induced activation of GluCl. Lastly, we stained adult, female A. aegypti and C. tarsalis for GluCl expression. Neuronal GluCl expression in these mosquitoes was similar to previously reported A

  10. CFTR and calcium-activated chloride channels in primary cultures of human airway gland cells of serous or mucous phenotype.

    PubMed

    Fischer, Horst; Illek, Beate; Sachs, Lorne; Finkbeiner, Walter E; Widdicombe, Jonathan H

    2010-10-01

    Using cell culture models, we have investigated the relative importance of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCC) in Cl secretion by mucous and serous cells of human airway glands. In transepithelial recordings in Ussing chambers, the CFTR inhibitor CFTR(inh)-172 abolished 60% of baseline Cl secretion in serous cells and 70% in mucous. Flufenamic acid (FFA), an inhibitor of CaCC, reduced baseline Cl secretion by ∼20% in both cell types. Methacholine and ATP stimulated Cl secretion in both cell types, which was largely blocked by treatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and partially by mucosal FFA or CFTR(inh)-172 with the exception of methacholine responses in mucous cells, which were not blocked by FFA and partially (∼60%) by CFTR(inh)-172. The effects of ionomycin on short-circuit current (I(sc)) were less than those of ATP or methacholine. Forskolin stimulated Cl secretion only if Cl in the mucosal medium was replaced by gluconate. In whole cell patch-clamp studies of single isolated cells, cAMP-induced Cl currents were ∼3-fold greater in serous than mucous cells. Ionomycin-induced Cl currents were 13 times (serous) or 26 times (mucous) greater than those generated by cAMP and were blocked by FFA. In serous cells, mRNA for transmembrane protein 16A (TMEM16A) was ∼10 times more abundant than mRNA for CFTR. In mucous cells it was ∼100 times more abundant. We conclude: 1) serous and mucous cells both make significant contributions to gland fluid secretion; 2) baseline Cl secretion in both cell types is mediated predominantly by CFTR, but CaCC becomes increasingly important after mediator-induced elevations of intracellular Ca; and 3) the high CaCC currents seen in patch-clamp studies and the high TMEM16A expression in intact polarized cells sheets are not reflected in transepithelial current recordings.

  11. The "small" conductance chloride channel in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias).

    PubMed

    Gögelein, H; Schlatter, E; Greger, R

    1987-06-01

    Besides the "larger" Cl- channel, with a single channel conductance of about 45 pS, a "small" channel was observed in the luminal membrane of the dogfish rectal gland. In cell excised (inside out) patches with NaCl solution on both sides, the latter channel had a single channel conductance of 11 +/- 1 pS (n = 21), and its current-voltage relationship was linear in the voltage range +90 to -90 mV. The open state probability increased moderately with negative clamp potentials. Ionic replacement studies revealed a high selectivity of Cl- over gluconate, sulfate, and iodide, whereas bromide was permeable to some extent. Also the channel is impermeable for Na+. The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate did not affect this "small" conductance Cl- channel. It can be concluded that the luminal membrane of stimulated rectal gland cells possesses two types of Cl- channels, which differ markedly in their characteristics.

  12. Kidney-specific chloride channel, OmClC-K, predominantly expressed in the diluting segment of freshwater-adapted tilapia kidney

    PubMed Central

    Miyazaki, Hiroaki; Kaneko, Toyoji; Uchida, Shinichi; Sasaki, Sei; Takei, Yoshio

    2002-01-01

    The kidney plays an important role in osmoregulation in freshwater teleosts, which are exposed to the danger of osmotic loss of Na+ and Cl−. However, ion-transport mechanisms in the kidney are poorly understood, and ion transporters of the fish nephron have not been identified thus far. From Mozambique tilapia, Oreochromis mossambicus, we have cloned a chloride channel, which is a homologue of the mammalian kidney-specific chloride channel, ClC-K. The cDNA of the channel, named OmClC-K, encodes a protein whose amino acid sequence has high homology to Xenopus and mammalian ClC-K (Xenopus ClC-K, 41.8%; rat ClC-K2, 40.9%; rat ClC-K1, 40.1%). The mRNA of OmClC-K was expressed exclusively in the kidney, and the expression level of mRNA was increased more in freshwater-adapted fish than seawater-adapted fish. The immunohistochemical study using a specific antibody showed that OmClC-K-positive cells were specifically located in the distal nephron segments. Immunoelectron microscopy further showed that immunoreaction of OmClC-K was recognizable on the structure of basolateral membrane infoldings in the distal tubule cells. The localization of OmClC-K and its induction in hypoosmotic media suggest that OmClC-K is involved in Cl− reabsorption in the distal tubule of freshwater-adapted tilapia. PMID:12427972

  13. The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels

    PubMed Central

    Chen, Yi-An; Peng, Yi-Jheng; Hu, Meng-Chun; Huang, Jing-Jia; Chien, Yun-Chia; Wu, June-Tai; Chen, Tsung-Yu; Tang, Chih-Yung

    2015-01-01

    Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels. PMID:26021757

  14. The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels.

    PubMed

    Chen, Yi-An; Peng, Yi-Jheng; Hu, Meng-Chun; Huang, Jing-Jia; Chien, Yun-Chia; Wu, June-Tai; Chen, Tsung-Yu; Tang, Chih-Yung

    2015-05-29

    Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels.

  15. Identification of a Novel Member of the Chloride Intracellular Channel Gene Family (CLIC5) That Associates with the Actin Cytoskeleton of Placental Microvilli

    PubMed Central

    Berryman, Mark; Bretscher, Anthony

    2000-01-01

    The chloride intracellular channel (CLIC) gene family has been implicated in chloride ion transport within various subcellular compartments. We report here the molecular, biochemical, and cellular characterization of a new member of this gene family termed CLIC5. CLIC5 was isolated from extracts of placental microvilli as a component of a multimeric complex consisting of several known cytoskeletal proteins, including actin, ezrin, α-actinin, gelsolin, and IQGAP1. We cloned human cDNAs and generated antibodies specific for CLIC5, CLIC1/NCC27, and CLIC4/huH1/p64H1. CLIC5 shares 52–76% overall identity with human CLIC1, CLIC2, CLIC3, and CLIC4. Northern blot analysis showed that CLIC5 has a distinct pattern of expression compared with CLIC1 and CLIC4. Immunoblot analysis of extracts from placental tissues demonstrated that CLIC4 and CLIC5 are enriched in isolated placental microvilli, whereas CLIC1 is not. Moreover, in contrast to CLIC1 and CLIC4, CLIC5 is associated with the detergent-insoluble cytoskeletal fraction of microvilli. Indirect immunofluorescence microscopy revealed that CLIC4 and CLIC5 are concentrated within the apical region of the trophoblast, whereas CLIC1 is distributed throughout the cytoplasm. These studies suggest that CLIC1, CLIC4, and CLIC5 play distinct roles in chloride transport and that CLIC5 interacts with the cortical actin cytoskeleton in polarized epithelial cells. PMID:10793131

  16. Niflumic acid inhibits chloride conductance of rat skeletal muscle by directly inhibiting the CLC-1 channel and by increasing intracellular calcium

    PubMed Central

    Liantonio, A; Giannuzzi, V; Picollo, A; Babini, E; Pusch, M; Conte Camerino, D

    2006-01-01

    Background and purpose: Given the crucial role of the skeletal muscle chloride conductance (gCl), supported by the voltage-gated chloride channel CLC-1, in controlling muscle excitability, the availability of ligands modulating CLC-1 are of potential medical as well as toxicological importance. Here, we focused our attention on niflumic acid (NFA), a molecule belonging to the fenamates group of non-steroidal anti-inflammatory drugs (NSAID). Experimental approach: Rat muscle Cl− conductance (gCl) and heterologously expressed CLC-1 currents were evaluated by means of current-clamp (using two-microelectrodes) and patch-clamp techniques, respectively. Fura-2 fluorescence was used to determine intracellular calcium concentration, [Ca2+]i, in native muscle fibres. Key results: NFA inhibited native gCl with an IC50 of 42 μM and blocked CLC-1 by interacting with an intracellular binding site. Additionally, NFA increased basal [Ca2+]i in myofibres by promoting a mitochondrial calcium efflux that was not dependent on cyclooxygenase or CLC-1. A structure-activity study revealed that the molecular conditions that mediate the two effects are different. Pretreatment with the Ca-dependent protein kinase C (PKC) inhibitor chelerythrine partially inhibited the NFA effect. Therefore, in addition to direct channel block, NFA also inhibits gCl indirectly by promoting PKC activation. Conclusions and Implications: These cellular effects of NFA on skeletal muscle demonstrate that it is possible to modify CLC-1 and consequently gCl directly by interacting with channel proteins and indirectly by interfering with the calcium-dependent regulation of the channel. The effect of NFA on mitochondrial calcium stores suggests that NSAIDs, widely used drugs, could have potentially dangerous side-effects. PMID:17128287

  17. Chloride channels of glycine and GABA receptors with blockers: Monte Carlo minimization and structure-activity relationships.

    PubMed Central

    Zhorov, B S; Bregestovski, P D

    2000-01-01

    GABA and glycine receptors (GlyRs) are pentameric ligand-gated ion channels that respond to the inhibitory neurotransmitters by opening a chloride-selective central pore lined with five M2 segments homologous to those of alpha(1) GlyR/ ARVG(2')LGIT(6')TVLTMTTQSSGSR. The activity of cyanotriphenylborate (CTB) and picrotoxinin (PTX), the best-studied blockers of the Cl(-) pores, depends essentially on the subunit composition of the receptors, in particular, on residues in positions 2' and 6' that form the pore-facing rings R(2') and R(6'). Thus, CTB blocks alpha(1) and alpha(1)/beta, but not alpha(2) GlyRs (Rundström, N., V. Schmieden, H. Betz, J. Bormann, and D. Langosch. 1994. Proc. Natl. Acad. Sci. U.S.A. 91:8950-8954). PTX blocks homomeric receptors (alpha(1) GlyR and rat rho(1) GABAR), but weakly antagonizes heteromeric receptors (alpha(1)/beta GlyR and rho(1)/rho(2) GABAR) (Pribilla, I., T. Takagi, D. Langosch, J. Bormann, and H. Betz. 1992. EMBO J. 11:4305-4311; Zhang D., Z. H. Pan, X. Zhang, A. D. Brideau, and S. A. Lipton. 1995. Proc. Natl. Acad. Sci. U.S.A. 92:11756-11760). Using as a template the kinked-helices model of the nicotinic acetylcholine receptor in the open state (Tikhonov, D. B., and B. S. Zhorov. 1998. Biophys. J. 74:242-255), we have built homology models of GlyRs and GABARs and calculated Monte Carlo-minimized energy profiles for the blockers pulled through the pore. The profiles have shallow minima at the wide extracellular half of the pore, a barrier at ring R(6'), and a deep minimum between rings R(6') and R(2') where the blockers interact with five M2s simultaneously. The star-like CTB swings necessarily on its way through ring R(6') and its activity inversely correlates with the barrier at R(6'): Thr(6')s and Ala(2')s in alpha(2) GlyR confine the swinging by increasing the barrier, while Gly(2')s in alpha(1) GlyR and Phe(6')s in beta GlyR shrink the barrier. PTX has an egg-like shape with an isopropenyl group at the elongated end and

  18. Contribution of calcium-activated chloride channel to elevated pulmonary artery pressure in pulmonary arterial hypertension induced by high pulmonary blood flow.

    PubMed

    Wang, Kai; Chen, Chuansi; Ma, Jianfa; Lao, Jinquan; Pang, Yusheng

    2015-01-01

    The correlation between calcium-activated chloride channel (CaCC) and pulmonary arterial hypertension (PAH) induced by high pulmonary blood flow remains uncertain. In this study, we investigated the possible role and effects of CaCC in this disease. Sixty rats were randomly assigned to normal, sham, and shunt groups. Rats in the shunt group underwent abdominal aorta and inferior vena cava shunt surgery. The pulmonary artery pressure was measured by catheterization. Pathological changes, right ventricle hypertrophy index (RVHI), arterial wall area/vessel area (W/V), and arterial wall thickness/vessel external diameter (T/D) were analyzed by optical microscopy. Electrophysiological characteristics of pulmonary arterial smooth muscle cells (PASMCs) were investigated using patch clamp technology. After 11 weeks of shunting, PAH and pulmonary vascular structural remodeling (PVSR) developed, accompanied by increased pulmonary pressure and pathological interstitial pulmonary changes. Compared with normal and sham groups, pulmonary artery pressure, RVHI, W/V, and T/D of the shunt group rats increased significantly. Electrophysiological results showed primary CaCC characteristics. Compared with normal and sham groups, membrane capacitance and current density of PASMCs in the shunt group increased significantly, which were subsequently attenuated following chloride channel blocker niflumic acid (NFA) treatment. To conclude, CaCC contributed to PAH induced by high pulmonary blood flow and may represent a potential target for treatment of PAH.

  19. Contribution of calcium-activated chloride channel to elevated pulmonary artery pressure in pulmonary arterial hypertension induced by high pulmonary blood flow

    PubMed Central

    Wang, Kai; Chen, Chuansi; Ma, Jianfa; Lao, Jinquan; Pang, Yusheng

    2015-01-01

    The correlation between calcium-activated chloride channel (CaCC) and pulmonary arterial hypertension (PAH) induced by high pulmonary blood flow remains uncertain. In this study, we investigated the possible role and effects of CaCC in this disease. Sixty rats were randomly assigned to normal, sham, and shunt groups. Rats in the shunt group underwent abdominal aorta and inferior vena cava shunt surgery. The pulmonary artery pressure was measured by catheterization. Pathological changes, right ventricle hypertrophy index (RVHI), arterial wall area/vessel area (W/V), and arterial wall thickness/vessel external diameter (T/D) were analyzed by optical microscopy. Electrophysiological characteristics of pulmonary arterial smooth muscle cells (PASMCs) were investigated using patch clamp technology. After 11 weeks of shunting, PAH and pulmonary vascular structural remodeling (PVSR) developed, accompanied by increased pulmonary pressure and pathological interstitial pulmonary changes. Compared with normal and sham groups, pulmonary artery pressure, RVHI, W/V, and T/D of the shunt group rats increased significantly. Electrophysiological results showed primary CaCC characteristics. Compared with normal and sham groups, membrane capacitance and current density of PASMCs in the shunt group increased significantly, which were subsequently attenuated following chloride channel blocker niflumic acid (NFA) treatment. To conclude, CaCC contributed to PAH induced by high pulmonary blood flow and may represent a potential target for treatment of PAH. PMID:25755701

  20. TMEM16A Inhibitors Reveal TMEM16A as a Minor Component of Calcium-activated Chloride Channel Conductance in Airway and Intestinal Epithelial Cells*

    PubMed Central

    Namkung, Wan; Phuan, Puay-Wah; Verkman, A. S.

    2011-01-01

    TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of ∼110,000 compounds revealed four novel chemical classes of small molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with an IC50 < 10 μm, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor, an aminophenylthiazole (T16Ainh-A01), had an IC50 of ∼1 μm. Two distinct types of inhibitors were identified. Some compounds, such as tannic acid and the arylaminothiophene CaCCinh-A01, fully inhibited CaCC current in human bronchial and intestinal cells. Other compounds, including T16Ainh-A01 and digallic acid, inhibited total CaCC current in these cells poorly, but blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia. The small molecule inhibitors identified here permit pharmacological dissection of TMEM16A/CaCC function and are potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production. PMID:21084298

  1. Rattlesnake Phospholipase A2 Increases CFTR-Chloride Channel Current and Corrects ∆F508CFTR Dysfunction: Impact in Cystic Fibrosis.

    PubMed

    Faure, Grazyna; Bakouh, Naziha; Lourdel, Stéphane; Odolczyk, Norbert; Premchandar, Aiswarya; Servel, Nathalie; Hatton, Aurélie; Ostrowski, Maciej K; Xu, Haijin; Saul, Frederick A; Moquereau, Christelle; Bitam, Sara; Pranke, Iwona; Planelles, Gabrielle; Teulon, Jacques; Herrmann, Harald; Roldan, Ariel; Zielenkiewicz, Piotr; Dadlez, Michal; Lukacs, Gergely L; Sermet-Gaudelus, Isabelle; Ollero, Mario; Corringer, Pierre-Jean; Edelman, Aleksander

    2016-07-17

    Deletion of Phe508 in the nucleotide binding domain (∆F508-NBD1) of the cystic fibrosis transmembrane regulator (CFTR; a cyclic AMP-regulated chloride channel) is the most frequent mutation associated with cystic fibrosis. This mutation affects the maturation and gating of CFTR protein. The search for new high-affinity ligands of CFTR acting as dual modulators (correctors/activators) presents a major challenge in the pharmacology of cystic fibrosis. Snake venoms are a rich source of natural multifunctional proteins, potential binders of ion channels. In this study, we identified the CB subunit of crotoxin from Crotalus durissus terrificus as a new ligand and allosteric modulator of CFTR. We showed that CB interacts with NBD1 of both wild type and ∆F508CFTR and increases their chloride channel currents. The potentiating effect of CB on CFTR activity was demonstrated using electrophysiological techniques in Xenopus laevis oocytes, in CFTR-HeLa cells, and ex vivo in mouse colon tissue. The correcting effect of CB was shown by functional rescue of CFTR activity after 24-h ΔF508CFTR treatments with CB. Moreover, the presence of fully glycosylated CFTR was observed. Molecular docking allowed us to propose a model of the complex involving of the ABCβ and F1-like ATP-binding subdomains of ΔF508-NBD1. Hydrogen-deuterium exchange analysis confirmed stabilization in these regions, also showing allosteric stabilization in two other distal regions. Surface plasmon resonance competition studies showed that CB disrupts the ∆F508CFTR-cytokeratin 8 complex, allowing for the escape of ∆F508CFTR from degradation. Therefore CB, as a dual modulator of ΔF508CFTR, constitutes a template for the development of new anti-CF agents.

  2. Ionotropic and metabotropic activation of a neuronal chloride channel by serotonin and dopamine in the leech Hirudo medicinalis

    PubMed Central

    Ali, Declan W; Catarsi, Stefano; Drapeau, Pierre

    1998-01-01

    Cl− channels on the pressure-sensitive (P) neuron in the leech are directly activated by synaptic release of serotonin (5-HT) and are indirectly stimulated by the cAMP second messenger pathway, suggesting an unusual dual regulation of the channels. We have investigated the mode of action of 5-HT and dopamine (DA) on a Cl− channel in adult P cells in culture by recording from cell-attached patches.5-HT increased Cl− channel activity only when included in the recording pipette and not when applied in the bath.Pipette or, more effectively, bath application of DA led to an increase in Cl− channel activity. This effect was blocked by the potent and specific dopaminergic (DA1) receptor blocker, SCH-23390.The stimulation by DA, but not by 5-HT, was also blocked by the cAMP-dependent protein kinase A (PKA) inhibitor Rp-cAMP and was mimicked by the membrane-permeant cAMP analogue dibutyryl cAMP (db-cAMP).Our results show that 5-HT directly gates a Cl− channel that is also activated by DA via the cAMP pathway. This study demonstrates that a ligand-gated channel can be independently operated by another transmitter acting via a second messenger pathway. PMID:9547394

  3. Multidimensional open-frameworks: combinations of one-dimensional channels and two-dimensional layers in novel BI/M oxo-chlorides.

    PubMed

    Lü, Minfeng; Aliev, Almaz; Olchowka, Jacob; Colmont, Marie; Huvé, Marielle; Wickleder, Claudia; Mentré, Olivier

    2014-01-06

    Here we discuss the synthesis and characterization of three novel bismuth oxo-chlorides ([Bi6Na0.5O7.5][Na0.5Cl3]channel[Cl]layer; [Bi17PbO22][Cl6]channel[Cl3]layer; [Bi9(Pb0.2Mn0.8)O12][Cl3]channel [Cl2]layer) which all show an original multidimensional crystal structure. It is formed of two-dimensional (2D)-layered blocks separated by Cl(-) layers. The blocks are porous with triangular one-dimensional (1D)-Cl(-) channels with various section sizes. This multidimensional feature is unique in the field of Bi and Pb oxo-halides, while so far only 1D or 2D halides units have been reported. The stability of the framework is allowed by Bi(3+)/M(n+) aliovalent substitution to balance charge neutrality. The channel and tunnel walls are formed by edge-sharing O(Bi,M)4 oxocentered tetrahedra, while the triangular tunnel junctions are achieved by O(Bi,M)5 pyramids. The three compounds are rather stable, but only [Bi6Na0.5O7.5][Na0.5Cl3]tunnel[Cl]layer was obtain as a single-phase material so that its photoluminecence properties have been investigated. It shows an unusual red bright luminescence with a maximum at 14150 cm(-1) at low temperatures due to Bi(3+) transitions that are well explained by the Bi-Cl bonding scheme.

  4. Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells

    NASA Astrophysics Data System (ADS)

    Rich, Devra P.; Anderson, Matthew P.; Gregory, Richard J.; Cheng, Seng H.; Paul, Sucharita; Jefferson, Douglas M.; McCann, John D.; Klinger, Katherine W.; Smith, Alan E.; Welsh, Michael J.

    1990-09-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (ΔF508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.

  5. Metal bridges illuminate transmembrane domain movements during gating of the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    El Hiani, Yassine; Linsdell, Paul

    2014-10-10

    Opening and closing of the cystic fibrosis transmembrane conductance regulator are controlled by ATP binding and hydrolysis by the cytoplasmic nucleotide-binding domains. Different conformational changes in the channel pore have been described during channel opening and closing; however, the relative importance of these changes to the process of gating the pore is not known. We have used patch clamp recording to identify high affinity Cd(2+) bridges formed between pairs of pore-lining cysteine residues introduced into different transmembrane α-helices (TMs). Seven Cd(2+) bridges were identified forming between cysteines in TMs 6 and 12. Interestingly, each of these Cd(2+) bridges apparently formed only in closed channels, and their formation stabilized the closed state. In contrast, a single Cd(2+) bridge identified between cysteines in TMs 1 and 12 stabilized the channel open state. Analysis of the pattern of Cd(2+) bridge formation in different channel states suggests that lateral separation and convergence of different TMs, rather than relative rotation or translation of different TMs, is the key conformational change that causes the channel pore to open and close.

  6. Metal Bridges Illuminate Transmembrane Domain Movements during Gating of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel*

    PubMed Central

    El Hiani, Yassine; Linsdell, Paul

    2014-01-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator are controlled by ATP binding and hydrolysis by the cytoplasmic nucleotide-binding domains. Different conformational changes in the channel pore have been described during channel opening and closing; however, the relative importance of these changes to the process of gating the pore is not known. We have used patch clamp recording to identify high affinity Cd2+ bridges formed between pairs of pore-lining cysteine residues introduced into different transmembrane α-helices (TMs). Seven Cd2+ bridges were identified forming between cysteines in TMs 6 and 12. Interestingly, each of these Cd2+ bridges apparently formed only in closed channels, and their formation stabilized the closed state. In contrast, a single Cd2+ bridge identified between cysteines in TMs 1 and 12 stabilized the channel open state. Analysis of the pattern of Cd2+ bridge formation in different channel states suggests that lateral separation and convergence of different TMs, rather than relative rotation or translation of different TMs, is the key conformational change that causes the channel pore to open and close. PMID:25143385

  7. Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension

    PubMed Central

    Forrest, Abigail S.; Joyce, Talia C.; Huebner, Marissa L.; Ayon, Ramon J.; Wiwchar, Michael; Joyce, John; Freitas, Natalie; Davis, Alison J.; Ye, Linda; Duan, Dayue D.; Singer, Cherie A.; Valencik, Maria L.; Greenwood, Iain A.

    2012-01-01

    Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca2+ levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca2+-activated Cl− channels (ClCa), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of ClCa channels in PH was investigated using the monocrotaline (MCT)-induced PH model in the rat. Three weeks postinjection with a single dose of MCT (50 mg/kg ip), the animals developed right ventricular hypertrophy (heart weight measurements) and changes in pulmonary arterial flow (pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole cell patch experiments revealed an increase in niflumic acid (NFA)-sensitive Ca2+-activated Cl− current [ICl(Ca)] density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats vs. aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in ICl(Ca) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for ClCa channels. The contraction to serotonin of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 μM), an l-type Ca2+ channel blocker, and NFA (30 or 100 μM, with or without 10 μM indomethacin to inhibit cyclooxygenases) or T16AInh-A01 (10 μM), TMEM16A/ClCa channel inhibitors, than that of control animals. In conclusion, augmented ClCa/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling of PA in this model of PH. TMEM16A-encoded channels may therefore represent a novel therapeutic target in this disease. PMID:23034390

  8. Parasitoid wasp sting: a cocktail of GABA, taurine, and beta-alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host.

    PubMed

    Moore, Eugene L; Haspel, Gal; Libersat, Frederic; Adams, Michael E

    2006-07-01

    The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal-giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI-TOF-MS revealed high levels of GABA (25 mM), and its receptor agonists beta-alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal-giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin-sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host.

  9. Nanomolar-Potency Aminophenyl-1,3,5-triazine Activators of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Channel for Prosecretory Therapy of Dry Eye Diseases.

    PubMed

    Lee, Sujin; Phuan, Puay-Wah; Felix, Christian M; Tan, Joseph-Anthony; Levin, Marc H; Verkman, Alan S

    2017-02-09

    Dry eye disorders are a significant health problem for which limited therapeutic options are available. CFTR is a major prosecretory chloride channel at the ocular surface. We previously identified, by high-throughput screening, aminophenyl-1,3,5-triazine CFTRact-K089 (1) that activated CFTR with EC50 ≈ 250 nM, which when delivered topically increased tear fluid secretion in mice and showed efficacy in an experimental dry eye model. Here, functional analysis of aminophenyl-1,3,5-triazine analogs elucidated structure-activity relationships for CFTR activation and identified substantially more potent analogs than 1. The most potent compound, 12, fully activated CFTR chloride conductance with EC50 ≈ 30 nM, without causing cAMP or calcium elevation. 12 was rapidly metabolized by hepatic microsomes, which supports its topical use. Single topical administration of 25 pmol of 12 increased tear volume in wild-type mice with sustained action for 8 h and was without effect in CFTR-deficient mice. Topically delivered 12 may be efficacious in human dry eye diseases.

  10. Calcium-calmodulin does not alter the anion permeability of the mouse TMEM16A calcium-activated chloride channel

    PubMed Central

    Yu, Yawei; Kuan, Ai-Seon

    2014-01-01

    The transmembrane protein TMEM16A forms a Ca2+-activated Cl− channel that is permeable to many anions, including SCN−, I−, Br−, Cl−, and HCO3−, and has been implicated in various physiological functions. Indeed, controlling anion permeation through the TMEM16A channel pore may be critical in regulating the pH of exocrine fluids such as the pancreatic juice. The anion permeability of the TMEM16A channel pore has recently been reported to be modulated by Ca2+-calmodulin (CaCaM), such that the pore of the CaCaM-bound channel shows a reduced ability to discriminate between anions as measured by a shift of the reversal potential under bi-ionic conditions. Here, using a mouse TMEM16A clone that contains the two previously identified putative CaM-binding motifs, we were unable to demonstrate such CaCaM-dependent changes in the bi-ionic potential. We confirmed the activity of CaCaM used in our study by showing CaCaM modulation of the olfactory cyclic nucleotide–gated channel. We suspect that the different bi-ionic potentials that were obtained previously from whole-cell recordings in low and high intracellular [Ca2+] may result from different degrees of bi-ionic potential shift secondary to a series resistance problem, an ion accumulation effect, or both. PMID:24981232

  11. cAMP-activated chloride channels in a CFTR-transfected pancreatic adenocarcinoma-derived cell line, pANS6.

    PubMed

    Smith, A N; Wardle, C J; Winpenny, J P; Verdon, B; Gray, M A; Argent, B E; Harris, A

    1995-06-09

    Pancreatic adenocarcinoma cell lines rarely express the CFTR gene, despite the high levels of CFTR protein that are present in primary pancreatic duct cells. We have attempted to generate a non-CF pancreatic adenocarcinoma cell line that stably produces high levels of CFTR mRNA and protein by transfecting a vector containing the CFTR cDNA, driven by a strong mammalian promoter, into the poorly differentiated pancreatic adenocarcinoma cell line, Panc-1. The pANS6 pancreatic duct cell line expresses substantial levels of CFTR mRNA, but little CFTR protein. Despite this we were able to detect low conductance chloride channels in 40% of patches, stimulated with cAMP, that have similar biophysical properties to CFTR.

  12. 5,5'-Dithio-bis(2-nitrobenzoic acid) modification of cysteine improves the crystal quality of human chloride intracellular channel protein 2

    SciTech Connect

    Mi Wei; Li Lanfen; Su Xiaodong

    2008-04-18

    Structural studies of human chloride intracellular channel protein 2 (CLIC2) had been hampered by the problem of generating suitable crystals primarily due to the protein containing exposed cysteines. Several chemical reagents were used to react with the cysteines on CLIC2 in order to modify the redox state of the protein. We have obtained high quality crystals that diffracted to better than 2.5 A at a home X-ray source by treating the protein with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). After solving the crystal structure of CLIC2, we found that the DTNB had reacted with the Cys{sup 114}, and made CLIC2 in a homogenous oxidized state. This study demonstrated that the DTNB modification drastically improved the crystallization of CLIC2, and it implied that this method may be useful for other proteins containing exposed cysteines in general.

  13. Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels.

    PubMed

    Peters, Christian J; Yu, Haibo; Tien, Jason; Jan, Yuh Nung; Li, Min; Jan, Lily Yeh

    2015-03-17

    TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative pore-lining basic residues significantly altered the IC50 of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore.

  14. Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

    PubMed Central

    Peters, Christian J.; Yu, Haibo; Tien, Jason; Jan, Yuh Nung; Li, Min; Jan, Lily Yeh

    2015-01-01

    TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative pore-lining basic residues significantly altered the IC50 of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore. PMID:25733897

  15. High-Level Expression, Functional Reconstitution, and Quaternary Structure of a Prokaryotic Clc-Type Chloride Channel

    PubMed Central

    Maduke, Merritt; Pheasant, Deborah J.; Miller, Christopher

    1999-01-01

    ClC-type anion-selective channels are widespread throughout eukaryotic organisms. BLAST homology searches reveal that many microbial genomes also contain members of the ClC family. An Escherichia coli–derived ClC Cl− channel homologue, “EriC,” the product of the yadQ gene, was overexpressed in E. coli and purified in milligram quantities in a single-step procedure. Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels. Cross-linking studies argue that EriC is a homodimer in both detergent micelles and reconstituted liposomes, a conclusion corroborated by gel filtration and analytical sedimentation experiments. PMID:10539975

  16. Inhibitory effect of taurine on veratridine-evoked D-[3H]aspartate release from murine corticostriatal slices: involvement of chloride channels and mitochondria.

    PubMed

    Molchanova, Svetlana M; Oja, Simo S; Saransaari, Pirjo

    2007-01-26

    We have previously shown that the inhibitory neuromodulator taurine attenuates the release of preloaded D-[3H]aspartate from murine corticostriatal slices evoked by ischemic conditions or by application of the sodium channel agonist veratridine. The release of D-[3H]aspartate (a non-metabolized analog of glutamate) was used as an index of glutamate release. The aim of the present study was to reveal the molecular mechanisms responsible for this inhibitory effect of taurine. It was shown that 10 mM taurine suppresses D-[3H]aspartate release evoked by 0.1 mM veratridine, but does not affect the high-K+ -(50 mM) or ouabain- (0.1 mM) evoked release. Taurine had no effect in Ca2+ -free medium when the synaptic exocytosis of D-[3H]aspartate was inhibited. Nor did it suppress the release from slices preloaded with the competitive glutamate uptake blocker DL-threo-beta-hydroxyaspartate (THBA), which inhibits D-[3H]aspartate release mediated by the reverse action of glutamate transporters. Omission of Cl- from the incubation medium reduced the effect of taurine, signifying the involvement of a Cl- channel. The glycine receptor antagonist strychnine and the GABA(A) receptor antagonist bicuculline did not block the taurine effect, although picrotoxin, a less specific blocker of agonist-gated chloride channels, completely prevented the effect of taurine on veratridine-induced D-[3H]aspartate release. The respiratory chain blocker rotenone or mitochondrial protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in combination with the mitochondrial ATPase inhibitor oligomycin, which inhibits the mitochondrial Ca2+ uniporter, also reduced the effect of taurine. The results obtained in the present study show that taurine acts specifically on the release of preloaded D-[3H]aspartate evoked by veratridine, but not on that evoked by other depolarizing agents, and affects the release mediated both by synaptic exocytosis and the reverse action of glutamate transporter. Taurine

  17. Sodium chloride salinity reduces Cd uptake by edible amaranth (Amaranthus mangostanus L.) via competition for Ca channels.

    PubMed

    Mei, XiuQin; Li, SongSong; Li, QuSheng; Yang, YuFeng; Luo, Xuan; He, BaoYan; Li, Hui; Xu, ZhiMin

    2014-07-01

    Soil salinity is known to enhance cadmium (Cd) accumulation in crops. However, the mechanism by which this occurs independent of the surrounding soil remains unclear. In this study, root adsorption and uptake of salt cations and Cd by edible amaranth under NaCl salinity stress were investigated in hydroponic cultures with 0, 40, 80, 120, and 160mM of NaCl and 27nM Cd. The dominant Cd species in the nutrient solution changed from free Cd(2+) to Cd chlorocomplexes as NaCl salinity increased. High salinity significantly reduced K, Ca, and Cd root adsorption and K, Ca, Mg, and Cd uptake. High salinity decreased root adsorption of Cd by 43 and 58 percent and Cd uptake by 32 and 36 percent in salt-tolerant and salt-sensitive cultivars, respectively. Transformation of Cd from free ion to chlorocomplexes is unlikely to have significantly affected Cd uptake by the plant because of the very low Cd concentrations involved. Application of Ca ion channel blocker significantly reduced Na, K, Ca, Mg, and Cd uptake by the roots, while blocking K ion channels significantly reduced Na and K uptake but not Ca, Mg, and Cd uptake. These results suggest that Na was absorbed by the roots through both Ca and K ion channels, while Cd was absorbed by the roots mainly through Ca ion channels and not K ion channels. Salinity caused a greater degree of reduction in Cd adsorption and uptake in the salt-sensitive cultivar than in the salt-tolerant cultivar. Thus, competition between Na and Cd for Ca ion channels can reduce Cd uptake at very low Cd concentrations in the nutrient solution.

  18. Identification of sites responsible for the potentiating effect of niflumic acid on ClC-Ka kidney chloride channels

    PubMed Central

    Zifarelli, G; Liantonio, A; Gradogna, A; Picollo, A; Gramegna, G; De Bellis, M; Murgia, AR; Babini, E; Conte Camerino, D; Pusch, M

    2010-01-01

    Background and purpose: ClC-K kidney Cl− channels are important for renal and inner ear transepithelial Cl− transport, and are potentially interesting pharmacological targets. They are modulated by niflumic acid (NFA), a non-steroidal anti-inflammatory drug, in a biphasic way: NFA activates ClC-Ka at low concentrations, but blocks the channel above ∼1 mM. We attempted to identify the amino acids involved in the activation of ClC-Ka by NFA. Experimental approach: We used site-directed mutagenesis and two-electrode voltage clamp analysis of wild-type and mutant channels expressed in Xenopus oocytes. Guided by the crystal structure of a bacterial CLC homolog, we screened 97 ClC-Ka mutations for alterations of NFA effects. Key results: Mutations of five residues significantly reduced the potentiating effect of NFA. Two of these (G167A and F213A) drastically altered general gating properties and are unlikely to be involved in NFA binding. The three remaining mutants (L155A, G345S and A349E) severely impaired or abolished NFA potentiation. Conclusions and implications: The three key residues identified (L155, G345, A349) are localized in two different protein regions that, based on the crystal structure of bacterial CLC homologs, are expected to be exposed to the extracellular side of the channel, relatively close to each other, and are thus good candidates for being part of the potentiating NFA binding site. Alternatively, the protein region identified mediates conformational changes following NFA binding. Our results are an important step towards the development of ClC-Ka activators for treating Bartter syndrome types III and IV with residual channel activity. PMID:20649569

  19. Activation of the Ano1 (TMEM16A) chloride channel by calcium is not mediated by calmodulin

    PubMed Central

    Zhu, Jinqiu; Qu, Zhiqiang; Cui, Yuan-Yuan; Hartzell, H. Criss

    2014-01-01

    The Ca2+-activated Cl channel anoctamin-1 (Ano1; Tmem16A) plays a variety of physiological roles, including epithelial fluid secretion. Ano1 is activated by increases in intracellular Ca2+, but there is uncertainty whether Ca2+ binds directly to Ano1 or whether phosphorylation or additional Ca2+-binding subunits like calmodulin (CaM) are required. Here we show that CaM is not necessary for activation of Ano1 by Ca2+ for the following reasons. (a) Exogenous CaM has no effect on Ano1 currents in inside-out excised patches. (b) Overexpression of Ca2+-insensitive mutants of CaM have no effect on Ano1 currents, whereas they eliminate the current mediated by the small-conductance Ca2+-activated K+ (SK2) channel. (c) Ano1 does not coimmunoprecipitate with CaM, whereas SK2 does. Furthermore, Ano1 binds very weakly to CaM in pull-down assays. (d) Ano1 is activated in excised patches by low concentrations of Ba2+, which does not activate CaM. In addition, we conclude that reversible phosphorylation/dephosphorylation is not required for current activation by Ca2+ because the current can be repeatedly activated in excised patches in the absence of ATP or other high-energy compounds. Although Ano1 is blocked by the CaM inhibitor trifluoperazine (TFP), we propose that TFP inhibits the channel in a CaM-independent manner because TFP does not inhibit Ano1 when applied to the cytoplasmic side of excised patches. These experiments lead us to conclude that CaM is not required for activation of Ano1 by Ca2+. Although CaM is not required for channel opening by Ca2+, work of other investigators suggests that CaM may have effects in modulating the biophysical properties of the channel. PMID:24420770

  20. Functional differences in pore properties between wild-type and cysteine-less forms of the CFTR chloride channel.

    PubMed

    Holstead, Ryan G; Li, Man-Song; Linsdell, Paul

    2011-10-01

    Studies of the structure and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have been advanced by the development of functional channel variants in which all 18 endogenous cysteine residues have been mutated ("cys-less" CFTR). However, cys-less CFTR has a slightly higher single-channel conductance than wild-type CFTR, raising questions as to the suitability of cys-less as a model of the wild-type CFTR pore. We used site-directed mutagenesis and patch-clamp recording to investigate the origin of this conductance difference and to determine the extent of functional differences between wild-type and cys-less CFTR channel permeation properties. Our results suggest that the conductance difference is the result of a single substitution, of C343: the point mutant C343S has a conductance similar to cys-less, whereas the reverse mutation, S343C in a cys-less background, restores wild-type conductance levels. Other cysteine substitutions (C128S, C225S, C376S, C866S) were without effect. Substitution of other residues for C343 suggested that conductance is dependent on amino acid side chain volume at this position. A range of other functional pore properties, including interactions with channel blockers (Au[CN] (2) (-) , 5-nitro-2-[3-phenylpropylamino]benzoic acid, suramin) and anion permeability, were not significantly different between wild-type and cys-less CFTR. Our results suggest that functional differences between these two CFTR constructs are of limited scale and scope and result from a small change in side chain volume at position 343. These results therefore support the use of cys-less as a model of the CFTR pore region.

  1. Permeant anions contribute to voltage dependence of ClC-2 chloride channel by interacting with the protopore gate

    PubMed Central

    Sánchez-Rodríguez, Jorge E; De Santiago-Castillo, José A; Arreola, Jorge

    2010-01-01

    It has been shown that the voltage (Vm) dependence of ClC Cl− channels is conferred by interaction of the protopore gate with H+ ions. However, in this paper we present evidence which indicates that permeant Cl− ions contribute to Vm-dependent gating of the broadly distributed ClC-2 Cl− channel. The apparent open probability (PA) of ClC-2 was enhanced either by changing the [Cl−]i from 10 to 200 mm or by keeping the [Cl−]i low (10 mm) and then raising [Cl−]o from 10 to 140 mm. Additionally, these changes in [Cl−] slowed down channel closing at positive Vm suggesting that high [Cl−] increased pore occupancy thus hindering closing of the protopore gate. The identity of the permeant anion was also important since the PA(Vm) curves were nearly identical with Cl− or Br− but shifted to negative voltages in the presence of SCN− ions. In addition, gating, closing rate and reversal potential displayed anomalous mole fraction behaviour in a SCN−/Cl− mixture in agreement with the idea that pore occupancy by different permeant anions modifies the Vm dependence ClC-2 gating. Based on the ec1-ClC anion pathway, we hypothesized that opening of the protopore gate is facilitated when Cl− ions dwell in the central binding site. In contrast, when Cl− ions dwell in the external binding site they prevent the gate from closing. Finally, this Cl−-dependent gating in ClC-2 channels is of physiological relevance since an increase in [Cl−]o enhances channel opening when the [Cl−]i is in the physiological range. PMID:20498235

  2. A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels

    PubMed Central

    Gradogna, Antonella; Babini, Elena; Picollo, Alessandra

    2010-01-01

    The two human CLC Cl− channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel β subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca2+ and H+ on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca2+]ext without full saturation up to 50 mM. However, in the absence of Ca2+, ClC-Ka currents were still 20% of currents in 10 mM [Ca2+]ext, demonstrating that Ca2+ is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H+]ext with a practically complete block at pH 6. Ca2+ and H+ act as gating modifiers without changing the single-channel conductance. Dose–response analysis suggested that two protons are necessary to induce block with an apparent pK of ∼7.1. A simple four-state allosteric model described the modulation by Ca2+ assuming a 13-fold higher Ca2+ affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca2+ and H+. A mutagenic screen of a large number of extracellularly accessible amino acids identified a pair of acidic residues (E261 and D278 on the loop connecting helices I and J), which are close to each other but positioned on different subunits of the channel, as a likely candidate for forming an intersubunit Ca2+-binding site. Single mutants E261Q and D278N greatly diminished and the double mutant E261Q/D278N completely abolished modulation by Ca2+. Several mutations of a histidine residue (H497) that is homologous to a histidine that is responsible for H+ block in ClC-2 did not yield functional channels. However, the triple mutant E261Q/D278N/H497M completely eliminated H+ -induced current block. We have thus identified a protein region that is involved in binding these physiologically important ligands and that is likely undergoing

  3. Chloride conducting light activated channel GtACR2 can produce both cessation of firing and generation of action potentials in cortical neurons in response to light.

    PubMed

    Malyshev, A Y; Roshchin, M V; Smirnova, G R; Dolgikh, D A; Balaban, P M; Ostrovsky, M A

    2017-02-15

    Optogenetics is a powerful technique in neuroscience that provided a great success in studying the brain functions during the last decade. Progress of optogenetics crucially depends on development of new molecular tools. Light-activated cation-conducting channelrhodopsin2 was widely used for excitation of cells since the emergence of optogenetics. In 2015 a family of natural light activated chloride channels GtACR was identified which appeared to be a very promising tool for using in optogenetics experiments as a cell silencer. Here we examined properties of GtACR2 channel expressed in the rat layer 2/3 pyramidal neurons by means of in utero electroporation. We have found that despite strong inhibition the light stimulation of GtACR2-positive neurons can surprisingly lead to generation of action potentials, presumably initiated in the axonal terminals. Thus, when using the GtACR2 in optogenetics experiments, its ability to induce action potentials should be taken into account. Our results also open an interesting possibility of using the GtACR2 both as cell silencer and cell activator in the same experiment varying the pattern of light stimulation.

  4. The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    Fischer, H; Machen, T E

    1996-12-01

    The role of the tyrosine kinase p60c-src on the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel was investigated with the cell-attached and excised patch clamp technique in conjunction with current noise analysis of recordings containing multiple channels per patch. Spectra of CFTR-generated current noise contained a low-frequency and a high-frequency Lorentzian noise component. In the cell-attached mode, the high-frequency Lorentzian was significantly dependent on the membrane potential, while the low-frequency Lorentzian was unaffected. Excision of forskolin-stimulated patches into ATP-containing solution significantly reduced the amplitude of the voltage-dependent high-frequency Lorentzian. Addition of the tyrosine kinase p60c-src to excised, active, CFTR-containing membrane patches increased mean currents by 54%, increased the corner frequency of the low-frequency Lorentzian, and recovered the high-frequency Lorentzian and its characteristics. Treatment with lambda-phosphatase inactivated src-induced currents and changes in gating. When active patches were excised under conditions in which patch-associated tyrosine phosphatases were blocked with sodium vanadate, the high-frequency gating remained relatively unchanged. The results suggest that CFTR's open probability and its voltage-dependent fast gate are dependent on tyrosine phosphorylation, and that membrane-associated tyrosine phosphatases are responsible for inactivation of the fast gate after patch excision.

  5. Inhibition of Calcium-Activated Chloride Channel ANO1/TMEM16A Suppresses Tumor Growth and Invasion in Human Lung Cancer

    PubMed Central

    Jia, Linghan; Liu, Wen; Guan, Lizhao; Lu, Min; Wang, KeWei

    2015-01-01

    Lung cancer or pulmonary carcinoma is primarily derived from epithelial cells that are thin and line on the alveolar surfaces of the lung for gas exchange. ANO1/TMEM16A, initially identified from airway epithelial cells, is a member of Ca2+-activated Cl- channels (CaCCs) that function to regulate epithelial secretion and cell volume for maintenance of ion and tissue homeostasis. ANO1/TMEM16A has recently been shown to be highly expressed in several epithelium originated carcinomas. However, the role of ANO1 in lung cancer remains unknown. In this study, we show that inhibition of calcium-activated chloride channel ANO1/TMEM16A suppresses tumor growth and invasion in human lung cancer. ANO1 is upregulated in different human lung cancer cell lines. Knocking-down ANO1 by small hairpin RNAs inhibited proliferation, migration and invasion of GLC82 and NCI-H520 cancel cells evaluated by CCK-8, would-healing, transwell and 3D soft agar assays. ANO1 protein is overexpressed in 77.3% cases of human lung adenocarcinoma tissues detected by immunohistochemistry. Furthermore, the tumor growth in nude mice implanted with GLC82 cells was significantly suppressed by ANO1 silencing. Taken together, our findings provide evidence that ANO1 overexpression contributes to tumor growth and invasion of lung cancer; and suppressing ANO1 overexpression may have therapeutic potential in lung cancer therapy. PMID:26305547

  6. Inhibition of Calcium-Activated Chloride Channel ANO1/TMEM16A Suppresses Tumor Growth and Invasion in Human Lung Cancer.

    PubMed

    Jia, Linghan; Liu, Wen; Guan, Lizhao; Lu, Min; Wang, KeWei

    2015-01-01

    Lung cancer or pulmonary carcinoma is primarily derived from epithelial cells that are thin and line on the alveolar surfaces of the lung for gas exchange. ANO1/TMEM16A, initially identified from airway epithelial cells, is a member of Ca2+-activated Cl- channels (CaCCs) that function to regulate epithelial secretion and cell volume for maintenance of ion and tissue homeostasis. ANO1/TMEM16A has recently been shown to be highly expressed in several epithelium originated carcinomas. However, the role of ANO1 in lung cancer remains unknown. In this study, we show that inhibition of calcium-activated chloride channel ANO1/TMEM16A suppresses tumor growth and invasion in human lung cancer. ANO1 is upregulated in different human lung cancer cell lines. Knocking-down ANO1 by small hairpin RNAs inhibited proliferation, migration and invasion of GLC82 and NCI-H520 cancel cells evaluated by CCK-8, would-healing, transwell and 3D soft agar assays. ANO1 protein is overexpressed in 77.3% cases of human lung adenocarcinoma tissues detected by immunohistochemistry. Furthermore, the tumor growth in nude mice implanted with GLC82 cells was significantly suppressed by ANO1 silencing. Taken together, our findings provide evidence that ANO1 overexpression contributes to tumor growth and invasion of lung cancer; and suppressing ANO1 overexpression may have therapeutic potential in lung cancer therapy.

  7. Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin-radixin-moesin network.

    PubMed

    Perez-Cornejo, Patricia; Gokhale, Avanti; Duran, Charity; Cui, Yuanyuan; Xiao, Qinghuan; Hartzell, H Criss; Faundez, Victor

    2012-06-26

    The newly discovered Ca(2+)-activated Cl(-) channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca(2+)-activated Cl(-) currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca(2+)-binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane.

  8. General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels.

    PubMed

    Marcet, Brice; Becq, Frédéric; Norez, Caroline; Delmas, Patrick; Verrier, Bernard

    2004-03-01

    1. Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is defective during cystic fibrosis (CF). Activators of the CFTR Cl(-) channel may be useful for therapy of CF. Here, we demonstrate that a range of general anesthetics like normal-alkanols (n-alkanols) and related compounds can stimulate the Cl(-) channel activity of wild-type CFTR and delF508-CFTR mutant. 2. The effects of n-alkanols like octanol on CFTR activity were measured by iodide ((125)I) efflux and patch-clamp techniques on three distinct cellular models: (1). CFTR-expressing Chinese hamster ovary cells, (2). human airway Calu-3 epithelial cells and (3). human airway JME/CF15 epithelial cells which express the delF508-CFTR mutant. 3. Our data show for the first time that n-alkanols activate both wild-type CFTR and delF508-CFTR mutant. Octanol stimulated (125)I efflux in a dose-dependent manner in CFTR-expressing cells (wild-type and delF508) but not in cell lines lacking CFTR. (125)I efflux and Cl(-) currents induced by octanol were blocked by glibenclamide but insensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, as expected for a CFTR Cl(-) current. 4. CFTR activation by octanol was neither due to cell-to-cell uncoupling properties of octanol nor to an intracellular cAMP increase. CFTR activation by octanol requires phosphorylation by protein kinase-A (PKA) since it was prevented by H-89, a PKA inhibitor. 5. n-Alkanols chain length was an important determinant for channel activation, with rank order of potencies: 1-heptanol<1-octanol<2-octanol<1-decanol. Our findings may be of valuable interest for developing novel therapeutic strategies for CF.

  9. Salmon lice (Lepeophtheirus salmonis) showing varying emamectin benzoate susceptibilities differ in neuronal acetylcholine receptor and GABA-gated chloride channel mRNA expression

    PubMed Central

    2013-01-01

    Background Caligid copepods, also called sea lice, are fish ectoparasites, some species of which cause significant problems in the mariculture of salmon, where the annual cost of infection is in excess of €300 million globally. At present, caligid control on farms is mainly achieved using medicinal treatments. However, the continued use of a restricted number of medicine actives potentially favours the development of drug resistance. Here, we report transcriptional changes in a laboratory strain of the caligid Lepeophtheirus salmonis (Krøyer, 1837) that is moderately (~7-fold) resistant to the avermectin compound emamectin benzoate (EMB), a component of the anti-salmon louse agent SLICE® (Merck Animal Health). Results Suppression subtractive hybridisation (SSH) was used to enrich transcripts differentially expressed between EMB-resistant (PT) and drug-susceptible (S) laboratory strains of L. salmonis. SSH libraries were subjected to 454 sequencing. Further L. salmonis transcript sequences were available as expressed sequence tags (EST) from GenBank. Contiguous sequences were generated from both SSH and EST sequences and annotated. Transcriptional responses in PT and S salmon lice were investigated using custom 15 K oligonucleotide microarrays designed using the above sequence resources. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains, these genes being enriched for functions such as calcium ion binding, chitin metabolism and muscle structure. γ-aminobutyric acid (GABA)-gated chloride channel (GABA-Cl) and neuronal acetylcholine receptor (nAChR) subunits showed significantly lower transcript levels in PT lice compared to S lice. Using RT-qPCR, the decrease in mRNA levels was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR. Salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 μg L-1 of EMB, a drug concentration tolerated by PT lice, but

  10. Identification and Functional Expression of a Glutamate- and Avermectin-Gated Chloride Channel from Caligus rogercresseyi, a Southern Hemisphere Sea Louse Affecting Farmed Fish

    PubMed Central

    Niemeyer, María Isabel; Marabolí, Vanessa; González-Nilo, F. Danilo; Teulon, Jacques; Sepúlveda, Francisco V.; Cid, L. Pablo

    2014-01-01

    Parasitic sea lice represent a major sanitary threat to marine salmonid aquaculture, an industry accounting for 7% of world fish production. Caligus rogercresseyi is the principal sea louse species infesting farmed salmon and trout in the southern hemisphere. Most effective control of Caligus has been obtained with macrocyclic lactones (MLs) ivermectin and emamectin. These drugs target glutamate-gated chloride channels (GluCl) and act as irreversible non-competitive agonists causing neuronal inhibition, paralysis and death of the parasite. Here we report the cloning of a full-length CrGluClα receptor from Caligus rogercresseyi. Expression in Xenopus oocytes and electrophysiological assays show that CrGluClα is activated by glutamate and mediates chloride currents blocked by the ligand-gated anion channel inhibitor picrotoxin. Both ivermectin and emamectin activate CrGluClα in the absence of glutamate. The effects are irreversible and occur with an EC50 value of around 200 nM, being cooperative (nH = 2) for ivermectin but not for emamectin. Using the three-dimensional structure of a GluClα from Caenorabditis elegans, the only available for any eukaryotic ligand-gated anion channel, we have constructed a homology model for CrGluClα. Docking and molecular dynamics calculations reveal the way in which ivermectin and emamectin interact with CrGluClα. Both drugs intercalate between transmembrane domains M1 and M3 of neighbouring subunits of a pentameric structure. The structure displays three H-bonds involved in this interaction, but despite similarity in structure only of two these are conserved from the C. elegans crystal binding site. Our data strongly suggest that CrGluClα is an important target for avermectins used in the treatment of sea louse infestation in farmed salmonids and open the way for ascertaining a possible mechanism of increasing resistance to MLs in aquaculture industry. Molecular modeling could help in the design of new, more efficient

  11. Inhibitory role of phosphatidylinositol 4,5-bisphosphate on TMEM16A-encoded calcium-activated chloride channels in rat pulmonary artery

    PubMed Central

    Pritchard, H A T; Leblanc, N; Albert, A P; Greenwood, I A

    2014-01-01

    Background and Purpose Calcium-activated chloride channels (CaCCs) are key depolarizing mechanisms that have an important role in vascular smooth muscle contraction. Here, we investigated whether these channels are regulated by phosphatidylinositol (4,5) bisphosphate [P(4,5)P2], a known regulator of various ion channels. Experimental Approach Calcium-activated Cl− currents (IClCa) were recorded by patch clamp electrophysiology of rat isolated pulmonary artery smooth muscle cells. TMEM16A protein-phosphoinositide interaction was studied by co-immunoprecipitation and phosphoinositide binding arrays on protein lysates from whole pulmonary arteries and HEK293 cells overexpressing TMEM16A, the molecular correlate. Key Results PI(4,5)P2 and other phospholipids were shown to bind directly to TMEM16A isolated from whole pulmonary artery (PA) and TMEM16A-eGFP expressed in HEK293 cells. Agents that reduced PI(4,5)P2 levels through different routes [PLC activation, PI4K inhibition, PI(4,5)P2 scavenging and absorption] all increased IClCa evoked by solutions containing clamped-free [Ca2+], whereas enrichment of activating solutions with PI(4,5)P2 inhibited IClca in PA smooth muscle cells with approximately 50% reduction at 1 μM. Conclusions and Implications These data are the first to show a negative regulation of TMEM16A-encoded CaCCs by PI(4,5)P2 and propose that control of PI(4,5)P2 levels is a key determinant of arterial physiology. PMID:24834965

  12. P2X4 Activation Modulates Volume-sensitive Outwardly Rectifying Chloride Channels in Rat Hepatoma Cells*

    PubMed Central

    Varela, Diego; Penna, Antonello; Simon, Felipe; Eguiguren, Ana Luisa; Leiva-Salcedo, Elías; Cerda, Oscar; Sala, Francisco; Stutzin, Andrés

    2010-01-01

    Volume-sensitive outwardly rectifying (VSOR) Cl− channels are critical for the regulatory volume decrease (RVD) response triggered upon cell swelling. Recent evidence indicates that H2O2 plays an essential role in the activation of these channels and that H2O2 per se activates the channels under isotonic isovolumic conditions. However, a significant difference in the time course for current onset between H2O2-induced and hypotonicity-mediated VSOR Cl− activation is observed. In several cell types, cell swelling induced by hypotonic challenges triggers the release of ATP to the extracellular medium, which in turn, activates purinergic receptors and modulates cell volume regulation. In this study, we have addressed the effect of purinergic receptor activation on H2O2-induced and hypotonicity-mediated VSOR Cl− current activation. Here we show that rat hepatoma cells (HTC) exposed to a 33% hypotonic solution responded by rapidly activating VSOR Cl− current and releasing ATP to the extracellular medium. In contrast, cells exposed to 200 μm H2O2 VSOR Cl− current onset was significantly slower, and ATP release was not detected. In cells exposed to either 11% hypotonicity or 200 μm H2O2, exogenous addition of ATP in the presence of extracellular Ca2+ resulted in a decrease in the half-time for VSOR Cl− current onset. Conversely, in cells that overexpress a dominant-negative mutant of the ionotropic receptor P2X4 challenged with a 33% hypotonic solution, the half-time for VSOR Cl− current onset was significantly slowed down. Our results indicate that, at high hypotonic imbalances, swelling-induced ATP release activates the purinergic receptor P2X4, which in turn modulates the time course of VSOR Cl− current onset in a extracellular Ca2+-dependent manner. PMID:20056605

  13. Uterine Contractility in the Nonpregnant Mouse: Changes During the Estrous Cycle and Effects of Chloride Channel Blockade.

    PubMed

    Dodds, Kelsi N; Staikopoulos, Vasiliki; Beckett, Elizabeth A H

    2015-06-01

    Mechanisms involved in the generation of spontaneous uterine contractions are not fully understood. Kit-expressing interstitial cells of Cajal are pacemakers of contractile rhythm in other visceral organs, and recent studies describe a role for Ca(2+)-activated Cl(-) currents as the initiating conductance in these cells. The existence and role of similar specialized pacemaker cells in the nonpregnant uterus remains undetermined. Spontaneous contractility patterns were characterized throughout the estrous cycle in isolated, nonpregnant mouse uteri using spatiotemporal mapping and tension recordings. During proestrus, estrus, and diestrus, contraction origin predominated in the oviduct end of the uterus, suggesting the existence of a dominant pacemaker site. Propagation speed of contractions during estrus and diestrus were significantly slower than in proestrus and metestrus. Five major patterns of activity were predominantly exhibited in particular stages: quiescent (diestrus), high-frequency phasic (proestrus), low-frequency phasic (estrus), multivariant (metestrus), and complex. Kit-immunopositive cells reminiscent of pacemaking ICCs were not consistently observed within the uterus. Niflumic acid (10 μM), anthracene-9-carboxylic acid (0.1-1 mM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (10 μM) each reduced the frequency of spontaneous contractions, suggesting involvement of Cl(-) channels in generating spontaneous uterine motor activity. It is unlikely that this conductance is generated by the Ca(2+)-activated Cl(-) channels, anoctamin-1 and CLCA4, as immunohistochemical labeling did not reveal protein expression within muscle or pacemaker cell networks. In summary, these results suggest that spontaneous uterine contractions may be generated by a Kit-negative pacemaker cell type or uterine myocytes, likely involving the activity of a yet-unidentified Cl(-) channel.

  14. Independent activation of ion conduction pores in the double-barreled calcium-activated chloride channel TMEM16A.

    PubMed

    Lim, Novandy K; Lam, Andy K M; Dutzler, Raimund

    2016-11-01

    The TMEM16 proteins constitute a family of membrane proteins with unusual functional breadth, including lipid scramblases and Cl(-) channels. Members of both these branches are activated by Ca(2+), acting from the intracellular side, and probably share a common architecture, which was defined in the recent structure of the lipid scramblase nhTMEM16. The structural features of subunits and the arrangement of Ca(2+)-binding sites in nhTMEM16 suggest that the dimeric protein harbors two locations for catalysis that are independent with respect to both activation and lipid conduction. Here, we ask whether a similar independence is observed in the Ca(2+)-activated Cl(-) channel TMEM16A. For this purpose, we generated concatenated constructs containing subunits with distinct activation and permeation properties. Our biochemical investigations demonstrate the integrity of concatemers after solubilization and purification. During investigation by patch-clamp electrophysiology, the functional behavior of constructs containing either two wild-type (WT) subunits or one WT subunit paired with a second subunit with compromised activation closely resembles TMEM16A. This resemblance extends to ion selectivity, conductance, and the concentration and voltage dependence of channel activation by Ca(2+) Constructs combining subunits with different potencies for Ca(2+) show a biphasic activation curve that can be described as a linear combination of the properties of its constituents. The functional independence is further supported by mutation of a putative pore-lining residue that changes the conduction properties of the mutated subunit. Our results strongly suggest that TMEM16A contains two ion conduction pores that are independently activated by Ca(2+) binding to sites that are embedded within the transmembrane part of each subunit.

  15. Ethanol potentiation of GABAergic transmission in cultured spinal cord neurons involves gamma-aminobutyric acidA-gated chloride channels

    SciTech Connect

    Mehta, A.K.; Ticku, M.K.

    1988-08-01

    The interaction of ethanol with gamma-aminobutyric acid (GABA)-mediated 36-Cl-influx and its modulation by various drugs was investigated in C57 mice spinal cord cultured neurons. Ethanol (5-100 mM) potentiated the effect of GABA on /sup 36/Cl-influx; whereas at concentrations greater than or equal to 50 mM ethanol activated Cl- channels directly. The effect of ethanol was specific for GABAA receptor-gated Cl- channels, as ethanol did not potentiate glycine-induced /sup 36/Cl-influx in the same neurons. Both the enhancing and direct effects of ethanol on /sup 36/Cl-influx were blocked by GABA antagonists like bicuculline, picrotoxinin and inverse agonists of the benzodiazepine site like the imidazodiazepine R015-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo (1,5 alpha), (1,4)benzodiazepine-3-carboxylate) and N-methyl-beta-carboline-3-carboxamide (FG-7142). Ethanol potentiating effect of GABA-induced /sup 36/Cl-influx was also reversed by methyl-6,7-dimethyl-4-ethyl-beta-carboline-3-carboxylate. The effects of the inverse agonists were blocked by the benzodiazepine receptor antagonist R015-1788. Both R015-4513 and FG-7142 reversed direct and GABA potentiating effects of ethanol effect at concentrations lower than those that exhibit inverse agonistic activity in the /sup 36/Cl-influx assay in cultured neurons. These results suggest that ethanol facilitation of GABAAergic transmission involves GABA receptor-gated Cl- channels and that this interaction may be responsible for some of the pharmacological effects of ethanol.

  16. A pure chloride channel mutant of CLC-5 causes Dent's disease via insufficient V-ATPase activation.

    PubMed

    Satoh, Nobuhiko; Yamada, Hideomi; Yamazaki, Osamu; Suzuki, Masashi; Nakamura, Motonobu; Suzuki, Atsushi; Ashida, Akira; Yamamoto, Daisuke; Kaku, Yoshitsugu; Sekine, Takashi; Seki, George; Horita, Shoko

    2016-07-01

    Dent's disease is characterized by defective endocytosis in renal proximal tubules (PTs) and caused by mutations in the 2Cl(-)/H(+) exchanger, CLC-5. However, the pathological role of endosomal acidification in endocytosis has recently come into question. To clarify the mechanism of pathogenesis for Dent's disease, we examined the effects of a novel gating glutamate mutation, E211Q, on CLC-5 functions and endosomal acidification. In Xenopus oocytes, wild-type (WT) CLC-5 showed outward-rectifying currents that were inhibited by extracellular acidosis, but E211Q and an artificial pure Cl(-) channel mutant, E211A, showed linear currents that were insensitive to extracellular acidosis. Moreover, depolarizing pulse trains induced a robust reduction in the surface pH of oocytes expressing WT CLC-5 but not E211Q or E211A, indicating that the E211Q mutant functions as a pure Cl(-) channel similar to E211A. In HEK293 cells, E211A and E211Q stimulated endosomal acidification and hypotonicity-inducible vacuolar-type H(+)-ATPase (V-ATPase) activation at the plasma membrane. However, the stimulatory effects of these mutants were reduced compared with WT CLC-5. Furthermore, gene silencing experiments confirmed the functional coupling between V-ATPase and CLC-5 at the plasma membrane of isolated mouse PTs. These results reveal for the first time that the conversion of CLC-5 from a 2Cl(-)/H(+) exchanger into a Cl(-) channel induces Dent's disease in humans. In addition, defective endosomal acidification as a result of insufficient V-ATPase activation may still be important in the pathogenesis of Dent's disease.

  17. GTP-Binding Proteins Inhibit cAMP Activation of Chloride Channels in Cystic Fibrosis Airway Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Schwiebert, Erik M.; Kizer, Neil; Gruenert, Dieter C.; Stanton, Bruce A.

    1992-11-01

    Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl^- secretion by airway epithelial cells. In CF, cAMP does not activate Cl^- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[β-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl^- currents and restore cAMP-activated Cl^- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[γ-thio]triphosphate and AlF^-_4 reduce Cl^- currents and inhibit cAMP from activating Cl^- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[β-thio]diphosphate and in normal cells, cAMP activates a Cl^- conductance that has properties similar to CF transmembrane-conductance regulator Cl^- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl^- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl^- secretion in CF.

  18. Mechanism of Block of Single Protopores of the Torpedo Chloride Channel Clc-0 by 2-(p-Chlorophenoxybutyric) Acid (Cpb)

    PubMed Central

    Pusch, Michael; Accardi, Alessio; Liantonio, Antonella; Ferrera, Loretta; De Luca, Annamaria; Camerino, Diana Conte; Conti, Franco

    2001-01-01

    We investigated in detail the mechanism of inhibition by the S(−) enantiomer of 2-(p-chlorophenoxy)butyric acid (CPB) of the Torpedo Cl− channel, ClC-0. The substance has been previously shown to inhibit the homologous skeletal muscle channel, CLC-1. ClC-0 is a homodimer with probably two independently gated protopores that are conductive only if an additional common gate is open. As a simplification, we used a mutant of ClC-0 (C212S) that has the common gate “locked open” (Lin, Y.W., C.W. Lin, and T.Y. Chen. 1999. J. Gen. Physiol. 114:1–12). CPB inhibits C212S currents only when applied to the cytoplasmic side, and single-channel recordings at voltages (V) between −120 and −80 mV demonstrate that it acts independently on individual protopores by introducing a long-lived nonconductive state with no effect on the conductance and little effect on the lifetime of the open state. Steady-state macroscopic currents at −140 mV are half-inhibited by ∼0.5 mM CPB, but the inhibition decreases with V and vanishes for V ≥ 40 mV. Relaxations of CPB inhibition after voltage steps are seen in the current responses as an additional exponential component that is much slower than the gating of drug-free protopores. For V ≤ −40 mV, where a significant inhibition is observable for the CPB concentrations used in this study (≤10 mM), the concentration dependence of its onset kinetics is consistent with CPB binding according to a bimolecular reaction. At all voltages, only the openings of drug-free protopores appear to contribute significantly to the current observed at any time. Lowering internal Cl− hastens significantly the apparent “on” rate, suggesting that internal Cl− antagonizes CPB binding to closed pores. Vice versa, lowering external Cl− reduces the apparent rate of CPB dissociation from open pores. We studied also the point mutant K519E (in the context of the C212S mutant) that has altered conduction properties and slower single protopore

  19. The Ca2+-activated chloride channel anoctamin-2 mediates spike-frequency adaptation and regulates sensory transmission in thalamocortical neurons

    PubMed Central

    Ha, Go Eun; Lee, Jaekwang; Kwak, Hankyul; Song, Kiyeong; Kwon, Jea; Jung, Soon-Young; Hong, Joohyeon; Chang, Gyeong-Eon; Hwang, Eun Mi; Shin, Hee-Sup; Lee, C. Justin; Cheong, Eunji

    2016-01-01

    Neuronal firing patterns, which are crucial for determining the nature of encoded information, have been widely studied; however, the molecular identity and cellular mechanisms of spike-frequency adaptation are still not fully understood. Here we show that spike-frequency adaptation in thalamocortical (TC) neurons is mediated by the Ca2+-activated Cl− channel (CACC) anoctamin-2 (ANO2). Knockdown of ANO2 in TC neurons results in significantly reduced spike-frequency adaptation along with increased tonic spiking. Moreover, thalamus-specific knockdown of ANO2 increases visceral pain responses. These results indicate that ANO2 contributes to reductions in spike generation in highly activated TC neurons and thereby restricts persistent information transmission. PMID:27991499

  20. The outwardly rectifying chloride channel in rat peritoneal mast cells is regulated by serine/threonine kinases and phosphatases.

    PubMed

    Seebeck, Jörg; Tritschler, Stefan; Roloff, Tim; Kruse, Marie-Luise; Schmidt, Wolfgang E; Ziegler, Albrecht

    2002-02-01

    A slowly activating, outwardly rectifying Cl- channel (ORCC) has been described in rat peritoneal mast cells (RPMCs). This channel is activated by intracellular application of cAMP, an effect that might be mediated by a PKA-type serine/threonine protein kinase. To test this hypothesis, whole-cell patch-clamp experiments (nystatin-perforated patch) were performed and 8-bromoadenosine 3',5'-cyclic monophosphothioate, Sp-enantiomer (Sp-8Br-cAMPS), a cell membrane-permeable activator of PKA, and three inhibitors of different serine/threonine protein phosphatases (okadaic acid, cantharidin, calyculin A), were tested. In RPMCs application of repetitive series of step hyper- and depolarizations (holding potential 0 mV, test potentials -80 to +80 mV, step size +20 mV) induced a slowly increasing, [half-maximal activation time ( t0.5) 11.0+/-1.1 min, Imax (at +80 mV) 18.7+/-3.1 pA pF-1], DIDS-sensitive, outwardly rectifying Cl- current I(Cl,OR). The activation of this current could be accelerated by Sp-8Br-cAMPS, okadaic acid or cantharidin in the extracellular solution. Co-application of Sp-8Br-cAMPS and okadaic acid increased Imax supra-additively. Calyculin A and higher concentrations of cantharidin inhibited the Cl- current via unknown mechanisms. Our findings suggest that I(Cl,OR) in RPMCs is activated by a PKA-type protein kinase, a process which is antagonized functionally by okadaic acid- and cantharidin-sensitive protein phosphatases.

  1. Multiple effects of anthracene-9-carboxylic acid on the TMEM16B/anoctamin2 calcium-activated chloride channel.

    PubMed

    Cherian, O Lijo; Menini, Anna; Boccaccio, Anna

    2015-04-01

    Ca(2+)-activated Cl(-) currents (CaCCs) play important roles in many physiological processes. Recent studies have shown that TMEM16A/anoctamin1 and TMEM16B/anoctamin2 constitute CaCCs in several cell types. Here we have investigated for the first time the extracellular effects of the Cl(-) channel blocker anthracene-9-carboxylic acid (A9C) and of its non-charged analogue anthracene-9-methanol (A9M) on TMEM16B expressed in HEK 293T cells, using the whole-cell patch-clamp technique. A9C caused a voltage-dependent block of outward currents and inhibited a larger fraction of the current as depolarization increased, whereas the non-charged A9M produced a small, not voltage dependent block of outward currents. A similar voltage-dependent block by A9C was measured both when TMEM16B was activated by 1.5 and 13μM Ca(2+). However, in the presence of 1.5μM Ca(2+) (but not in 13μM Ca(2+)), A9C also induced a strong potentiation of tail currents measured at -100mV after depolarizing voltages, as well as a prolongation of the deactivation kinetics. On the contrary, A9M did not produce potentiation of tail currents, showing that the negative charge is required for potentiation. Our results provide the first evidence that A9C has multiple effects on TMEM16B and that the negative charge of A9C is necessary both for voltage-dependent block and for potentiation. Future studies are required to identify the molecular mechanisms underlying these complex effects of A9C on TMEM16B. Understanding these mechanisms will contribute to the elucidation of the structure and functional properties of TMEM16B channels.

  2. Dehydrocostuslactone, a sesquiterpene lactone activates wild-type and ΔF508 mutant CFTR chloride channel.

    PubMed

    Wang, Xue; Zhang, Yao-Fang; Yu, Bo; Yang, Shuang; Luan, Jian; Liu, Xin; Yang, Hong

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) represents the main cAMP-activated Cl⁻ channel expressed in the apical membrane of serous epithelial cells. Both deficiency and overactivation of CFTR may cause fluid and salt secretion related diseases. The aim of this study was to identify natural compounds that are able to stimulate wild-type (wt) and ΔF508 mutant CFTR channel activities in CFTR-expressing Fischer rat thyroid (FRT) cells. We found that dehydrocostuslactone [DHC, (3aS, 6aR, 9aR, 9bS)-decahydro-3,6,9-tris (methylene) azuleno [4,5-b] furan-2(3H)-one)] dose dependently potentiates both wt and ΔF508 mutant CFTR-mediated iodide influx in cell-based fluorescent assays and CFTR-mediated Cl⁻ currents in short-circuit current studies, and the activations could be reversed by the CFTR inhibitor CFTRinh-172. Maximal CFTR-mediated apical Cl⁻ current secretion in CFTR-expressing FRT cells was stimulated by 100 μM DHC. Determination of intracellular cAMP content showed that DHC modestly but significantly increased cAMP level in FRT cells, but cAMP elevation effects contributed little to DHC-stimulated iodide influx. DHC also stimulated CFTR-mediated apical Cl⁻ current secretion in FRT cells expressing ΔF508-CFTR. Subsequent studies demonstrated that activation of CFTR by DHC is forskolin dependent. DHC represents a new class of CFTR potentiators that may have therapeutic potential in CFTR-related diseases.

  3. Chloride channel ClC- 2 enhances intestinal epithelial tight junction barrier function via regulation of caveolin-1 and caveolar trafficking of occludin.

    PubMed

    Nighot, Prashant K; Leung, Lana; Ma, Thomas Y

    2017-03-01

    Previous studies have demonstrated that the chloride channel ClC-2 plays a critical role in intestinal epithelial tight junction (TJ) barrier function via intracellular trafficking of TJ protein occludin. To study the mechanism of ClC-2-mediated TJ barrier function and intracellular trafficking of occludin, we established ClC-2 over-expressing Caco-2 cell line (Caco-2(CLCN2)) by full length ClC-2 ORF transfection. ClC-2 over-expression (Caco-2(CLCN2)) significantly enhanced TJ barrier (increased TER by ≥2 times and reduced inulin flux by 50%) compared to control Caco-2(pEZ) cells. ClC-2 over-expression (Caco-2(CLCN2)) increased occludin protein level compared to control Caco-2(pEZ) cells. Surface biotinylation assay revealed reduced steady state endocytosis of occludin in Caco-2(CLCN2) cells. Furthermore, ClC-2 over-expression led to reduction in caveolin-1 protein level and diminishment of caveolae assembly. Caveolae disruption increased TJ permeability in control but not ClC-2 over-expressing Caco-2(CLCN2) cells. Selective ClC-2 channel blocker GaTx2 caused an increase in caveolin-1 protein level and reduced occludin level. Delivery of cell permeable caveolin-1 scaffolding domain reduced the occludin protein level. Over all, these results suggest that ClC- 2 enhances TJ barrier function in intestinal epithelial cells via regulation of caveolin-1 and caveolae-mediated trafficking of occludin.

  4. Regulatory R region of the CFTR chloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions.

    PubMed

    Bozoky, Zoltan; Krzeminski, Mickael; Muhandiram, Ranjith; Birtley, James R; Al-Zahrani, Ateeq; Thomas, Philip J; Frizzell, Raymond A; Ford, Robert C; Forman-Kay, Julie D

    2013-11-19

    Intrinsically disordered proteins play crucial roles in regulatory processes and often function as protein interaction hubs. Here, we present a detailed characterization of a full-length disordered hub protein region involved in multiple dynamic complexes. We performed NMR, CD, and fluorescence binding studies on the nonphosphorylated and highly PKA-phosphorylated human cystic fibrosis transmembrane conductance regulator (CFTR) regulatory region, a ∼200-residue disordered segment involved in phosphorylation-dependent regulation of channel trafficking and gating. Our data provide evidence for dynamic, phosphorylation-dependent, multisite interactions of various segments of the regulatory region for its intra- and intermolecular partners, including the CFTR nucleotide binding domains 1 and 2, a 42-residue peptide from the C terminus of CFTR, the SLC26A3 sulphate transporter and antisigma factor antagonist (STAS) domain, and 14-3-3β. Because of its large number of binding partners, multivalent binding of individually weak sites facilitates rapid exchange between free and bound states to allow the regulatory region to engage with different partners and generate a graded or rheostat-like response to phosphorylation. Our results enrich the understanding of how disordered binding segments interact with multiple targets. We present structural models consistent with our data that illustrate this dynamic aspect of phospho-regulation of CFTR by the disordered regulatory region.

  5. Up-Regulation of Interleukin-9 and the Interleukin-9-Associated Calcium-Activated Chloride Channel hCLCA1 in Nasal Mucosa Following In Vivo Allergen Challenge

    PubMed Central

    2007-01-01

    Interleukin (IL)-9 is a pleiotropic T helper 2-type cytokine that has been shown to be up-regulated in allergic airway disease, including asthma. IL-9 has been demonstrated to be a potent stimulus for the production and secretion of mucus from airway epithelial cells via induction of a calcium-activated chloride channel, hCLCA1. The objective of this study was to investigate the expression of IL-9 and hCLCA1 following allergen challenge in the nasal mucosa of allergic rhinitis patients. Nasal biopsies were obtained from allergic rhinitis patients out of allergen season both before (baseline) and after local antigen challenge with either ragweed or diluent (control). Immunohistochemistry and in situ hybridization were used to assess IL-9 protein and hCLCA1 messenger ribonucleic acid. Eosinophils and T cells were detected using immunohistochemistry. IL-9 and hCLCA1 were very low at baseline, and expression was significantly up-regulated following ragweed challenge. Whereas the number of eosinophils increased after allergen challenge, T-cell counts did not change significantly. The results of this study demonstrate the relationship between specific allergen challenge and expression of both IL-9 and hCLCA1, suggesting a possible mechanism for the increased production of mucus from airway epithelial cells in allergic rhinitis. PMID:20525149

  6. Differences in the biophysical properties of the benzodiazepine/gamma-aminobutyric acid receptor chloride channel complex in the long-sleep and short-sleep mouse lines.

    PubMed

    McIntyre, T D; Trullas, R; Skolnick, P

    1988-08-01

    Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.

  7. Synthesis and evaluation of 5,6-disubstituted thiopyrimidine aryl aminothiazoles as inhibitors of the calcium-activated chloride channel TMEM16A/Ano1.

    PubMed

    Piechowicz, Katarzyna A; Truong, Eric C; Javed, Kashif M; Chaney, Rachelle R; Wu, Johnny Y; Phuan, Puay W; Verkman, Alan S; Anderson, Marc O

    2016-12-01

    Transmembrane protein 16A (TMEM16A), also called Ano1, is a Ca(2+) activated Cl(-) channel expressed widely in mammalian epithelia, as well as in vascular smooth muscle and some tumors and electrically excitable cells. TMEM16A inhibitors have potential utility for treatment of disorders of epithelial fluid and mucus secretion, hypertension, some cancers and other diseases. 4-Aryl-2-amino thiazole T16Ainh-01 was previously identified by high-throughput screening. Here, a library of 47 compounds were prepared that explored the 5,6-disubstituted pyrimidine scaffold found in T16Ainh-01. TMEM16A inhibition activity was measured using fluorescence plate reader and short-circuit current assays. We found that very little structural variation of T16Ainh-01 was tolerated, with most compounds showing no activity at 10 μM. The most potent compound in the series, 9bo, which substitutes 4-methoxyphenyl in T16Ainh-01 with 2-thiophene, had IC50 ∼1 μM for inhibition of TMEM16A chloride conductance.

  8. Mapping of long-range INS promoter interactions reveals a role for calcium-activated chloride channel ANO1 in insulin secretion.

    PubMed

    Xu, Zhixiong; Lefevre, Gaelle M; Gavrilova, Oksana; Foster St Claire, Mark B; Riddick, Gregory; Felsenfeld, Gary

    2014-11-25

    We used circular chromatin conformation capture (4C) to identify a physical contact in human pancreatic islets between the region near the insulin (INS) promoter and the ANO1 gene, lying 68 Mb away on human chromosome 11, which encodes a Ca(2+)-dependent chloride ion channel. In response to glucose, this contact was strengthened and ANO1 expression increased, whereas inhibition of INS gene transcription by INS promoter targeting siRNA decreased ANO1 expression, revealing a regulatory effect of INS promoter on ANO1 expression. Knockdown of ANO1 expression caused decreased insulin secretion in human islets, establishing a physical proximity-dependent feedback loop involving INS transcription, ANO1 expression, and insulin secretion. To explore a possible role of ANO1 in insulin metabolism, we carried out experiments in Ano1(+/-) mice. We observed reduced serum insulin levels and insulin-to-glucose ratios in high-fat diet-fed Ano1(+/-) mice relative to Ano1(+/+) mice fed the same diet. Our results show that determination of long-range contacts within the nucleus can be used to detect novel and physiologically relevant mechanisms. They also show that networks of long-range physical contacts are important to the regulation of insulin metabolism.

  9. Synthesis and evaluation of 5,6-disubstituted thiopyrimidine aryl aminothiazoles as inhibitors of the calcium-activated chloride channel TMEM16A/Ano1

    PubMed Central

    Piechowicz, Katarzyna A.; Truong, Eric C.; Javed, Kashif M.; Chaney, Rachelle R.; Wu, Johnny Y.; Phuan, Puay W.; Verkman, Alan S.; Anderson, Marc O.

    2016-01-01

    Transmembrane protein 16A (TMEM16A), also called Ano1, is a Ca2+ activated Cl− channel expressed widely in mammalian epithelia, as well as in vascular smooth muscle and some tumors and electrically excitable cells. TMEM16A inhibitors have potential utility for treatment of disorders of epithelial fluid and mucus secretion, hypertension, some cancers and other diseases. 4-Aryl-2-amino thiazole T16Ainh-01 was previously identified by high-throughput screening. Here, a library of 47 compounds were prepared that explored the 5,6-disubstituted pyrimidine scaffold found in T16Ainh-01. TMEM16A inhibition activity was measured using fluorescence plate reader and short-circuit current assays. We found that very little structural variation of T16Ainh-01 was tolerated, with most compounds showing no activity at 10 µM. The most potent compound in the series, 9bo, which substitutes 4-methoxyphenyl in T16Ainh-01 with 2-thiophene, had IC50 ~1 µM for inhibition of TMEM16A chloride conductance. PMID:26796863

  10. Mapping of long-range INS promoter interactions reveals a role for calcium-activated chloride channel ANO1 in insulin secretion

    PubMed Central

    Xu, Zhixiong; Lefevre, Gaelle M.; Gavrilova, Oksana; Foster St. Claire, Mark B.; Riddick, Gregory; Felsenfeld, Gary

    2014-01-01

    We used circular chromatin conformation capture (4C) to identify a physical contact in human pancreatic islets between the region near the insulin (INS) promoter and the ANO1 gene, lying 68 Mb away on human chromosome 11, which encodes a Ca2+-dependent chloride ion channel. In response to glucose, this contact was strengthened and ANO1 expression increased, whereas inhibition of INS gene transcription by INS promoter targeting siRNA decreased ANO1 expression, revealing a regulatory effect of INS promoter on ANO1 expression. Knockdown of ANO1 expression caused decreased insulin secretion in human islets, establishing a physical proximity-dependent feedback loop involving INS transcription, ANO1 expression, and insulin secretion. To explore a possible role of ANO1 in insulin metabolism, we carried out experiments in Ano1+/− mice. We observed reduced serum insulin levels and insulin-to-glucose ratios in high-fat diet–fed Ano1+/− mice relative to Ano1+/+ mice fed the same diet. Our results show that determination of long-range contacts within the nucleus can be used to detect novel and physiologically relevant mechanisms. They also show that networks of long-range physical contacts are important to the regulation of insulin metabolism. PMID:25385647

  11. The Porcine Chloride Channel Calcium-Activated Family Member pCLCA4a Mirrors Lung Expression of the Human hCLCA4

    PubMed Central

    Plog, Stephanie; Grötzsch, Tanja; Klymiuk, Nikolai; Kobalz, Ursula; Gruber, Achim D.

    2012-01-01

    Pig models of cystic fibrosis (CF) have recently been established that are expected to mimic the human disease closer than mouse models do. The human CLCA (originally named chloride channels, calcium-activated) member hCLCA4 is considered a potential modifier of disease severity in CF, but its murine ortholog, mCLCA6, is not expressed in the mouse lung. Here, we have characterized the genomic structure, protein processing, and tissue expression patterns of the porcine ortholog to hCLCA4, pCLCA4a. The genomic structure and cellular protein processing of pCLCA4a were found to closely mirror those of hCLCA4 and mCLCA6. Similar to human lung, pCLCA4a mRNA was strongly expressed in porcine lungs, and the pCLCA4a protein was immunohistochemically detected on the apical membranes of tracheal and bronchial epithelial cells. This stands in sharp contrast to mouse mCLCA6, which has been detected exclusively in intestinal epithelia but not the murine lung. The results may add to the understanding of species-specific differences in the CF phenotype and support the notion that the CF pig model may be more suitable than murine models to study the role of hCLCA4. PMID:22205680

  12. Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel.

    PubMed

    Betto, Giulia; Cherian, O Lijo; Pifferi, Simone; Cenedese, Valentina; Boccaccio, Anna; Menini, Anna

    2014-06-01

    At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca(2+)-activated Cl(-) channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio sequence determined by substituting Cl(-) with other anions (PX/PCl) was SCN(-) > I(-) > NO3 (-) > Br(-) > Cl(-) > F(-) > gluconate. When external Cl(-) was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 µM Ca(2+) were modified according to the sequence of permeability ratios, with anions more permeant than Cl(-) slowing both activation and deactivation and anions less permeant than Cl(-) accelerating them. Moreover, replacement of external Cl(-) with gluconate, or sucrose, shifted the voltage dependence of steady-state activation (G-V relation) to more positive potentials, whereas substitution of extracellular or intracellular Cl(-) with SCN(-) shifted G-V to more negative potentials. Dose-response relationships for Ca(2+) in the presence of different extracellular anions indicated that the apparent affinity for Ca(2+) at +100 mV increased with increasing permeability ratio. The apparent affinity for Ca(2+) in the presence of intracellular SCN(-) also increased compared with that in Cl(-). Our results provide the first evidence that TMEM16B gating is modulated by permeant anions and provide the basis for future studies aimed at identifying the molecular determinants of TMEM16B ion selectivity and gating.

  13. Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel

    PubMed Central

    Betto, Giulia; Cherian, O. Lijo; Pifferi, Simone; Cenedese, Valentina; Menini, Anna

    2014-01-01

    At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca2+-activated Cl− channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio sequence determined by substituting Cl− with other anions (PX/PCl) was SCN− > I− > NO3− > Br− > Cl− > F− > gluconate. When external Cl− was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 µM Ca2+ were modified according to the sequence of permeability ratios, with anions more permeant than Cl− slowing both activation and deactivation and anions less permeant than Cl− accelerating them. Moreover, replacement of external Cl− with gluconate, or sucrose, shifted the voltage dependence of steady-state activation (G-V relation) to more positive potentials, whereas substitution of extracellular or intracellular Cl− with SCN− shifted G-V to more negative potentials. Dose–response relationships for Ca2+ in the presence of different extracellular anions indicated that the apparent affinity for Ca2+ at +100 mV increased with increasing permeability ratio. The apparent affinity for Ca2+ in the presence of intracellular SCN− also increased compared with that in Cl−. Our results provide the first evidence that TMEM16B gating is modulated by permeant anions and provide the basis for future studies aimed at identifying the molecular determinants of TMEM16B ion selectivity and gating. PMID:24863931

  14. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    SciTech Connect

    Taguchi, J.; Kuriyama, K. )

    1990-05-01

    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

  15. Molecular determinants of agonist selectivity in glutamate-gated chloride channels which likely explain the agonist selectivity of the vertebrate glycine and GABAA-ρ receptors.

    PubMed

    Blarre, Thomas; Bertrand, Hugues-Olivier; Acher, Francine C; Kehoe, JacSue

    2014-01-01

    Orthologous Cys-loop glutamate-gated chloride channels (GluClR's) have been cloned and described electrophysiologically and pharmacologically in arthropods and nematodes (both members of the invertebrate ecdysozoan superphylum). Recently, GluClR's from Aplysia californica (a mollusc from the lophotrochozoan superphylum) have been cloned and similarly studied. In spite of sharing a common function, the ecdysozoan and lophotrochozoan receptors have been shown by phylogenetic analyses to have evolved independently. The recent crystallization of the GluClR from C. elegans revealed the binding pocket of the nematode receptor. An alignment of the protein sequences of the nematode and molluscan GluClRs showed that the Aplysia receptor does not contain all of the residues defining the binding mode of the ecdysozoan receptor. That the two receptors have slightly different binding modes is not surprising since earlier electrophysiological and pharmacological experiments had suggested that they were differentially responsive to certain agonists. Knowledge of the structure of the C. elegans GluClR has permitted us to generate a homology model of the binding pocket of the Aplysia receptor. We have analyzed the differences between the two binding modes and evaluated the relative significance of their non-common residues. We have compared the GluClRs electrophysiologically and pharmacologically and we have used site-directed mutagenesis on both receptor types to test predictions made from the model. Finally, we propose an explanation derived from the model for why the nematode receptors are gated only by glutamate, whereas the molluscan receptors can also be activated by β-alanine, GABA and taurine. Like the Aplysia receptor, the vertebrate glycine and GABAA-ρ receptors also respond to these other agonists. An alignment of the sequences of the molluscan and vertebrate receptors shows that the reasons we have given for the ability of the other agonists to activate the Aplysia

  16. Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1)*

    PubMed Central

    Musrap, Natasha; Tuccitto, Alessandra; Karagiannis, George S.; Saraon, Punit; Batruch, Ihor; Diamandis, Eleftherios P.

    2015-01-01

    Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving patient outcome, and may result in the identification of novel therapeutic targets. Thus, we attempted to gain insight into the proteomic changes that occur during anchorage-independent cancer cell aggregation. As such, an ovarian cancer cell line, OV-90, was cultured in adherent and non-adherent conditions using stable isotope labeling with amino acids in cell culture (SILAC). Anchorage-dependent cells (OV-90AD) were grown in tissue culture flasks, whereas anchorage-independent cells (OV-90AI) were grown in suspension using the hanging-drop method. Cellular proteins from both conditions were then identified using LC-MS/MS, which resulted in the quantification of 1533 proteins. Of these, 13 and 6 proteins were up-regulated and down-regulated, respectively, in aggregate-forming cells compared with cells grown as monolayers. Relative gene expression and protein expression of candidates were examined in other cell line models of aggregate formation (TOV-112D and ES-2), which revealed an increased expression of calcium-activated chloride channel regulator 1 (CLCA1). Moreover, inhibitor and siRNA transfection studies demonstrated an apparent effect of CLCA1 on cancer cell aggregation. Further elucidation of the role of CLCA1 in the pathogenesis of ovarian cancer is warranted. PMID:26004777

  17. Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1).

    PubMed

    Musrap, Natasha; Tuccitto, Alessandra; Karagiannis, George S; Saraon, Punit; Batruch, Ihor; Diamandis, Eleftherios P

    2015-07-10

    Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving patient outcome, and may result in the identification of novel therapeutic targets. Thus, we attempted to gain insight into the proteomic changes that occur during anchorage-independent cancer cell aggregation. As such, an ovarian cancer cell line, OV-90, was cultured in adherent and non-adherent conditions using stable isotope labeling with amino acids in cell culture (SILAC). Anchorage-dependent cells (OV-90AD) were grown in tissue culture flasks, whereas anchorage-independent cells (OV-90AI) were grown in suspension using the hanging-drop method. Cellular proteins from both conditions were then identified using LC-MS/MS, which resulted in the quantification of 1533 proteins. Of these, 13 and 6 proteins were up-regulated and down-regulated, respectively, in aggregate-forming cells compared with cells grown as monolayers. Relative gene expression and protein expression of candidates were examined in other cell line models of aggregate formation (TOV-112D and ES-2), which revealed an increased expression of calcium-activated chloride channel regulator 1 (CLCA1). Moreover, inhibitor and siRNA transfection studies demonstrated an apparent effect of CLCA1 on cancer cell aggregation. Further elucidation of the role of CLCA1 in the pathogenesis of ovarian cancer is warranted.

  18. Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules.

    PubMed

    Christensen, Erik I; Devuyst, Olivier; Dom, Geneviève; Nielsen, Rikke; Van der Smissen, Patrick; Verroust, Pierre; Leruth, Michèle; Guggino, William B; Courtoy, Pierre J

    2003-07-08

    Loss of the renal endosome-associated chloride channel, ClC-5, in Dent's disease and knockout (KO) mice strongly inhibits endocytosis of filtered proteins by kidney proximal tubular cells (PTC). The underlying mechanism remains unknown. We therefore tested whether this endocytic failure could primarily reflect a loss of reabsorption by the multiligand receptors, megalin, and cubilin, caused by a trafficking defect. Impaired protein endocytosis in PTC of ClC-5 KO mice was demonstrated by (i) a major decreased uptake of injected 125I-beta 2-microglobulin, but not of the fluid-phase tracer, FITC-dextran, (ii) reduced labeling of endosomes by injected peroxidase and for the endogenous megalin/cubilin ligands, vitamin D- and retinol-binding proteins, and (iii) urinary appearance of low-molecular-weight proteins and the selective cubilin ligand, transferrin. Contrasting with preserved mRNA levels, megalin and cubilin abundance was significantly decreased in kidney extracts of KO mice. Percoll gradients resolving early and late endosomes (Rab5a, Rab7), brush border (villin, aminopeptidase M), and a dense peak comprising lysosomes (acid hydrolases) showed a disappearance of the brush border component for megalin and cubilin in KO mice. Quantitative ultrastructural immunogold labeling confirmed the overall decrease of megalin and cubilin in PTC and their selective loss at the brush border. In contrast, total contents of the rate-limiting endocytic catalysts, Rab5a and Rab7, were unaffected. Thus, impaired protein endocytosis caused by invalidation of ClC-5 primarily reflects a trafficking defect of megalin and cubilin in PTC.

  19. Cystic fibrosis transmembrane regulator inhibitors CFTR(inh)-172 and GlyH-101 target mitochondrial functions, independently of chloride channel inhibition.

    PubMed

    Kelly, Mairead; Trudel, Stephanie; Brouillard, Franck; Bouillaud, Frederick; Colas, Julien; Nguyen-Khoa, Thao; Ollero, Mario; Edelman, Aleksander; Fritsch, Janine

    2010-04-01

    Two highly potent and selective cystic fibrosis (CF) transmembrane regulator (CFTR) inhibitors have been identified by high-throughput screening: the thiazolidinone CFTR(inh)-172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]- 2-thioxo-4-thiazolidinone] and the glycine hydrazide GlyH-101 [N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide]. Inhibition of the CFTR chloride channel by these compounds has been suggested to be of pharmacological interest in the treatment of secretory diarrheas and polycystic kidney disease. In addition, functional inhibition of CFTR by CFTR(inh)-172 has been proposed to be sufficient to mimic the CF inflammatory profile. In the present study, we investigated the effects of the two compounds on reactive oxygen species (ROS) production and mitochondrial membrane potential in several cell lines: the CFTR-deficient human lung epithelial IB3-1 (expressing the heterozygous F508del/W1282X mutation), the isogenic CFTR-corrected C38, and HeLa and A549 as non-CFTR-expressing controls. Both inhibitors were able to induce a rapid increase in ROS levels and depolarize mitochondria in the four cell types, suggesting that these effects are independent of CFTR inhibition. In HeLa cells, these events were associated with a decrease in the rate of oxygen consumption, with GlyH-101 demonstrating a higher potency than CFTR(inh)-172. The impact of CFTR inhibitors on inflammatory parameters was also tested in HeLa cells. CFTR(inh)-172, but not GlyH-101, induced nuclear translocation of nuclear factor-kappaB (NF-kappaB). CFTR(inh)-172 slightly decreased interleukin-8 secretion, whereas GlyH-101 induced a slight increase. These results support the conclusion that CFTR inhibitors may exert nonspecific effects regarding ROS production, mitochondrial failure, and activation of the NF-kappaB signaling pathway, independently of CFTR inhibition.

  20. Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ.

    PubMed

    Camerino, Giulia Maria; Bouchè, Marina; De Bellis, Michela; Cannone, Maria; Liantonio, Antonella; Musaraj, Kejla; Romano, Rossella; Smeriglio, Piera; Madaro, Luca; Giustino, Arcangela; De Luca, Annamaria; Desaphy, Jean-François; Camerino, Diana Conte; Pierno, Sabata

    2014-12-01

    In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process.

  1. Layer by layer growth of silver chloride nanoparticle within the pore channels of SBA-15/SO3H mesoporous silica (AgClNP/SBA-15/SO3K): Synthesis, characterization and antibacterial properties

    NASA Astrophysics Data System (ADS)

    Rostamnia, Sadegh; Doustkhah, Esmail; Estakhri, Saba; Karimi, Ziba

    2016-02-01

    The growth of silver chloride nanoparticles within the pore channels of functionalized SBA-15 mesoporous was achieved by sequential dipping steps in alternating bath of potassium chloride and silver nitrate under ultrasound irradiation at pH=9. The effects of sequential dipping steps in growth of the AgCl nanoparticles have been studied. The growth and formation of AgCl nanoparticles inside the sulfonated SBA-15 were characterized by X-ray diffraction (XRD), Fourier transformation infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Antibacterial activity of the synthesized materials was investigated against Escherichia coli (E.coli) using the conventional diffusion-disc method. The materials showed high antibacterial activity.

  2. Chloride Test

    MedlinePlus

    ... of the imbalance. In persons with too much base, urine chloride measurements can tell the healthcare practitioner whether the cause ... healthcare practitioner will look at whether the chloride measurement changes ... an acid-base imbalance and helps to guide treatment. ^ Back to ...

  3. Discrete-state representation of ion permeation coupled to fast gating in a model of CLC-chloride channels: analytic estimation of the state-to-state rate constants.

    PubMed

    Coalson, Rob D; Cheng, Mary Hongying

    2011-09-01

    Analytical estimation of state-to-state rate constants is carried out for a recently developed discrete state model of chloride ion motion in a CLC chloride channel (Coalson and Cheng, J. Phys. Chem. B 2010, 114, 1424). In the original presentation of this model, the same rate constants were evaluated via three-dimensional Brownian dynamics simulations. The underlying dynamical theory is an appropriate single- or multiparticle three-dimensional Smoluchowski equation. Taking advantage of approximate geometric symmetries (based on the details of the model channel geometry), well-known formulas for state-to-state transition rates are appealed to herein and adapted as necessary to the problem at hand. Rates of ionic influx from a bulk electrolyte reservoir to the nearest binding site within the channel pore are particularly challenging to compute analytically because they reflect multi-ion interactions (as opposed to single-ion dynamics). A simple empirical correction factor is added to the single-ion rate constant formula in this case to account for the saturation of influx rate constants with increasing bulk Cl(-) concentration. Overall, the agreement between all analytically estimated rate constants is within a factor of 2 of those computed via three-dimensional Brownian dynamics simulations, and often better than this. Current-concentration curves obtained using rate constants derived from these two different computational approaches agree to within 25%.

  4. Salt, chloride, bleach, and innate host defense

    PubMed Central

    Wang, Guoshun; Nauseef, William M.

    2015-01-01

    Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense. PMID:26048979

  5. Salt, chloride, bleach, and innate host defense.

    PubMed

    Wang, Guoshun; Nauseef, William M

    2015-08-01

    Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense.

  6. Effects of lorazepam tolerance and withdrawal on GABA[sub A] receptor operated chloride channels in mice selected for differences in ethanol withdrawal severity

    SciTech Connect

    Allan, A.M.; Baier, L.D.; Zhang, Xiaoying )

    1992-01-01

    Withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) mice were treated with 5 mg/kg lorazepam for 7 days via implanted osmotic mini pumps. Following chronic drug treatment, brains were assayed for GABA-mediated chloride flux (GABA-Cl[sup [minus

  7. Influence of salinity on the localization and expression of the CFTR chloride channel in the ionocytes of Dicentrarchus labrax during ontogeny.

    PubMed

    Bodinier, Charlotte; Boulo, Viviane; Lorin-Nebel, Catherine; Charmantier, Guy

    2009-03-01

    The expression and localization of the cystic fibrosis transmembrane conductance regulator (CFTR) were determined in four osmoregulatory tissues during the ontogeny of the sea-bass Dicentrarchus labrax acclimated to fresh water and sea water. At hatch in sea water, immunolocalization showed an apical CFTR in the digestive tract and integumental ionocytes. During the ontogeny, although CFTR was consistently detected in the digestive tract, it shifted from the integument to the gills. In fresh water, CFTR was not present in the integument and the gills, suggesting the absence of chloride secretion. In the kidney, the CFTR expression was brief from D4 to D35, prior to the larva-juvenile transition. CFTR was apical in the renal tubules, suggesting a chloride secretion at both salinities, and it was basolateral only in sea water in the collecting ducts, suggesting chloride absorption. In the posterior intestine, CFTR was located differently from D4 depending on salinity. In sea water, the basolateral CFTR may facilitate ionic absorption, perhaps in relation to water uptake. In fresh water, CFTR was apical in the gut, suggesting chloride secretion. Increased osmoregulatory ability was acquired just before metamorphosis, which is followed by the sea-lagoon migration.

  8. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic Acid (DIDS) Ameliorates Ischemia-Hypoxia-Induced White Matter Damage in Neonatal Rats through Inhibition of the Voltage-Gated Chloride Channel ClC-2

    PubMed Central

    Zhao, Baixiong; Quan, Hongyu; Ma, Teng; Tian, Yanping; Cai, Qiyan; Li, Hongli

    2015-01-01

    Chronic cerebral hypoperfusion is believed to cause white matter lesions (WMLs), leading to cognitive impairment. Previous studies have shown that inflammation and apoptosis of oligodendrocytes (OLs) are involved in the pathogenesis of WMLs, but effective treatments have not been studied. In this study, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a chloride (Cl−) channel blocker, was injected into chronic cerebral ischemia-hypoxia rat models at different time points. Our results showed that DIDS significantly reduced the elevated mRNA levels and protein expression of chloride channel 2 (ClC-2) in neonatal rats induced by ischemia-hypoxia. Meanwhile, DIDS application significantly decreased the concentrations of reactive oxygen species (ROS); and the mRNA levels of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha TNF-α in neonatal rats with hypoxic-ischemic damage. Myelin staining was weaker in neonatal rats with hypoxic-ischemic damage compared to normal controls in corpus callosum and other white matter, which was ameliorated by DIDS. Furthermore, the elevated number of caspase-3 and neural/glial antigen 2 (NG-2) double-labeled positive cells was attenuated by DIDS after ischemia anoxic injury. Administration of DIDS soon after injury alleviated damage to OLs much more effectively in white matter. In conclusion, our study suggests that early application of DIDS after ischemia-hypoxia injury may partially protect developing OLs. PMID:25961953

  9. Dual regulation of the native ClC-K2 chloride channel in the distal nephron by voltage and pH

    PubMed Central

    Pinelli, Laurent; Nissant, Antoine; Edwards, Aurélie; Paulais, Marc

    2016-01-01

    ClC-K2, a member of the ClC family of Cl− channels and transporters, forms the major basolateral Cl− conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl− absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl−, and Ca2+ on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca2+ strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl− has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl−/HCO3− exchange in type B intercalated cells. PMID:27574292

  10. Dual regulation of the native ClC-K2 chloride channel in the distal nephron by voltage and pH.

    PubMed

    Pinelli, Laurent; Nissant, Antoine; Edwards, Aurélie; Lourdel, Stéphane; Teulon, Jacques; Paulais, Marc

    2016-09-01

    ClC-K2, a member of the ClC family of Cl(-) channels and transporters, forms the major basolateral Cl(-) conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl(-) absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl(-), and Ca(2+) on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca(2+) strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl(-) has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl(-)/HCO3 (-) exchange in type B intercalated cells.

  11. Hydrogen chloride

    Integrated Risk Information System (IRIS)

    Hydrogen chloride ; CASRN 7647 - 01 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogeni

  12. Mepiquat chloride

    Integrated Risk Information System (IRIS)

    Mepiquat chloride ; CASRN 24307 - 26 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogen

  13. Acetyl chloride

    Integrated Risk Information System (IRIS)

    Acetyl chloride ; CASRN 75 - 36 - 5 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  14. Methyl chloride

    Integrated Risk Information System (IRIS)

    EPA / 635 / R01 / 003 TOXICOLOGICAL REVIEW OF METHYL CHLORIDE ( CAS No . 74 - 87 - 3 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) June 2001 U.S . Environmental Protection Agency Washington , DC DISCLAIMER This document has been reviewed in accordance with U.

  15. Vinyl chloride

    Integrated Risk Information System (IRIS)

    EPA / 635R - 00 / 004 TOXICOLOGICAL REVIEW OF VINYL CHLORIDE ( CAS No . 75 - 01 - 4 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) May 2000 U.S . Environmental Protection Agency Washington , DC DISCLAIMER This document has been reviewed in accordance with U.S

  16. Ethyl chloride

    Integrated Risk Information System (IRIS)

    Ethyl chloride ; CASRN 75 - 00 - 3 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  17. Benzyl chloride

    Integrated Risk Information System (IRIS)

    Benzyl chloride ; CASRN 100 - 44 - 7 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic E

  18. Allyl chloride

    Integrated Risk Information System (IRIS)

    Allyl chloride ; CASRN 107 - 05 - 1 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  19. Voltage-dependent and -independent titration of specific residues accounts for complex gating of a ClC chloride channel by extracellular protons

    PubMed Central

    Niemeyer, María Isabel; Cid, L Pablo; Yusef, Yamil R; Briones, Rodolfo; Sepúlveda, Francisco V

    2009-01-01

    The ClC transport protein family comprises both Cl− ion channel and H+/Cl− and H+/NO3− exchanger members. Structural studies on a bacterial ClC transporter reveal a pore obstructed at its external opening by a glutamate side-chain which acts as a gate for Cl− passage and in addition serves as a staging post for H+ exchange. This same conserved glutamate acts as a gate to regulate Cl− flow in ClC channels. The activity of ClC-2, a genuine Cl− channel, has a biphasic response to extracellular pH with activation by moderate acidification followed by abrupt channel closure at pH values lower than ∼7. We have now investigated the molecular basis of this complex gating behaviour. First, we identify a sensor that couples extracellular acidification to complete closure of the channel. This is extracellularly-facing histidine 532 at the N-terminus of transmembrane helix Q whose neutralisation leads to channel closure in a cooperative manner. We go on to show that acidification-dependent activation of ClC-2 is voltage dependent and probably mediated by protonation of pore gate glutamate 207. Intracellular Cl− acts as a voltage-independent modulator, as though regulating the pKa of the protonatable residue. Our results suggest that voltage dependence of ClC-2 is given by hyperpolarisation-dependent penetration of protons from the extracellular side to neutralise the glutamate gate deep within the channel, which allows Cl− efflux. This is reminiscent of a partial exchanger cycle, suggesting that the ClC-2 channel evolved from its transporter counterparts. PMID:19153159

  20. The Muscle Chloride Channel ClC-1 Has a Double-Barreled Appearance that Is Differentially Affected in Dominant and Recessive Myotonia

    PubMed Central

    Saviane, Chiara; Conti, Franco; Pusch, Michael

    1999-01-01

    Single-channel recordings of the currents mediated by the muscle Cl− channel, ClC-1, expressed in Xenopus oocytes, provide the first direct evidence that this channel has two equidistant open conductance levels like the Torpedo ClC-0 prototype. As for the case of ClC-0, the probabilities and dwell times of the closed and conducting states are consistent with the presence of two independently gated pathways with ≈ 1.2 pS conductance enabled in parallel via a common gate. However, the voltage dependence of the common gate is different and the kinetics are much faster than for ClC-0. Estimates of single-channel parameters from the analysis of macroscopic current fluctuations agree with those from single-channel recordings. Fluctuation analysis was used to characterize changes in the apparent double-gate behavior of the ClC-1 mutations I290M and I556N causing, respectively, a dominant and a recessive form of myotonia. We find that both mutations reduce about equally the open probability of single protopores and that mutation I290M yields a stronger reduction of the common gate open probability than mutation I556N. Our results suggest that the mammalian ClC-homologues have the same structure and mechanism proposed for the Torpedo channel ClC-0. Differential effects on the two gates that appear to modulate the activation of ClC-1 channels may be important determinants for the different patterns of inheritance of dominant and recessive ClC-1 mutations. PMID:10051520

  1. Relaxation of endothelin-1-induced pulmonary arterial constriction by niflumic acid and NPPB: mechanism(s) independent of chloride channel block.

    PubMed

    Kato, K; Evans, A M; Kozlowski, R Z

    1999-03-01

    We investigated the effects of the Cl- channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) on endothelin-1 (ET-1)-induced constriction of rat small pulmonary arteries (diameter 100-400 microm) in vitro, following endothelium removal. ET-1 (30 nM) induced a sustained constriction of rat pulmonary arteries in physiological salt solution. Arteries preconstricted with ET-1 were relaxed by niflumic acid (IC50: 35.8 microM) and NPPB (IC50: 21.1 microM) in a reversible and concentration-dependent manner. However, at concentrations known to block Ca++-activated Cl- channels, DIDS (channel blockers. When L-type Ca++ channels were blocked by nifedipine (10 microM), the ET-1-induced (30 nM) constriction was inhibited by only 5.8%. However, niflumic acid (30 microM) and NPPB (30 microM) inhibited the ET-1-induced constriction by approximately 53% and approximately 60%, respectively, both in the continued presence of nifedipine and in Ca++-free physiological salt solution. The Ca++ ionophore A23187 (10 microM) also evoked a sustained constriction of pulmonary arteries. Surprisingly, the A23187-induced constriction was also inhibited in a reversible and concentration-dependent manner by niflumic acid (IC50: 18.0 microM) and NPPB (IC50: 8.8 microM), but not by DIDS (channel blockade. One possibility is that these compounds may block the Ca++-dependent contractile processes.

  2. The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop

    PubMed Central

    Cenedese, Valentina; Betto, Giulia; Celsi, Fulvio; Cherian, O. Lijo; Pifferi, Simone

    2012-01-01

    Ca2+-activated Cl− channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure–function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca2+ dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild-type and mutant TMEM16B channels expressed in HEK 293T cells using the whole cell voltage-clamp technique in the presence of various intracellular Ca2+ concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates 386EEEEE390 and 399EYE401. The EYE deletion did not significantly modify the apparent Ca2+ dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca2+ affinity but modified the voltage dependence, shifting the conductance–voltage relations toward more positive voltages. These findings indicate that glutamates E367 and 386EEEEE390 in the first intracellular putative loop play an important role in the voltage dependence of TMEM16B, thus providing an initial structure–function study for this channel. PMID:22412191

  3. Mechanism of block of single protopores of the Torpedo chloride channel ClC-0 by 2-(p-chlorophenoxy)butyric acid (CPB).

    PubMed

    Pusch, M; Accardi, A; Liantonio, A; Ferrera, L; De Luca, A; Camerino, D C; Conti, F

    2001-07-01

    We investigated in detail the mechanism of inhibition by the S(-) enantiomer of 2-(p-chlorophenoxy)butyric acid (CPB) of the Torpedo Cl(-)channel, ClC-0. The substance has been previously shown to inhibit the homologous skeletal muscle channel, CLC-1. ClC-0 is a homodimer with probably two independently gated protopores that are conductive only if an additional common gate is open. As a simplification, we used a mutant of ClC-0 (C212S) that has the common gate "locked open" (Lin, Y.W., C.W. Lin, and T.Y. Chen. 1999. J. Gen. Physiol. 114:1-12). CPB inhibits C212S currents only when applied to the cytoplasmic side, and single-channel recordings at voltages (V) between -120 and -80 mV demonstrate that it acts independently on individual protopores by introducing a long-lived nonconductive state with no effect on the conductance and little effect on the lifetime of the open state. Steady-state macroscopic currents at -140 mV are half-inhibited by approximately 0.5 mM CPB, but the inhibition decreases with V and vanishes for V > or = 40 mV. Relaxations of CPB inhibition after voltage steps are seen in the current responses as an additional exponential component that is much slower than the gating of drug-free protopores. For V = 60 mV) with an IC50 of approximately 30-40 mM. Altogether, these findings support a model for the mechanism of CPB inhibition in which the drug competes with Cl(-) for binding to a site of the pore where it blocks permeation. CPB binds preferentially to closed channels, and thereby also strongly alters the gating of the single protopore. Since the affinity of CPB for open WT pores is extremely low, we cannot decide in this case if it acts also as an open pore blocker. However, the experiments with the mutant K519E strongly support this interpretation. CPB block may become a useful tool to study the pore of ClC channels. As a first application, our results provide additional evidence for a double-barreled structure of ClC-0 and ClC-1.

  4. Cryptococcus neoformans Ca(2+) homeostasis requires a chloride channel/antiporter Clc1 in JEC21, but not in H99.

    PubMed

    Li, Dong; Zhang, Xiaojiao; Li, Zhongming; Yang, Jiao; Pan, Jiao; Zhu, Xudong

    2012-02-01

    CLC-type chloride/proton antiporters are required for copper/iron homeostasis in fungi. A relationship between CLCs and Ca(2+) homeostasis has not been found before. Here we demonstrate the requirement of the antiporter CLC1 for Ca(2+) homeostasis/signaling in Cryptococcus neoformans. The deletion of CLC1 in JEC21 resulted in a mutant hypersensitive to cyclosporine A, an inhibitor of calcineurin. Intracellular Ca(2+) deficiency in the mutant Tx1 was confirmed with Fluo-3 staining epi-fluorescence microscopy. Tx1 failed to grow at elevated temperature and in SDS and displayed defects in cell wall integrity and cell separation. This defective phenotype is because of Ca(2+) deficiency that was restorable by exogenous Ca(2+) . In contrast, H99 CLC1 was dispensable for Ca(2+) homeostasis and had no comparable defective consequences if deleted, suggesting divergent roles of CLCs in Ca(2+) homeostasis. Distinct Ca(2+) homeostasis mechanisms may contribute the virulence difference between the two strains. This work reveals a novel action of CLC antiporters in fungi and may provide information as to the evolution of pathogenicity among cryptococcal strains.

  5. Luminal cholinergic signalling in airway lining fluid: a novel mechanism for activating chloride secretion via Ca2+-dependent Cl− and K+ channels

    PubMed Central

    Hollenhorst, Monika I; Lips, Katrin S; Wolff, Miriam; Wess, Jürgen; Gerbig, Stefanie; Takats, Zoltan; Kummer, Wolfgang; Fronius, Martin

    2012-01-01

    BACKGROUND AND PURPOSE Recent studies detected the expression of proteins involved in cholinergic metabolism in airway epithelial cells, although the function of this non-neuronal cholinergic system is not known in detail. Thus, this study focused on the effect of luminal ACh as a regulator of transepithelial ion transport in epithelial cells. EXPERIMENTAL APPROACH RT-PCR experiments were performed using mouse tracheal epithelial cells for ChAT and organic cation transporter (OCT) transcripts. Components of tracheal airway lining fluid were analysed with desorption electrospray ionization (DESI) MS. Effects of nicotine on mouse tracheal epithelial ion transport were examined with Ussing-chamber experiments. KEY RESULTS Transcripts encoding ChAT and OCT1–3 were detected in mouse tracheal epithelial cells. The DESI experiments identified ACh in the airway lining fluid. Luminal ACh induced an immediate, dose-dependent increase in the transepithelial ion current (EC50: 23.3 µM), characterized by a transient peak and sustained plateau current. This response was not affected by the Na+-channel inhibitor amiloride. The Cl−-channel inhibitor niflumic acid or the K+-channel blocker Ba2+ attenuated the ACh effect. The calcium ionophore A23187 mimicked the ACh effect. Luminal nicotine or muscarine increased the ion current. Experiments with receptor gene-deficient animals revealed the participation of muscarinic receptor subtypes M1 and M3. CONCLUSIONS AND IMPLICATIONS The presence of luminal ACh and activation of transepithelial ion currents by luminal ACh receptors identifies a novel non-neuronal cholinergic pathway in the airway lining fluid. This pathway could represent a novel drug target in the airways. PMID:22300281

  6. Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is dependent on GABAA receptors and niflumic acid-sensitive chloride channels.

    PubMed

    Babot, Zoila; Cristòfol, Rosa; Suñol, Cristina

    2005-01-01

    Excitotoxic neuronal death has been linked to neurological and neurodegenerative diseases. Several studies have sought to clarify the involvement of Cl(-) channels in neuronal excitotoxicity using either N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainic acid agonists. In this work we induced excitotoxic death in primary cultures of cerebellar granule cells by means of endogenously released glutamate. Excitotoxicity was provoked by exposure to high extracellular K(+) concentrations ([K(+)](o)) for 5 min. Under these conditions, a Ca(2+)-dependent release of glutamate was evoked. When extracellular glutamate concentration rose to between 2 and 4 microM, cell viability was significantly reduced by 30-40%. The NMDA receptor antagonists (MK-801 and D-2-amino-5-phosphonopentanoic acid) prevented cell death. Exposure to high [K(+)](o) produced a (36)Cl(-) influx which was significantly reduced by picrotoxinin. In addition, the GABA(A) receptor antagonists (bicuculline, picrotoxinin and SR 95531) protected cells from high [K(+)](o)-triggered excitotoxicity and reduced extracellular glutamate concentration. The Cl(-) channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid also exerted a neuroprotective effect and reduced extracellular glutamate concentration, even though they did not reduce high [K(+)](o)-induced (36)Cl(-) influx. Primary cultures of cerebellar granule cells also contain a population of GABAergic neurons that released GABA in response to high [K(+)](o). Chronic treatment of primary cultures with kainic acid abolished GABA release and rendered granule cells insensitive to high [K(+)](o) exposure, even though NMDA receptors were functional. Altogether, these results demonstrate that, under conditions of membrane depolarization, low micromolar concentrations of extracellular glutamate might induce an excitotoxic process through both NMDA and GABA(A) receptors and niflumic acid-sensitive Cl

  7. Potassium and ANO1/TMEM16A chloride channel profiles distinguish atypical and typical smooth muscle cells from interstitial cells in the mouse renal pelvis

    PubMed Central

    Iqbal, Javed; Tonta, Mary A; Mitsui, Retsu; Li, Qun; Kett, Michelle; Li, Jinhua; Parkington, Helena C; Hashitani, Hikaru; Lang, Richard J

    2012-01-01

    BACKGROUND AND PURPOSE Although atypical smooth muscle cells (SMCs) in the proximal renal pelvis are thought to generate the pacemaker signals that drive pyeloureteric peristalsis, their location and electrical properties remain obscure. EXPERIMENTAL APPROACH Standard patch clamp, intracellular microelectrode and immunohistochemistry techniques were used. To unequivocally identify SMCs, transgenic mice with enhanced yellow fluorescent protein (eYFP) expressed in cells containing α-smooth muscle actin (α-SMA) were sometimes used. KEY RESULTS Atypical SMCs were distinguished from typical SMCs by the absence of both a transient 4-aminopyridine-sensitive K+ current (IKA) and spontaneous transient outward currents (STOCs) upon the opening of large-conductance Ca2+-activated K+ (BK) channels. Many typical SMCs displayed a slowly activating, slowly decaying Cl- current blocked by niflumic acid (NFA). Immunostaining for KV4.3 and ANO1/ TMEM16A Cl- channel subunits co-localized with α-SMA immunoreactive product predominately in the distal renal pelvis. Atypical SMCs fired spontaneous inward currents that were either selective for Cl- and blocked by NFA, or cation-selective and blocked by La3+. α-SMA- interstitial cells (ICs) were distinguished by the presence of a Xe991-sensitive KV7 current, BK channel STOCs and Cl- selective, NFA-sensitive spontaneous transient inward currents (STICs). Intense ANO1/ TMEM16A and KV7.5 immunostaining was present in Kit-α-SMA- ICs in the suburothelial and adventitial regions of the renal pelvis. CONCLUSIONS AND IMPLICATIONS We conclude that KV4.3+α-SMA+ SMCs are typical SMCs that facilitate muscle wall contraction, that ANO1/ TMEM16A and KV7.5 immunoreactivity may be selective markers of Kit- ICs and that atypical SMCs which discharge spontaneous inward currents are the pelviureteric pacemakers. PMID:22014103

  8. Berberine Reduces cAMP-Induced Chloride Secretion in T84 Human Colonic Carcinoma Cells through Inhibition of Basolateral KCNQ1 Channels

    PubMed Central

    Alzamora, Rodrigo; O’Mahony, Fiona; Ko, Wing-Hung; Yip, Tiffany Wai-Nga; Carter, Derek; Irnaten, Mustapha; Harvey, Brian Joseph

    2011-01-01

    Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl− secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl− secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC50 80 ± 8 μM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K+ current by 88%, suggesting inhibition of KCNQ1 K+ channels. Berberine did not affect either apical Cl− conductance or basolateral Na+–K+-ATPase activity. Berberine stimulated p38 MAPK, PKCα and PKA, but had no effect on p42/p44 MAPK and PKCδ. However, berberine pre-treatment prevented stimulation of p42/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl− secretion was partially blocked by HBDDE (∼65%), an inhibitor of PKCα and to a smaller extent by inhibition of p38 MAPK with SB202190 (∼15%). Berberine treatment induced an increase in association between PKCα and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl− secretion through inhibition of basolateral KCNQ1 channels responsible for K+ recycling via a PKCα-dependent pathway. PMID:21747769

  9. Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: Involvement of the effector proteins in the regulatory mechanism

    SciTech Connect

    Saxena, Sunil K. . E-mail: ssaxena@stevens.edu; Kaur, Simarna

    2006-07-21

    Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein-protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) and Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein-protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein-protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.

  10. Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop

    PubMed Central

    Xiao, Qinghuan; Yu, Kuai; Perez-Cornejo, Patricia; Cui, Yuanyuan; Arreola, Jorge; Hartzell, H. Criss

    2011-01-01

    Ca2+-activated Cl− channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca2+ has recently become possible with the discovery that Ano1 (TMEM16a) is an essential subunit of CaCCs. Ano1 can be gated by Ca2+ or by voltage in the absence of Ca2+, but Ca2+- and voltage-dependent gating are very closely coupled. Here we identify a region in the first intracellular loop that is crucial for both Ca2+ and voltage sensing. Deleting 448EAVK in the first intracellular loop dramatically decreases apparent Ca2+ affinity. In contrast, mutating the adjacent amino acids 444EEEE abolishes intrinsic voltage dependence without altering the apparent Ca2+affinity. Voltage-dependent gating of Ano1 measured in the presence of intracellular Ca2+ was facilitated by anions with high permeability or by an increase in [Cl−]e. Our data show that the transition between closed and open states is governed by Ca2+ in a voltage-dependent manner and suggest that anions allosterically modulate Ca2+-binding affinity. This mechanism provides a unified explanation of CaCC channel gating by voltage and ligand that has long been enigmatic. PMID:21555582

  11. Chloride influx provokes lamellipodium formation in microglial cells.

    PubMed

    Zierler, Susanna; Frei, Eva; Grissmer, Stephan; Kerschbaum, Hubert H

    2008-01-01

    Lamellipodium extension and retraction is the driving force for cell migration. Although several studies document that activation of chloride channels are essential in cell migration, little is known about their contribution in lamellipodium formation. To address this question, we characterized chloride channels and transporters by whole cell recording and RT-PCR, respectively, as well as quantified lamellipodium formation in murine primary microglial cells as well as the microglial cell-line, BV-2, using time-lapse microscopy. The repertoire of chloride conducting pathways in BV-2 cells included, swelling-activated chloride channels as well as the KCl cotransporters, KCC1, KCC2, KCC3, and KCC4. Swelling-activated chloride channels were either activated by a hypoosmotic solution or by a high KCl saline, which promotes K(+) and Cl(-) influx instead of efflux by KCCs. Conductance through swelling-activated chloride channels was completely blocked by flufenamic acid (200 microM), SITS (1 mM) and DIOA (10 microM). By exposing primary microglial cells or BV-2 cells to a high KCl saline, we observed a local swelling, which developed into a prominent lamellipodium. Blockade of chloride influx by flufenamic acid (200 microM) or DIOA (10 microM) as well as incubation of cells in a chloride-free high K(+) saline suppressed formation of a lamellipodium. We assume that local swellings, established by an increase in chloride influx, are a general principle in formation of lamellipodia in eukaryotic cells.

  12. Hypotonicity activates a native chloride current in Xenopus oocytes

    PubMed Central

    1994-01-01

    Xenopus oocytes are frequently utilized for in vivo expression of cellular proteins, especially ion channel proteins. A thorough understanding of the endogenous conductances and their regulation is paramount for proper characterization of expressed channel proteins. Here we detail a novel chloride current (ICl.swell) responsive to hypotonicity in Xenopus oocytes using the two-electrode voltage clamp technique. Reducing the extracellular osmolarity by 50% elicited a calcium-independent chloride current having an anion conductivity sequence identical with swelling-induced chloride currents observed in epithelial cells. The hypotonicity-activated current was blocked by chloride channel blockers, trivalent lanthanides, and nucleotides. G- protein, cAMP-PKA, and arachidonic acid signaling cascades were not involved in ICl.swell activation. ICl.swell is distinct from both stretch-activated nonselective cation channels and the calcium- activated chloride current in oocytes and may play a critical role in volume regulation in Xenopus oocytes. PMID:8189203

  13. Recovery of mucosal barrier function in ischemic porcine ileum and colon is stimulated by a novel agonist of the ClC-2 chloride channel, lubiprostone.

    PubMed

    Moeser, Adam J; Nighot, Prashant K; Engelke, Kory J; Ueno, Ryuji; Blikslager, Anthony T

    2007-02-01

    Previous studies utilizing an ex vivo porcine model of intestinal ischemic injury demonstrated that prostaglandin (PG)E(2) stimulates repair of mucosal barrier function via a mechanism involving Cl(-) secretion and reductions in paracellular permeability. Further experiments revealed that the signaling mechanism for PGE(2)-induced mucosal recovery was mediated via type-2 Cl(-) channels (ClC-2). Therefore, the objective of the present study was to directly investigate the role of ClC-2 in mucosal repair by evaluating mucosal recovery in ischemia-injured intestinal mucosa treated with the selective ClC-2 agonist lubiprostone. Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers, and short-circuit current (I(sc)) and transepithelial electrical resistance (TER) were measured in response to lubiprostone. Application of 0.01-1 microM lubiprostone to ischemia-injured mucosa induced concentration-dependent increases in TER, with 1 microM lubiprostone stimulating a twofold increase in TER (DeltaTER = 26 Omega.cm(2); P < 0.01). However, lubiprostone (1 microM) stimulated higher elevations in TER despite lower I(sc) responses compared with the nonselective secretory agonist PGE(2) (1 microM). Furthermore, lubiprostone significantly (P < 0.05) reduced mucosal-to-serosal fluxes of (3)H-labeled mannitol to levels comparable to those of normal control tissues and restored occludin localization to tight junctions. Activation of ClC-2 with the selective agonist lubiprostone stimulated elevations in TER and reductions in mannitol flux in ischemia-injured intestine associated with structural changes in tight junctions. Prostones such as lubiprostone may provide a selective and novel pharmacological mechanism of accelerating recovery of acutely injured intestine compared with the nonselective action of prostaglandins such as PGE(2).

  14. Abnormal passive chloride absorption in cystic fibrosis jejunum functionally opposes the classic chloride secretory defect.

    PubMed

    Russo, Michael A; Hogenauer, Christoph; Coates, Stephen W; Santa Ana, Carol A; Porter, Jack L; Rosenblatt, Randall L; Emmett, Michael; Fordtran, John S

    2003-07-01

    Due to genetic defects in apical membrane chloride channels, the cystic fibrosis (CF) intestine does not secrete chloride normally. Depressed chloride secretion leaves CF intestinal absorptive processes unopposed, which results in net fluid hyperabsorption, dehydration of intestinal contents, and a propensity to inspissated intestinal obstruction. This theory is based primarily on in vitro studies of jejunal mucosa. To determine if CF patients actually hyperabsorb fluid in vivo, we measured electrolyte and water absorption during steady-state perfusion of the jejunum. As expected, chloride secretion was abnormally low in CF, but surprisingly, there was no net hyperabsorption of sodium or water during perfusion of a balanced electrolyte solution. This suggested that fluid absorption processes are reduced in CF jejunum, and further studies revealed that this was due to a marked depression of passive chloride absorption. Although Na+-glucose cotransport was normal in the CF jejunum, absence of passive chloride absorption completely blocked glucose-stimulated net sodium absorption and reduced glucose-stimulated water absorption 66%. This chloride absorptive abnormality acts in physiological opposition to the classic chloride secretory defect in the CF intestine. By increasing the fluidity of intraluminal contents, absence of passive chloride absorption may reduce the incidence and severity of intestinal disease in patients with CF.

  15. Influence of salinity on the localization of Na+/K +-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and CFTR anion channel in chloride cells of the Hawaiian goby (Stenogobius hawaiiensis)

    USGS Publications Warehouse

    McCormick, S.D.; Sundell, K.; Bjornsson, Bjorn Thrandur; Brown, C.L.; Hiroi, J.

    2003-01-01

    Na+/K+-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR) are the three major transport proteins thought to be involved in chloride secretion in teleost fish. If this is the case, the levels of these transporters should be high in chloride cells of seawater-acclimated fish. We therefore examined the influence of salinity on immunolocalization of Na +/K+-ATPase, NKCC and CFTR in the gills of the Hawaiian goby (Stenogobius hawaiiensis). Fish were acclimated to freshwater and 20??? and 30??? seawater for 10 days. Na+/K +-ATPase and NKCC were localized specifically to chloride cells and stained throughout most of the cell except for the nucleus and the most apical region, indicating a basolateral/tubular distribution. All Na+/K +-ATPase-positive chloride cells were also positive for NKCC in all salinities. Salinity caused a slight increase in chloride cell number and size and a slight decrease in staining intensity for Na+/K +-ATPase and NKCC, but the basic pattern of localization was not altered. Gill Na+/K+-ATPase activity was also not affected by salinity. CFTR was localized to the apical surface of chloride cells, and only cells staining positive for Na+/K+-ATPase were CFTR-positive. CFTR-positive cells greatly increased in number (5-fold), area stained (53%) and intensity (29%) after seawater acclimation. In freshwater, CFTR immunoreactivity was light and occurred over a broad apical surface on chloride cells, whereas in seawater there was intense immunoreactivity around the apical pit (which was often punctate in appearance) and a light subapical staining. The results indicate that Na+/K +-ATPase, NKCC and CFTR are all present in chloride cells and support current models that all three are responsible for chloride secretion by chloride cells of teleost fish.

  16. Influence of salinity on the localization of Na+/K+-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and CFTR anion channel in chloride cells of the Hawaiian goby (Stenogobius hawaiiensis).

    PubMed

    McCormick, Stephen D; Sundell, Kristina; Björnsson, Björn Thrandur; Brown, Christopher L; Hiroi, Junya

    2003-12-01

    Na+/K+-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR) are the three major transport proteins thought to be involved in chloride secretion in teleost fish. If this is the case, the levels of these transporters should be high in chloride cells of seawater-acclimated fish. We therefore examined the influence of salinity on immunolocalization of Na+/K+-ATPase, NKCC and CFTR in the gills of the Hawaiian goby (Stenogobius hawaiiensis). Fish were acclimated to freshwater and 20 per thousand and 30 per thousand seawater for 10 days. Na+/K+-ATPase and NKCC were localized specifically to chloride cells and stained throughout most of the cell except for the nucleus and the most apical region, indicating a basolateral/tubular distribution. All Na+/K+-ATPase-positive chloride cells were also positive for NKCC in all salinities. Salinity caused a slight increase in chloride cell number and size and a slight decrease in staining intensity for Na+/K+-ATPase and NKCC, but the basic pattern of localization was not altered. Gill Na+/K+-ATPase activity was also not affected by salinity. CFTR was localized to the apical surface of chloride cells, and only cells staining positive for Na+/K+-ATPase were CFTR-positive. CFTR-positive cells greatly increased in number (5-fold), area stained (53%) and intensity (29%) after seawater acclimation. In freshwater, CFTR immunoreactivity was light and occurred over a broad apical surface on chloride cells, whereas in seawater there was intense immunoreactivity around the apical pit (which was often punctate in appearance) and a light subapical staining. The results indicate that Na+/K+-ATPase, NKCC and CFTR are all present in chloride cells and support current models that all three are responsible for chloride secretion by chloride cells of teleost fish.

  17. CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING IN CENTER, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING IN CENTER, CALCIUM CHLORIDE STORAGE BUILDING ON RIGHT WITH SA (SODA ASH) BUILDING IN RIGHT BACKGROUND. - Solvay Process Company, Calcium Chloride Plant, Between Willis & Milton Avenues, Solvay, Onondaga County, NY

  18. Antidepressants and seizure-interactions at the GABA-receptor chloride-ionophore complex

    SciTech Connect

    Malatynska, E.; Knapp, R.J.; Ikeda, M.; Yamamura, H.I.

    1988-01-01

    Convulsive seizures are a potential side effect of antidepressant drug treatment and can be produced by all classes of antidepressants. It is also know that some convulsant and anticonvulsant drug actions are mediated by the GABA-receptor chloride-ionophore complex. Drugs acting at this complex appear to induce convulsions by inhibiting chloride conductance through the associated chloride channel. Using the method of GABA-stimulated /sup 36/Cl-uptake by rat cerebral cortical vesicles, we show that some antidepressant drugs can inhibit the GABA-receptor chloride uptake, and that the degree of chloride channel inhibition by these drugs correlates with the frequency of convulsive seizures induced by them.

  19. Cl-out is a novel cooperative optogenetic tool for extruding chloride from neurons.

    PubMed

    Alfonsa, Hannah; Lakey, Jeremy H; Lightowlers, Robert N; Trevelyan, Andrew J

    2016-11-17

    Chloride regulation affects brain function in many ways, for instance, by dictating the GABAergic reversal potential, and thereby influencing neuronal excitability and spike timing. Consistent with this, there is increasing evidence implicating chloride in a range of neurological conditions. Investigations about these conditions, though, are made difficult by the limited range of tools available to manipulate chloride levels. In particular, there has been no way to actively remove chloride from neurons; we now describe an optogenetic strategy, 'Cl-out', to do exactly this. Cl-out achieves its effect by the cooperative action of two different component opsins: the proton pump, Archaerhodopsin and a chloride channel opsin. The removal of chloride happens when both are activated together, using Archaerhodopsin as an optical voltage clamp to provide the driving force for chloride removal through the concurrently opened, chloride channels. We further show that this novel optogenetic strategy can reverse an in vitro epileptogenic phenotype.

  20. Cl-out is a novel cooperative optogenetic tool for extruding chloride from neurons

    PubMed Central

    Alfonsa, Hannah; Lakey, Jeremy H.; Lightowlers, Robert N.; Trevelyan, Andrew J.

    2016-01-01

    Chloride regulation affects brain function in many ways, for instance, by dictating the GABAergic reversal potential, and thereby influencing neuronal excitability and spike timing. Consistent with this, there is increasing evidence implicating chloride in a range of neurological conditions. Investigations about these conditions, though, are made difficult by the limited range of tools available to manipulate chloride levels. In particular, there has been no way to actively remove chloride from neurons; we now describe an optogenetic strategy, ‘Cl-out', to do exactly this. Cl-out achieves its effect by the cooperative action of two different component opsins: the proton pump, Archaerhodopsin and a chloride channel opsin. The removal of chloride happens when both are activated together, using Archaerhodopsin as an optical voltage clamp to provide the driving force for chloride removal through the concurrently opened, chloride channels. We further show that this novel optogenetic strategy can reverse an in vitro epileptogenic phenotype. PMID:27853135

  1. Role of Quercetin in Modulating Chloride Transport in the Intestine

    PubMed Central

    Yu, Bo; Jiang, Yu; Jin, Lingling; Ma, Tonghui; Yang, Hong

    2016-01-01

    Epithelial chloride channels provide the pathways for fluid secretion in the intestine. Cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCCs) are the main chloride channels in the luminal membrane of enterocytes. These transmembrane proteins play important roles in many physiological processes. In this study, we have identified a flavonoid quercetin as a modulator of CaCC chloride channel activity. Fluorescence quenching assay showed that quercetin activated Cl− transport in a dose-dependent manner, with EC50 ~37 μM. Short-circuit current analysis confirmed that quercetin activated CaCC-mediated Cl− currents in HT-29 cells that can be abolished by CaCCinh-A01. Ex vivo studies indicated that application of quercetin to mouse ileum and colon on serosal side resulted in activation of CFTR and CaCC-mediated Cl− currents. Notably, we found that quercetin exhibited inhibitory effect against ANO1 chloride channel activity in ANO1-expressing FRT cells and decreased mouse intestinal motility. Quercetin-stimulated short-circuit currents in mouse ileum was multi-component, which included elevation of Ca2+ concentration through L-type calcium channel and activation of basolateral NKCC, Na+/K+-ATPase, and K+ channels. In vivo studies further revealed that quercetin promoted fluid secretion in mouse ileum. The modulatory effect of quercetin on CaCC chloirde channels may therefore represent a potential therapeutic strategy for treating CaCC-related diseases like constipation, secretory diarrhea and hypertension. The inverse effects of quercetin on CaCCs provided evidence that ANO1 and intestinal epithelial CaCCs are different calcium-activated chloride channels. PMID:27932986

  2. Twenty years of fluorescence imaging of intracellular chloride

    PubMed Central

    Arosio, Daniele; Ratto, Gian Michele

    2014-01-01

    Chloride homeostasis has a pivotal role in controlling neuronal excitability in the adult brain and during development. The intracellular concentration of chloride is regulated by the dynamic equilibrium between passive fluxes through membrane conductances and the active transport mediated by importers and exporters. In cortical neurons, chloride fluxes are coupled to network activity by the opening of the ionotropic GABAA receptors that provides a direct link between the activity of interneurons and chloride fluxes. These molecular mechanisms are not evenly distributed and regulated over the neuron surface and this fact can lead to a compartmentalized control of the intracellular concentration of chloride. The inhibitory drive provided by the activity of the GABAA receptors depends on the direction and strength of the associated currents, which are ultimately dictated by the gradient of chloride, the main charge carrier flowing through the GABAA channel. Thus, the intracellular distribution of chloride determines the local strength of ionotropic inhibition and influences the interaction between converging excitation and inhibition. The importance of chloride regulation is also underlined by its involvement in several brain pathologies, including epilepsy and disorders of the autistic spectra. The full comprehension of the physiological meaning of GABAergic activity on neurons requires the measurement of the spatiotemporal dynamics of chloride fluxes across the membrane. Nowadays, there are several available tools for the task, and both synthetic and genetically encoded indicators have been successfully used for chloride imaging. Here, we will review the available sensors analyzing their properties and outlining desirable future developments. PMID:25221475

  3. Piezo2 is the principal mechanotransduction channel for proprioception.

    PubMed

    Woo, Seung-Hyun; Lukacs, Viktor; de Nooij, Joriene C; Zaytseva, Dasha; Criddle, Connor R; Francisco, Allain; Jessell, Thomas M; Wilkinson, Katherine A; Patapoutian, Ardem

    2015-12-01

    Proprioception, the perception of body and limb position, is mediated by proprioceptors, specialized mechanosensory neurons that convey information about the stretch and tension experienced by muscles, tendons, skin and joints. In mammals, the molecular identity of the stretch-sensitive channel that mediates proprioception is unknown. We found that the mechanically activated nonselective cation channel Piezo2 was expressed in sensory endings of proprioceptors innervating muscle spindles and Golgi tendon organs in mice. Two independent mouse lines that lack Piezo2 in proprioceptive neurons showed severely uncoordinated body movements and abnormal limb positions. Moreover, the mechanosensitivity of parvalbumin-expressing neurons that predominantly mark proprioceptors was dependent on Piezo2 expression in vitro, and the stretch-induced firing of proprioceptors in muscle-nerve recordings was markedly reduced in Piezo2-deficient mice. Together, our results indicate that Piezo2 is the major mechanotransducer of mammalian proprioceptors.

  4. Phosphonium chloride for thermal storage

    NASA Technical Reports Server (NTRS)

    Sutton, J. G.; Heimlich, P. F.; Tepper, E. H.

    1972-01-01

    Development of systems for storage of thermal energy is discussed. Application of phosphonium chloride for heat storage through reversible dissociation is described. Chemical, physical, and thermodynamic properties of phosphonium chloride are analyzed and dangers in using phosphonium chloride are explained.

  5. Alcohol intoxication: Ion channels and genetics

    SciTech Connect

    Harris, A.R.; Allan, A.M. )

    1989-04-01

    Acute in vitro exposure to ethanol and other intoxicant-anesthetics activates {gamma}-aminobutyric acid (GABA)-stimulated chloride channels and inhibits voltage-dependent calcium and sodium channels of isolated brain membranes. The question of whether these neurochemical actions are responsible for intoxication in vivo has been addressed using animal populations displaying genetic differences in sensitivity to alcohol and benzodiazepine intoxication. These genetic approaches include inbred strains, selected lines, recombinant inbred strains, and heterogeneous stocks. Genetic differences in ion channel function provide strong evidence for a role of the GABA-stimulated chloride channel in ethanol and benzodiazepine intoxication; the role of calcium and sodium channels is less clear.

  6. Chloride flux in phagocytes.

    PubMed

    Wang, Guoshun

    2016-09-01

    Phagocytes, such as neutrophils and macrophages, engulf microbes into phagosomes and launch chemical attacks to kill and degrade them. Such a critical innate immune function necessitates ion participation. Chloride, the most abundant anion in the human body, is an indispensable constituent of the myeloperoxidase (MPO)-H2 O2 -halide system that produces the potent microbicide hypochlorous acid (HOCl). It also serves as a balancing ion to set membrane potentials, optimize cytosolic and phagosomal pH, and regulate phagosomal enzymatic activities. Deficient supply of this anion to or defective attainment of this anion by phagocytes is linked to innate immune defects. However, how phagocytes acquire chloride from their residing environment especially when they are deployed to epithelium-lined lumens, and how chloride is intracellularly transported to phagosomes remain largely unknown. This review article will provide an overview of chloride protein carriers, potential mechanisms for phagocytic chloride preservation and acquisition, intracellular chloride supply to phagosomes for oxidant production, and methods to measure chloride levels in phagocytes and their phagosomes.

  7. CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING ON LEFT, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING ON LEFT, CALCIUM CHLORIDE STORAGE BUILDING ON RIGHT OF CENTER WITH TOP OF SA (SODA ASH) BUILDING IN RIGHT BACKGROUND. - Solvay Process Company, Calcium Chloride Plant, Between Willis & Milton Avenues, Solvay, Onondaga County, NY

  8. Chloride in diet

    MedlinePlus

    Institute of Medicine. Food and Nutrition Board. Dietary Reference Intakes for Water, Potassium, Sodium, Chloride, and Sulfate. National Academy Press, Washington, DC: 2005. PMID: 101209392 www.ncbi.nlm.nih.gov/nlmcatalog/101209392 Mason JB. Vitamins, trace ...

  9. Hydrogen chloride test set

    NASA Technical Reports Server (NTRS)

    Workman, G. L.

    1976-01-01

    Detector uses tertiary amine, which makes reaction fairly specific for relatively small highly polarized hydrogen chloride molecule. Reaction is monitored by any microbalance capable of measuring extremely small mass differences in real time.

  10. Lithium Sulfuryl Chloride Battery.

    DTIC Science & Technology

    Primary batteries , Electrochemistry, Ionic current, Electrolytes, Cathodes(Electrolytic cell), Anodes(Electrolytic cell), Thionyl chloride ...Phosphorus compounds, Electrical conductivity, Calibration, Solutions(Mixtures), Electrical resistance, Performance tests, Solvents, Lithium compounds

  11. Mercuric chloride poisoning

    MedlinePlus

    ... Mercuric chloride is a very poisonous form of mercury. It is a type of mercury salt. There are different types of mercury poisonings . This article discusses poisoning from swallowing mercuric ...

  12. Strontium-89 Chloride

    MedlinePlus

    ... ever had bone marrow disease, blood disorders, or kidney disease.you should know that strontium-89 chloride may interfere with the normal menstrual cycle (period) in women and may stop sperm production ...

  13. Osmoregulated Chloride Currents in Hemocytes from Mytilus galloprovincialis

    PubMed Central

    Piazza, Veronica; Sbrana, Francesca; Vassalli, Massimo; Faimali, Marco

    2016-01-01

    We investigated the biophysical properties of the transport mediated by ion channels in hemocytes from the hemolymph of the bivalve Mytilus galloprovincialis. Besides other transporters, mytilus hemocytes possess a specialized channel sensitive to the osmotic pressure with functional properties similar to those of other transport proteins present in vertebrates. As chloride fluxes may play an important role in the regulation of cell volume in case of modifications of the ionic composition of the external medium, we focused our attention on an inwardly-rectifying voltage-dependent, chloride-selective channel activated by negative membrane potentials and potentiated by the low osmolality of the external solution. The chloride channel was slightly inhibited by micromolar concentrations of zinc chloride in the bath solution, while the antifouling agent zinc pyrithione did not affect the channel conductance at all. This is the first direct electrophysiological characterization of a functional ion channel in ancestral immunocytes of mytilus, which may bring a contribution to the understanding of the response of bivalves to salt and contaminant stresses. PMID:27936151

  14. Osmoregulated Chloride Currents in Hemocytes from Mytilus galloprovincialis.

    PubMed

    Bregante, Monica; Carpaneto, Armando; Piazza, Veronica; Sbrana, Francesca; Vassalli, Massimo; Faimali, Marco; Gambale, Franco

    2016-01-01

    We investigated the biophysical properties of the transport mediated by ion channels in hemocytes from the hemolymph of the bivalve Mytilus galloprovincialis. Besides other transporters, mytilus hemocytes possess a specialized channel sensitive to the osmotic pressure with functional properties similar to those of other transport proteins present in vertebrates. As chloride fluxes may play an important role in the regulation of cell volume in case of modifications of the ionic composition of the external medium, we focused our attention on an inwardly-rectifying voltage-dependent, chloride-selective channel activated by negative membrane potentials and potentiated by the low osmolality of the external solution. The chloride channel was slightly inhibited by micromolar concentrations of zinc chloride in the bath solution, while the antifouling agent zinc pyrithione did not affect the channel conductance at all. This is the first direct electrophysiological characterization of a functional ion channel in ancestral immunocytes of mytilus, which may bring a contribution to the understanding of the response of bivalves to salt and contaminant stresses.

  15. Effects of AP39, a novel triphenylphosphonium derivatised anethole dithiolethione hydrogen sulfide donor, on rat haemodynamic parameters and chloride and calcium Cav3 and RyR2 channels.

    PubMed

    Tomasova, Lenka; Pavlovicova, Michaela; Malekova, Lubica; Misak, Anton; Kristek, Frantisek; Grman, Marian; Cacanyiova, Sona; Tomasek, Milan; Tomaskova, Zuzana; Perry, Alexis; Wood, Mark E; Lacinova, Lubica; Ondrias, Karol; Whiteman, Matthew

    2015-04-30

    H2S donor molecules have the potential to be viable therapeutic agents. The aim of this current study was (i) to investigate the effects of a novel triphenylphosphonium derivatised dithiolethione (AP39), in the presence and absence of reduced nitric oxide bioavailability and (ii) to determine the effects of AP39 on myocardial membrane channels; CaV3, RyR2 and Cl(-). Normotensive, L-NAME- or phenylephrine-treated rats were administered Na2S, AP39 or control compounds (AP219 and ADT-OH) (0.25-1 µmol kg(-1)i.v.) and haemodynamic parameters measured. The involvement of membrane channels T-type Ca(2+) channels CaV3.1, CaV3.2 and CaV3.3 as well as Ca(2+) ryanodine (RyR2) and Cl(-) single channels derived from rat heart sarcoplasmic reticulum were also investigated. In anaesthetised Wistar rats, AP39 (0.25-1 µmol kg(-1) i.v) transiently decreased blood pressure, heart rate and pulse wave velocity, whereas AP219 and ADT-OH and Na2S had no significant effect. In L-NAME treated rats, AP39 significantly lowered systolic blood pressure for a prolonged period, decreased heart rate and arterial stiffness. In electrophysiological studies, AP39 significantly inhibited Ca(2+) current through all three CaV3 channels. AP39 decreased RyR2 channels activity and increased conductance and mean open time of Cl(-) channels. This study suggests that AP39 may offer a novel therapeutic opportunity in conditions whereby (•)NO and H2S bioavailability are deficient such as hypertension, and that CaV3, RyR2 and Cl(-) cardiac membrane channels might be involved in its biological actions.

  16. CHLORIDE RETENTION IN EXPERIMENTAL HYDRONEPHROSIS

    PubMed Central

    Keith, Norman M.; Pulford, D. Schuyler

    1923-01-01

    1. In acute experimental hydronephrosis chloride retention occurs as well as retention of water, urea, and phenolsulfonephthalein. 2. If both water and chlorides are retained there may be no appreciable rise in the plasma chloride content. 3. When chlorides are retained, but not water, the chloride content of the plasma rises strikingly. 4. After the removal of the ureteral obstruction in acute hydronephrosis all renal functions, water, urea, and chloride excretion, may be rapidly restored in equal degree, or the chlorides may be retained temporarily while there is free excretion of water and urea. 5. In chronic hydronephrosis adequate daily excretion of urea and chlorides may be maintained by a compensatory polyuria. 6. Chloride retention or an abnormal chloride excretion may occur in certain renal lesions when there is no change in the urea, phenolsulfonephthalein, or water excretion. PMID:19868720

  17. Cyclic Voltammetry of Silver Chloride in Lithium Chloride-Potassium Chloride Eutectic.

    DTIC Science & Technology

    TRY), Fused salts, Silver, Reduction(Chemistry), Dissolving, ChloridesSilver chloride, Cyclic voltammetry , *VoltammetryThe technique of cyclic ... voltammetry was employed to study the deposition and dissolution of silver metal at platinum wire electrodes in molten lithium chloride-potassium chloride

  18. Chloride removal from vitrification offgas

    SciTech Connect

    Slaathaug, E.J.

    1995-06-01

    This study identified and investigated techniques of selectively purging chlorides from the low-level waste (LLW) vitrification process with the purge stream acceptable for burial on the Hanford Site. Chlorides will be present in high concentration in several individual feeds to the LLW Vitrification Plant. The chlorides are highly volatile in combustion type melters and are readily absorbed by wet scrubbing of the melter offgas. The Tank Waste Remediation System (TWRS) process flow sheets show that the resulting chloride rich scrub solution is recycled back to the melter. The chlorides must be purged from the recycle loop to prevent the buildup of excessively high chloride concentrations.

  19. Pharmacological analysis of epithelial chloride secretion mechanisms in adult murine airways.

    PubMed

    Gianotti, Ambra; Ferrera, Loretta; Philp, Amber R; Caci, Emanuela; Zegarra-Moran, Olga; Galietta, Luis J V; Flores, Carlos A

    2016-06-15

    Defective epithelial chloride secretion occurs in humans with cystic fibrosis (CF), a genetic defect due to loss of function of CFTR, a cAMP-activated chloride channel. In the airways, absence of an active CFTR causes a severe lung disease. In mice, genetic ablation of CFTR function does not result in similar lung pathology. This may be due to the expression of an alternative chloride channel which is activated by calcium. The most probable protein performing this function is TMEM16A, a calcium-activated chloride channel (CaCC). Our aim was to assess the relative contribution of CFTR and TMEM16A to chloride secretion in adult mouse trachea. For this purpose we tested pharmacological inhibitors of chloride channels in normal and CF mice. The amplitude of the cAMP-activated current was similar in both types of animals and was not affected by a selective CFTR inhibitor. In contrast, a CaCC inhibitor (CaCCinh-A01) strongly blocked the cAMP-activated current as well as the calcium-activated chloride secretion triggered by apical UTP. Although control experiments revealed that CaCCinh-A01 also shows inhibitory activity on CFTR, our results indicate that transepithelial chloride secretion in adult mouse trachea is independent of CFTR and that another channel, possibly TMEM16A, performs both cAMP- and calcium-activated chloride transport. The prevalent function of a non-CFTR channel may explain the absence of a defect in chloride transport in CF mice.

  20. The role of chloride in the lens of the eye.

    PubMed

    Zhang, J J; Jacob, T J

    1997-03-01

    Until recently, the investigation of membrane ion transport mechanisms and their relationship with cataract formation has mainly focused on sodium, potassium and calcium. The specific role of chloride in solute transport within the lens has been given little attention. Rather, chloride was considered as simply the counterion to sodium. The purpose of this review is to emphasize the importance of chloride and its involvement in the membrane ion transport systems within the lens. We summarize the general physiological and chemical properties of the chloride ion in the lens, with reference to the regional ion fluxes generated by its special anatomical and electrical structure. We also present our current knowledge of the principal ion transport mechanisms associated with chloride, with particular emphasis on Cl- channels, and discuss their possible physiological significance. Maintenance of a constant cell volume is an evolutionarily ancient homeostatic process and we present some important findings associated with volume regulatory mechanisms in the whole lens and in single lens cells. Finally, we review and discuss the link between cataract formation and chloride channel dysfunction.

  1. Moxifloxacinium chloride monohydrate

    PubMed Central

    Qian, Jing-Jing; Gu, Jian-Ming; Shen, Jin; Hu, Xiu-Rong; Wu, Su-Xiang

    2011-01-01

    The title compound {systematic name: 7-[(1S,6S)-8-aza-2-azonia­bicyclo­[4.3.0]non-8-yl]-1-cyclo­propyl-6-fluoro-8-meth­oxy-4-oxo-1,4-dihydro­quinoline-3-carb­oxy­lic acid chloride monohydrate}, C21H25FN3O4 +·Cl−·H2O, crystallizes with two moxi­floxa­cinium cations, two chloride ions and two uncoordinated water mol­ecules in the unit cell. The crystal structure has a pseudo-inversion center except for the chloride ions. In both moxi­floxa­cinium cations, the quinoline rings are approximately planar, the maximum atomic deviations being 0.107 (3) and 0.118 (3) Å. The piperidine rings adopt a chair conformation while the pyrrolidine rings display a half-chair conformation. In the crystal, the carboxyl groups, the protonated piperidyl groups, the uncoordinated water mol­ecule and chloride anions participate in O—H⋯O, O—H⋯Cl and N—H⋯Cl hydrogen bonding; weak inter­molecular C—H⋯O and C—H⋯Cl hydrogen bonding is also present in the crystal structure. PMID:22058817

  2. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells

    NASA Astrophysics Data System (ADS)

    Saha, Sonali; Prakash, Ved; Halder, Saheli; Chakraborty, Kasturi; Krishnan, Yamuna

    2015-07-01

    The concentration of chloride ions in the cytoplasm and subcellular organelles of living cells spans a wide range (5-130 mM), and is tightly regulated by intracellular chloride channels or transporters. Chloride-sensitive protein reporters have been used to study the role of these chloride regulators, but they are limited to a small range of chloride concentrations and are pH-sensitive. Here, we show that a DNA nanodevice can precisely measure the activity and location of subcellular chloride channels and transporters in living cells in a pH-independent manner. The DNA nanodevice, called Clensor, is composed of sensing, normalizing and targeting modules, and is designed to localize within organelles along the endolysosomal pathway. It allows fluorescent, ratiometric sensing of chloride ions across the entire physiological regime. We used Clensor to quantitate the resting chloride concentration in the lumen of acidic organelles in Drosophila melanogaster. We showed that lumenal lysosomal chloride, which is implicated in various lysosomal storage diseases, is regulated by the intracellular chloride transporter DmClC-b.

  3. Chloride transport in functionally active phagosomes isolated from Human neutrophils

    PubMed Central

    Aiken, Martha L.; Painter, Richard G.; Zhou, Yun; Wang, Guoshun

    2012-01-01

    Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC–dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport

  4. Dynamic [Cl(-)](i) measurement with chloride sensing quantum dots nanosensor in epithelial cells.

    PubMed

    Wang, Yuchi; Mao, Hua; Wong, Lid B

    2010-02-05

    We have synthesized a chloride sensing quantum dots (QD) nanosensor, Cl-QD, for the dynamic measurements of chloride ion concentration in the millimolar range, a sensitivity that is applicable to most physiological intracellular chloride ion concentration ([Cl(-)](i)) measurements in epithelial cells. The Cl-QD is synthesized by conjugating an anion receptor, 1-(2-mercapto-ethyl)-3-phenyl-thiourea (MEPTU) to a water soluble CdSe/ZnS QD at an emission wavelength of 620 nm. Upon binding of chloride ions to the Cl-QD, a photo-induced electron transfer mechanism caused the fluorescence of the QD to quench. This resulted in an inversely proportional relationship between the chloride ion concentration and the fluorescence intensity of the Cl-QD. We have utilized this Cl-QD to measure [Cl(-)](i) in T84 and CF-PAC cultured cells, with either the C1C-2 or CFTR chloride channels being manipulated by pharmacological chloride channel activators and inhibitors. Activations of C1C-2 and CFTR chloride channels in T84 by the respective lubiprostone and genistein caused predictive increases in the fluorescence of the Cl-QD, i.e., a decrease of [Cl(-)](i). Conversely, glibenclamide, a chloride channel inhibitor, applied to the CF-PAC cells caused a predictable decrease in the fluorescence of Cl-QD due to the increase of [Cl(-)](i). These are the first data in using QD-based chloride ion sensors for dynamic measurements of intracellular chloride ion concentrations in epithelial cells.

  5. Dynamic [Cl-]i measurement with chloride sensing quantum dots nanosensor in epithelial cells

    NASA Astrophysics Data System (ADS)

    Wang, Yuchi; Mao, Hua; Wong, Lid B.

    2010-02-01

    We have synthesized a chloride sensing quantum dots (QD) nanosensor, Cl-QD, for the dynamic measurements of chloride ion concentration in the millimolar range, a sensitivity that is applicable to most physiological intracellular chloride ion concentration ([Cl-]i) measurements in epithelial cells. The Cl-QD is synthesized by conjugating an anion receptor, 1-(2-mercapto-ethyl)-3-phenyl-thiourea (MEPTU) to a water soluble CdSe/ZnS QD at an emission wavelength of 620 nm. Upon binding of chloride ions to the Cl-QD, a photo-induced electron transfer mechanism caused the fluorescence of the QD to quench. This resulted in an inversely proportional relationship between the chloride ion concentration and the fluorescence intensity of the Cl-QD. We have utilized this Cl-QD to measure [Cl-]i in T84 and CF-PAC cultured cells, with either the C1C-2 or CFTR chloride channels being manipulated by pharmacological chloride channel activators and inhibitors. Activations of C1C-2 and CFTR chloride channels in T84 by the respective lubiprostone and genistein caused predictive increases in the fluorescence of the Cl-QD, i.e., a decrease of [Cl-]i. Conversely, glibenclamide, a chloride channel inhibitor, applied to the CF-PAC cells caused a predictable decrease in the fluorescence of Cl-QD due to the increase of [Cl-]i. These are the first data in using QD-based chloride ion sensors for dynamic measurements of intracellular chloride ion concentrations in epithelial cells.

  6. A Quick Reference on Chloride.

    PubMed

    Bohn, Andrea A; de Morais, Helio Autran

    2017-03-01

    Chloride is an essential element, playing important roles in digestion, muscular activity, regulation of body fluids, and acid-base balance. As the most abundant anion in extracellular fluid, chloride plays a major role in maintaining electroneutrality. Chloride is intrinsically linked to sodium in maintaining osmolality and fluid balance and has an inverse relationship with bicarbonate in maintaining acid-base balance. It is likely because of these close ties that chloride does not get the individual attention it deserves; we can use these facts to simplify and interpret changes in serum chloride concentrations.

  7. Fungal colonization with Pneumocystis correlates to increasing chloride channel accessory 1 (hCLCA1) suggesting a pathway for up-regulation of airway mucus responses, in infant lungs

    PubMed Central

    Pérez, Francisco J.; Ponce, Carolina A.; Rojas, Diego A.; Iturra, Pablo A.; Bustamante, Rebeca I.; Gallo, Myriam; Hananias, Karime; Vargas, Sergio L.

    2014-01-01

    Fungal colonization with Pneumocystis is associated with increased airway mucus in infants during their primary Pneumocystis infection, and to severity of COPD in adults. The pathogenic mechanisms are under investigation. Interestingly, increased levels of hCLCA1 – a member of the calcium-sensitive chloride conductance family of proteins that drives mucus hypersecretion – have been associated with increased mucus production in patients diagnosed with COPD and in immunocompetent rodents with Pneumocystis infection. Pneumocystis is highly prevalent in infants; therefore, the contribution of Pneumocystis to hCLCA1 expression was examined in autopsied infant lungs. Respiratory viruses that may potentially increase mucus, were also examined. hCLCA1 expression was measured using actin-normalized Western-blot, and the burden of Pneumocystis organisms was quantified by qPCR in 55 autopsied lungs from apparently healthy infants who died in the community. Respiratory viruses were diagnosed using RT-PCR for RSV, metapneumovirus, influenza, and parainfluenza viruses; and by PCR for adenovirus. hCLCA1 levels in virus positive samples were comparable to those in virus-negative samples. An association between Pneumocystis and increased hCLCA1 expression was documented (P=0.028). Additionally, increasing Pneumocystis burden correlated with increasing hCLCA1 protein expression levels (P=0.017). Results strengthen the evidence of Pneumocystis-associated up-regulation of mucus-related airway responses in infant lungs. Further characterization of this immunocompetent host-Pneumocystis-interaction, including assessment of potential clinical significance, is warranted. PMID:25379375

  8. ClC-3 Chloride Channel Proteins Regulate the Cell Cycle by Up-regulating cyclin D1-CDK4/6 through Suppressing p21/p27 Expression in Nasopharyngeal Carcinoma Cells

    PubMed Central

    Ye, Dong; Luo, Hai; Lai, Zhouyi; Zou, Lili; Zhu, Linyan; Mao, Jianwen; Jacob, Tim; Ye, Wencai; Wang, Liwei; Chen, Lixin

    2016-01-01

    It was shown in this study that knockdown of ClC-3 expression by ClC-3 siRNA prevented the activation of hypotonicity-induced chloride currents, and arrested cells at the G0/G1 phase in nasopharyngeal carcinoma CNE-2Z cells. Reconstitution of ClC-3 expression with ClC-3 expression plasmids could rescue the cells from the cell cycle arrest caused by ClC-3 siRNA treatments. Transfection of cells with ClC-3 siRNA decreased the expression of cyclin D1, cyclin dependent kinase 4 and 6, and increased the expression of cyclin dependent kinase inhibitors (CDKIs), p21 and p27. Pretreatments of cells with p21 and p27 siRNAs depleted the inhibitory effects of ClC-3 siRNA on the expression of CDK4 and CDK6, but not on that of cyclin D1, indicating the requirement of p21 and p27 for the inhibitory effects of ClC-3 siRNA on CDK4 and CDK6 expression. ClC-3 siRNA inhibited cells to progress from the G1 phase to the S phase, but pretreatments of cells with p21 and p27 siRNAs abolished the inhibitory effects of ClC-3 siRNA on the cell cycle progress. Our data suggest that ClC-3 may regulate cell cycle transition between G0/G1 and S phases by up-regulation of the expression of CDK4 and CDK6 through suppression of p21 and p27 expression. PMID:27451945

  9. ClC-3 Chloride Channel Proteins Regulate the Cell Cycle by Up-regulating cyclin D1-CDK4/6 through Suppressing p21/p27 Expression in Nasopharyngeal Carcinoma Cells.

    PubMed

    Ye, Dong; Luo, Hai; Lai, Zhouyi; Zou, Lili; Zhu, Linyan; Mao, Jianwen; Jacob, Tim; Ye, Wencai; Wang, Liwei; Chen, Lixin

    2016-07-25

    It was shown in this study that knockdown of ClC-3 expression by ClC-3 siRNA prevented the activation of hypotonicity-induced chloride currents, and arrested cells at the G0/G1 phase in nasopharyngeal carcinoma CNE-2Z cells. Reconstitution of ClC-3 expression with ClC-3 expression plasmids could rescue the cells from the cell cycle arrest caused by ClC-3 siRNA treatments. Transfection of cells with ClC-3 siRNA decreased the expression of cyclin D1, cyclin dependent kinase 4 and 6, and increased the expression of cyclin dependent kinase inhibitors (CDKIs), p21 and p27. Pretreatments of cells with p21 and p27 siRNAs depleted the inhibitory effects of ClC-3 siRNA on the expression of CDK4 and CDK6, but not on that of cyclin D1, indicating the requirement of p21 and p27 for the inhibitory effects of ClC-3 siRNA on CDK4 and CDK6 expression. ClC-3 siRNA inhibited cells to progress from the G1 phase to the S phase, but pretreatments of cells with p21 and p27 siRNAs abolished the inhibitory effects of ClC-3 siRNA on the cell cycle progress. Our data suggest that ClC-3 may regulate cell cycle transition between G0/G1 and S phases by up-regulation of the expression of CDK4 and CDK6 through suppression of p21 and p27 expression.

  10. Oxomemazine hydro-chloride.

    PubMed

    Siddegowda, M S; Butcher, Ray J; Akkurt, Mehmet; Yathirajan, H S; Ramesh, A R

    2011-08-01

    IN THE TITLE COMPOUND [SYSTEMATIC NAME: 3-(5,5-dioxo-phen-othia-zin-10-yl)-N,N,2-trimethyl-propanaminium chloride], C(18)H(23)N(2)O(2)S(+)·Cl(-), the dihedral angle between the two outer aromatic rings of the phenothia-zine unit is 30.5 (2)°. In the crystal, the components are linked by N-H⋯Cl and C-H⋯Cl hydrogen bonds and C-H⋯π inter-actions.

  11. Stimulation of Wild-Type, F508del- and G551D-CFTR Chloride Channels by Non-Toxic Modified pyrrolo[2,3-b]pyrazine Derivatives.

    PubMed

    Dannhoffer, Luc; Billet, Arnaud; Jollivet, Mathilde; Melin-Heschel, Patricia; Faveau, Christelle; Becq, Frédéric

    2011-01-01

    Cystic fibrosis (CF) is a major inherited disorder involving abnormalities of fluid and electrolyte transport in a number of different organs due to abnormal function of cystic fibrosis transmembrane conductance regulator (CFTR) protein. We recently identified a family of CFTR activators, which contains the hit: RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]-pyrrolo[2,3-b]pyrazine]. Here, we further evaluated the effect of the chemical modifications of the RP107-OH radical on CFTR activation. The replacement of the OH radical by a fluorine atom at position 2 (RP193) or 4 (RP185) significantly decreased the toxicity of the compounds without altering the ability to activate CFTR, especially for RP193. The non-toxic compound RP193 has no effect on cAMP production but stimulates the channel activity of wild-type CFTR in stably transfected CHO cells, in human bronchial epithelial NuLi-1 cells, and in primary culture of human bronchial epithelial cells (HBEC). Whole-cell and single patch-clamp recordings showed that RP193 induced a linear, time- and voltage-independent current, which was fully inhibited by two different and selective CFTR inhibitors (CFTRinh-172 and GP(inh)5a). Moreover, RP193 stimulates CFTR in temperature-rescued CuFi-1 (F508del/F508del) HBEC and in CHO cells stably expressing G551D-CFTR. This study shows that it is feasible to reduce cytotoxicity of chemical compounds without affecting their potency to activate CFTR and to rescue the class 2 F508del-CFTR and class 3 G551D-CFTR CF mutant activities.

  12. An intracellular anion channel critical for pigmentation.

    PubMed

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-12-16

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation.

  13. Benzalkonium Chloride and Glaucoma

    PubMed Central

    Kaufman, Paul L.; Kiland, Julie A.

    2014-01-01

    Abstract Glaucoma patients routinely take multiple medications, with multiple daily doses, for years or even decades. Benzalkonium chloride (BAK) is the most common preservative in glaucoma medications. BAK has been detected in the trabecular meshwork (TM), corneal endothelium, lens, and retina after topical drop installation and may accumulate in those tissues. There is evidence that BAK causes corneal and conjunctival toxicity, including cell loss, disruption of tight junctions, apoptosis and preapoptosis, cytoskeleton changes, and immunoinflammatory reactions. These same effects have been reported in cultured human TM cells exposed to concentrations of BAK found in common glaucoma drugs and in the TM of primary open-angle glaucoma donor eyes. It is possible that a relationship exists between chronic exposure to BAK and glaucoma. The hypothesis that BAK causes/worsens glaucoma is being tested experimentally in an animal model that closely reflects human physiology. PMID:24205938

  14. Benzalkonium chloride and glaucoma.

    PubMed

    Rasmussen, Carol A; Kaufman, Paul L; Kiland, Julie A

    2014-01-01

    Glaucoma patients routinely take multiple medications, with multiple daily doses, for years or even decades. Benzalkonium chloride (BAK) is the most common preservative in glaucoma medications. BAK has been detected in the trabecular meshwork (TM), corneal endothelium, lens, and retina after topical drop installation and may accumulate in those tissues. There is evidence that BAK causes corneal and conjunctival toxicity, including cell loss, disruption of tight junctions, apoptosis and preapoptosis, cytoskeleton changes, and immunoinflammatory reactions. These same effects have been reported in cultured human TM cells exposed to concentrations of BAK found in common glaucoma drugs and in the TM of primary open-angle glaucoma donor eyes. It is possible that a relationship exists between chronic exposure to BAK and glaucoma. The hypothesis that BAK causes/worsens glaucoma is being tested experimentally in an animal model that closely reflects human physiology.

  15. Role of chloride transport proteins in the vasorelaxant action of nitroprusside in isolated rat aorta.

    PubMed

    Valero, Marta; Pereboom, Désirée; Garay, Ricardo P; Alda, José Octavio

    2006-12-28

    Chloride ions play a key role in smooth muscle contraction, but little is known concerning their role in smooth muscle relaxation. Here we investigated the effect of chloride transport inhibitors on the vasorelaxant responses to nitroprusside in isolated and endothelium-denuded rat aorta, precontracted with phenylephrine 1 muM. Incubation of aortic rings in NO(3)(-) media strongly potentiated the vasorelaxant responses to nitroprusside. Bumetanide, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and acetazolamide strongly potentiated the vasorelaxant responses to nitroprusside (by 70-100%). EC(50) were 2.3+/-0.5 microM for bumetanide, 26+/-15 microM for DIDS and 510+/-118 microM for acetazolamide (n=6 for condition). Niflumic acid, a selective inhibitor of ClCa (calcium-activated chloride channels), potentiated nitroprusside relaxation to a similar extent as chloride transport inhibitors, in a non-additive manner. Zinc and nickel ions, both modestly potentiated nitroprusside vasorelaxation (by 20-30%). Cobaltum had negligible effect on nitroprusside vasorelaxation. CPA (p-chlorophenoxy-acetic acid), an inhibitor of volume-sensitive chloride channels (ClC), slightly potentiated nitroprusside vasorelaxation (by 15%), and the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel inhibitors CFTR(inh)172 (5-[(4-Carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone), DPC (diphenylamine-2,2'-dicarboxylic acid) and glibenclamide were without significant effect. In conclusion, inhibition of chloride transport proteins strongly potentiates the vasorelaxant responses to nitroprusside in isolated rat aorta. This effect seems mediated by chloride depletion and inhibition of a chloride channel activated by both, calcium and cyclic GMP (cGMP).

  16. Measurement of atmospheric vinyl chloride.

    PubMed

    Lande, S S

    1979-02-01

    Methods for atmospheric vinyl chloride measurement have been reviewed. The lowest detection limits and most specific measurement are achieved by scrubbing atmospheric samples with activated charcoal, desorbing the vinyl chloride, and assaying it by gas chromatography (GC). NIOSH currently recommends collecting samples using tubes packed with 150 mg of coconut shell charcoal, desorbing with carbon disulfide, and analyzing by GC equipped with flame-ionization detection (FID); the method is capable of detecting less than 1 ppm vinyl chloride and has an apparent recovery of abo the ppb level with no loss of accuracy or precision. Some field methods, such as infrared analysis and conductivity measurement, are capable of detecting 1 ppm or lower but are subject to interferences by other contaminants; th-y could be useful for evaluating sources of vinyl chloride leaks and for continuous monitoring. Permeation tubes are superior to gravimetric or volumetric methods for generating atmospheres of known vinyl chloride concentration.

  17. Antiviral effect of lithium chloride.

    PubMed

    Cernescu, C; Popescu, L; Constantinescu, S; Cernescu, S

    1988-01-01

    Studies in human embryo fibroblasts infected with measles or herpes simplex virus showed a reduction in virus yield when cultures were pretreated with 1-10 mM lithium chloride doses. Maximum effect was obtained by a 1 h treatment with 10 mM lithium chloride, preceding viral infection by 19-24 hours. A specific antiviral effect against measles virus was manifest immediately after culture pretreatment. Intermittent treatment with 10 mM lithium chloride of cultures persistently infected with measles or herpes virus obtained from human myeloid K-562 cell line shows a reduction in the extracellular virus yield. In the K-562/herpes virus system, the culture treatment with lithium chloride and acyclovir (10 microM) has an additive inhibitory effect on virus production. The paper is focused on the mechanism of lithium chloride antiviral action and the expediency of lithium therapy in SSPE (subacute sclerosing panencephalitis).

  18. Myotonic discharges discriminate chloride from sodium muscle channelopathies.

    PubMed

    Drost, Gea; Stunnenberg, Bas C; Trip, Jeroen; Borm, George; McGill, Kevin C; Ginjaar, Ieke H B; van der Kooi, Arendina W; Zwarts, Machiel J; van Engelen, Baziel G M; Faber, Catharina G; Stegeman, Dick F; Lateva, Zoia

    2015-01-01

    Non-dystrophic myotonic syndromes represent a heterogeneous group of clinically quite similar diseases sharing the feature of myotonia. These syndromes can be separated into chloride and sodium channelopathies, with gene-defects in chloride or sodium channel proteins of the sarcolemmal membrane. Myotonia has its basis in an electrical instability of the sarcolemmal membrane. In the present study we examine the discriminative power of the resulting myotonic discharges for these disorders. Needle electromyography was performed by an electromyographer blinded for genetic diagnosis in 66 non-dystrophic myotonia patients (32 chloride and 34 sodium channelopathy). Five muscles in each patient were examined. Individual trains of myotonic discharges were extracted and analyzed with respect to firing characteristics. Myotonic discharge characteristics in the rectus femoris muscle almost perfectly discriminated chloride from sodium channelopathy patients. The first interdischarge interval as a single variable was longer than 30 ms in all but one of the chloride channelopathy patients and shorter than 30 ms in all of the sodium channelopathy patients. This resulted in a detection rate of over 95%. Myotonic discharges of a single muscle can be used to better guide toward a molecular diagnosis in non-dystrophic myotonic syndromes.

  19. Cation-chloride cotransporters in neuronal development, plasticity and disease

    PubMed Central

    Kaila, Kai; Price, Theodore J.; Payne, John A.; Puskarjov, Martin; Voipio, Juha

    2015-01-01

    Electrical activity in neurons requires a seamless functional coupling between plasmalemmal ion channels and ion transporters. Although ion channels have been studied intensively for several decades, research on ion transporters is in its infancy. In recent years, it has become evident that one family of ion transporters, cation-chloride cotransporters (CCCs), and in particular K+–Cl− cotransporter 2 (KCC2), have seminal roles in shaping GABAergic signalling and neuronal connectivity. Studying the functions of these transporters may lead to major paradigm shifts in our understanding of the mechanisms underlying brain development and plasticity in health and disease. PMID:25234263

  20. Lithium-Thionyl Chloride Battery.

    DTIC Science & Technology

    1981-04-01

    EEEElhIhEEEEEE 1111 1 - MI(CRO( fy Hl ff1Sf UIIIUN Ift I IA I~t Research and Development Technical Report DELET - TR - 78 - 0563 - F Cq LITHIUM - THIONYL CHLORIDE ...2b(1110) S. TYPE OF REPORT & PERIOD COVERED Lithium - Thionyl Chloride Battery -10/1/78 - 11/30/80 6. PNING ORG. REPORT NUMBER Z %A a.~as B.,OWRACT OR...block number) Inorganic Electrolyte battery, Thionyl Chloride , lithium , high rate D cell, high rate flat cylindrical cell, laser designator battery. C//i